Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Clinical features, cardiac phenotype and translocation breakpoint structure in DGAP105. ( A ) Facial features at 4 years of age included mild hypertelorism, bilateral epicanthal folds, downslanting palpebral fissures, strabismus and a broad nose with a smooth philtrum and thin vermillion border. ( B ) Ideograms depicting 46,XY,t(1;5)(p36.11;q31.2)dn . Arrows mark locations of AHDC1 and MATR3 breakpoints. ( C and D ) Echocardiograms at age of 2 days. (C) Ductal view showing distal aortic arch, CoA just distal to the left subclavian artery (LSCA), accompanied by a prominent posterior unfolding (‘posterior shelf’) and PDA. (D) Aortic arch view showing ascending aorta (5.7-mm diameter), hypoplastic aortic arch (4.4-mm diameter) and CoA posterior shelf. ( E ) Summary of the 1p36.11 and 5q31.2 breakpoints in DGAP105. The 1p36.11 breakpoint disrupts AHDC1 intron 1, whereas the 5q31.2 breakpoint disrupts MATR3 exon 15 in the 3′ UTR. BACs used in FISH analyses are indicated.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Translocation Assay

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Analysis of human MATR3 transcripts and protein expression in control and DGAP105 lymphoblasts. ( A ) Schematic of the MATR3 exon 13–15 region with the chromosomal translocation breakpoint in patient DGAP105 marked by dotted line. The proximal AAUAAA polyadenylation signal and the distal AAUAAA polyadenylation signal site are shown flanking the breakpoint in the 3′ UTR. TAA denotes the stop codon in exon 15. ( B ) MATR3 3′ RACE products in human control (Lane 1) and DGAP105 lymphoblast (Lanes 2, 3), and control human fetal heart tissue (Lanes 4, 5). RT ‘+’ or ‘−’ denote inclusion or omission of reverse transcriptase in the cDNA synthesis. The large product (1589 bp, arrow) uses the distal polyadenylation signal and predominates in control human lymphoblasts (Lane 1). In contrast, the short product (963 bp, arrow) predominates in DGAP105 lymphoblasts (Lane 2) and in control human fetal heart (Lane 4) and represents MATR3 transcripts that use the proximal polyadenylation signal. ( C ) Northern blot analysis of adult human tissues shows MATR3 transcripts of ∼3.5 and ∼2.9 kb. In heart and skeletal muscle, the 2.9-kb transcript predominates and likely corresponds to the 3′ RACE product using the proximal polyadenylation signal. In brain and other tissues, the 3.5-kb transcript predominates and corresponds to the 3′ RACE product using the distal polyadenylation signal. ( D ) Western blot analysis of protein isolated from DGAP105 and three control lymphoblast lines, showing up-regulation of Matrin 3 in DGAP105 compared with controls. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars represent the mean fold expression of four independent experiments ± SEM, corrected for loading, and normalized to Control 2; * P < 0.05 between DGAP105 and mean of the three control lines via paired Student's t test.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Expressing, Control, Translocation Assay, Reverse Transcription, cDNA Synthesis, Northern Blot, Western Blot, Isolation

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Analysis of mouse Matr3 and Ahdc1 transcripts expression in developing heart. ( A ) RT–PCR analyses of Matr3 and Ahdc1 . ( a ) Semi-quantitative RT–PCR analyses show strong Matr 3 expression in developing mouse heart, limb and brain at E11.5, 16.5 stages, ( b ) with down-regulation at the newborn (NB) and adult stages. In contrast, Ahdc1 expression is only weakly detected in limb and brain at E11.5 and 16.5. ( B ) Section in situ hybridizations at E11.5 for mouse Matr3 and Ahdc1. Matr3 is expressed in CNS, pharyngeal arches, limb buds and in the developing heart (enlarged section), whereas Ahdc1 expression was undetectable in heart (enlarged section). Sense controls (not shown) showed no expression.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, In Situ

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Analysis of the Matr3 Gt-ex13 gene trap allele. ( A ) Structure of mouse Matr3 wild-type and Gt-ex13 gene trap mutant alleles. Wild-type mouse Matr3 encodes an 846-amino acid protein. Intron 12 (2749 bp) and exon 13 (223 bp) are shown. Matr3 Gt-ex-13 gene trap allele inserts a β-Geo gene trap vector at position 118 bp in exon 13, which will generate Matr3-β-geo fusion transcripts. PCR genotyping primers depict WT-F1 (in exon 13) and WT-R1 (in intron 13) for the wild-type allele, and Mu-F1 (in exon 13) and Mu-R1 (in gene trap vector) for the mutant allele. Primers used in 3′ RACE are summarized on Materials and Methods. ( B ) E3.5 PCR genotyping shows 394-bp wild-type and 492-bp mutant alleles for wild-type (+/+), heterozygous (+/−) and homozygous (−/−) embryos. ( C ) Matr3 GT-ex13 3′ RACE analysis of mouse E14.5 brain and heart tissues detects a novel Matr3-β-Geo fusion transcript (∼4 kb) in heterozygotes. The long Matr3 3′ RACE product (1647 bp), the only form detected in brain, is reduced in heterozygous brain. Both long and short Matr3 3′ RACE products (1647 and 1025 bp) are reduced in heterozygous heart. ( D ) Western blot analysis of Matrin 3 protein isolated from wild-type and heterozygous mouse E15.5 brain and heart tissues. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars are fold ± SEM expression level from mean of three independent experiments, corrected for loading, and normalized to a value of 1.0 for wild-type heart. The small increase in expression in Matr3 Gt-ex13/+ heart is not statistically significant.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Mutagenesis, Plasmid Preparation, Western Blot, Isolation, Control, Expressing

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Early embryonic lethality in Matr3 GT-ex13 homozygous mouse embryos

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques:

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: X-Gal staining of Matr3 Gt-ex13 heterozygotes. ( A ) X-Gal staining of primitive heart (arrow) in E10.5 Matr3 heterozygote, and in CNS (brain, spinal cord), pharyngeal arches and limb bud. ( B ) X-Gal staining in primitive heart of E8.5 Matr3 heterozygote. Arrow depicts dorsal mesocardium; BC, bulbus cordis; CVC, common ventricular chamber. ( C ) Negative control wild-type E9.5 embryo with eosin counterstain. ( D ) X-Gal staining in wall (arrow) of atrial chamber (AC), bulbus cordis (BC) and ( E ) myocardium and endocardium, and ( F ) interventricular septum (IVS) of newborn (NB) Matr3 GT-ex13 heterozygote heart (arrows).

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Staining, Negative Control

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Matrin 3 protein cardiovascular expression in newborn mice. ( A ) Matrin 3 expression in wild-type newborn heart. ( B ) Pulmonary valve (PV) from (A) shows Matrin 3 in interstitial and endocardial cells. ( C ) Matrin 3 in cardiomyocyte nuclei (arrow). ( D–F ) Immunostaining in arterial vascular smooth muscle and endothelial cells for Matrin 3 (D), PECAM (E) and merged (F). Matrin 3 is expressed in both arterial smooth muscle cell nuclei (green) external to PECAM-1 endothelial cell membrane staining (red) and internal to the PECAM-1 domain, in endothelial cell nuclei. This is better seen in ( G–I ) with Matrin 3 and PECAM in small venules, which are largely devoid of vascular smooth muscle. Arrows in (I) denote Matrin 3 in endothelial cell nuclei. Scale bar: 40 μm (D–F); 5 μm (G–I).

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Expressing, Immunostaining, Membrane, Staining

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Subaortic VSD and DORV phenotypes in Matr3 Gt-ex13 heterozygotes. Transverse serial sections through the hearts of E18.5 wild type ( A and B ), and two different representative Matr3 Gt-ex13 heterozygotes (embryo 1 in C and D and embryo 2 in E and F ), each sectioned at a cranial and caudal level, illustrating the DORV with subaortic VSD phenotype in Matr3 Gt-ex13 heterozygotes (see Table ). (A and B) Wild-type sections show left and right ventricles separated by the interventricular septum, the two atrioventricular (tricuspid, mitral) valves and the aortic valve (pulmonic valve not seen in this view). Heterozygous sections do not closely resemble wild-type sections in overall cardiac configuration because, in addition to specific cardiac anomalies, affected newborn Matr3 GT-ex13 heterozygote hearts are frequently maloriented and exhibit an abnormal ‘boot shape’, with the cardiac apex pointing horizontally to the animal's left (see insets, A and C). (C) Subaortic VSD is directly inferior to and aligned with the aortic valve. (D) The aortic valve significantly overrides the right ventricle, which together with the normal communication of right ventricle to pulmonary artery (data not shown), establishes DORV. (E) In this specimen, an unusually close continuity exists between the tricuspid and aortic valves. (F) Subaortic VSD and DORV are shown. AoV, aortic valve; LA and LV, left atrium and ventricle; MV, mitral valve; RA and RV, right atrium and ventricle; TV, tricuspid valve; VSD, ventricular septal defect. Scale bar: 500 μm.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques:

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Congenital heart defects in Matr3 GT-ex13 embryonic and newborn heterozygotes

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Laser Capture Microdissection

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Semilunar heart valve defects in Matr 3 Gt-ex-13 heterozygotes. ( A ) Diagram of ascending aorta, aortic arch and descending aorta, showing plane of section for aortic valve analysis. Note left and right coronary ostia (openings) below the fibrous annulus (ring) that demarcates the valve [Cleveland Clinic Foundation (CCF), with permission]. ( B ) Wild-type newborn aortic valve (AoV) showing tri-leaflet (*) morphology in open configuration. Commissural attachments to the annulus are marked (arrows). ( C ) Matr3 GT-ex13 heterozygote newborn bicuspid AoV (BAV) showing two leaflets in closed configuration. ( D ) Wild-type newborn pulmonic valve (PuV) showing tri-leaflet morphology in open configuration. ( E ) Matr3 GT-ex13 heterozygote newborn bicuspid PuV (BPV) showing two leaflets in closed configuration.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques:

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Aortic arch abnormalities in Matr3 Gt-ex13 heterozygotes. ( A and B ) Newborn wild-type aortic arch vasculature, showing pre- (A) and post-corrosion (B) cast analysis. ( C–L ) Matr3 heterozygous newborns with various outflow tract defects. (C and D) Tubular hypoplasia and CoA. The deformed aortic arch is uniformly narrowed (segment between arrowheads), and a CoA (arrow) lies distal to the LSA near a closed DAo. (E and F) CoA (arrow) just distal to the LSA and at the level of the closed DAo also called a ‘juxaductal CoA’. (G and H) Interrupted aortic arch just distal to the LSA, with a strand of tissue joining the two segments (‘atretic aortic arch’; arrow). A VSD with left to right shunting is also present, as evident by red polymer in both ventricles. A large PDA (arrowhead) is the sole source of blood to the lower half of the body. (I and J) A wide PDA (arrowhead) and VSD are present. Following LV injection, both ventricles and the PT contain red polymer; the PT is connected to the PDA that joins the DAo. (K and L) Dual-color corrosion casting shows admixture of red (injected into LV) and blue polymers (injected into RV) in both ventricles, confirming the presence of a VSD (arrow, K). Both polymers are also present in the pulmonary trunk and aorta. A small PDA is present (arrowheads, K and L). AAo, ascending aorta; BA, brachiocephalic artery; DAo, descending aorta; IAA, interrupted aortic arch; LV, left ventricle; PT, pulmonary trunk; RCC/LCC, right/left common carotid arteries; RSA/LSA, right/left subclavian arteries; RV, right ventricle; VSD, ventricular septal defect.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Polymer, Injection

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: N-terminal sequences in matrin 3 mediate phase separation into droplet-like structures that recruit TDP43 variants lacking RNA binding elements.

doi: 10.1038/s41374-019-0260-7

Figure Lengend Snippet: Fig. 2 MATR3:YFP lacking RRM1 or RRM2 displays a greater tendency towards formation of spherical droplet- like intranuclear structures. Mouse C2C12 cells were grown on glass coverslips and transiently transfected with pEF. Bos vectors encoding WT MATR3:YFP lacking RRM1 (a−d) or RRM2 (e−h). At 24 h posttransfection, the cells were fixed and imaged with an Olympus epifluorescence microscope (×40 original magnification). The images shown are representative of images seen in three independent transfections, viewing 20−40 cells on each cover slip

Article Snippet: Flag- MATR3 del RRM1 (plasmid #32881) and Flag- MATR3 delRRM2 (plasmid #32882) vectors were obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids were amplified by PCR, excluding the Flag tag, and cloned into a pEF-BOS.

Techniques: Transfection, Microscopy

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: N-terminal sequences in matrin 3 mediate phase separation into droplet-like structures that recruit TDP43 variants lacking RNA binding elements.

doi: 10.1038/s41374-019-0260-7

Figure Lengend Snippet: Fig. 3 N-terminal fragments of MATR3 targeted to the nucleus spontaneously form spherical droplet-like structures. a−c Mouse C2C12 cells were grown on glass coverslips and transiently transfected with pEF.Bos vectors encoding NLS-N397-MATR3:YFP. At 24 h posttransfection, the cells were fixed and imaged with an Olympus epifluorescence microscope (×40 original magnification). The images shown are representative of images seen in three independent trans- fections, viewing at least 50 cells on each cover slip. a Merged images, b YFP channel alone, c Dapi channel alone

Article Snippet: Flag- MATR3 del RRM1 (plasmid #32881) and Flag- MATR3 delRRM2 (plasmid #32882) vectors were obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids were amplified by PCR, excluding the Flag tag, and cloned into a pEF-BOS.

Techniques: Transfection, Microscopy

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: N-terminal sequences in matrin 3 mediate phase separation into droplet-like structures that recruit TDP43 variants lacking RNA binding elements.

doi: 10.1038/s41374-019-0260-7

Figure Lengend Snippet: Fig. 4 Variable effects of disease mutations on the morphology of NLS-N397- MATR3:YFP within the nucleus. Mouse C2C12 cells were grown on glass coverslips and transiently transfected with pEF.Bos vectors encoding NLS- N397-MATR3:YFP with the mutations indicated in each panel. At 24 h posttransfection, the cells were fixed and imaged with an Olympus epifluorescence microscope. a−d Images captured at ×20 magnification. e, f Images captured at ×40 magnification. The images shown are representative of images seen in three independent transfections, viewing at least 50 cells on each cover slip

Article Snippet: Flag- MATR3 del RRM1 (plasmid #32881) and Flag- MATR3 delRRM2 (plasmid #32882) vectors were obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids were amplified by PCR, excluding the Flag tag, and cloned into a pEF-BOS.

Techniques: Transfection, Microscopy

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: N-terminal sequences in matrin 3 mediate phase separation into droplet-like structures that recruit TDP43 variants lacking RNA binding elements.

doi: 10.1038/s41374-019-0260-7

Figure Lengend Snippet: Fig. 5 Colocalization of MATR3:YFP-WT-ΔRRM2 and TDP43: mCherry variants with disabled RRM1 domains. Mouse C2C12 cells were cotransfected with MATR3:YFP and TDP43:mCher constructs as indicated below. The images shown are representative of what was found in at least two separate transfection experiments analyzing 50–100 transfected cells in each experiment. a−d Images were cap- tured with a Nikon A1RMP imaging system (original magnification ×60 with ×2 digital enlargement; scale bars are approximations). Each image is from a single z-plane. a, b WT MATR3:YFPΔRRM coex- pressed with TDP43:mCherΔRRM1 or TDP43:mCherRRM1-M. c, d F115C or S85C MATR3:YFPΔRRM coexpressed with TDP43: mCherRRM1-M. e WT MATR3:YFPΔRRM coexpressed with WT TDP43:mCher at original magnification (×60). Expression plasmids were generated as described in Materials and methods

Article Snippet: Flag- MATR3 del RRM1 (plasmid #32881) and Flag- MATR3 delRRM2 (plasmid #32882) vectors were obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids were amplified by PCR, excluding the Flag tag, and cloned into a pEF-BOS.

Techniques: Construct, Transfection, Imaging, Expressing, Generated

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Genes influenced by over-expression of MATR3:YFP (no change in expression by YFP alone).

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Expressing, Over Expression

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Summary of spectral count data for the top 50 interacting proteins ranked by fold enrichment for MATR3:AviTag relative to controls.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques:

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: A graphic representation of the interactome for WT-MATR3 in human HEK293 cells. Proteomic mass spectrometry data from Supplemental Table and the software program Cytoscape (version 3.5.1) were used to generate a graphic representation of MATR3 interaction. The intensity of shading of the oval containing the gene name is linked to the spectral counting data in Supplemental Table ; the lighter the shading the higher the fold increase in spectral counts for a given protein relative to controls (untransfected cells; no expression of Avi-tagged MATR3). Proteins marked with an asterisk are interactors also identified by other studies (see Supplemental Table ). The localization or function of the identified proteins was determined by three sources { www.uniprot.org } and , . Using quantification by label-free spectral counting, HNRNPM and numerous RNA processing factors including ADAR, PDBP3, SFRS14, and PTBP1 appear to be the strongest interactors with MATR3.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Mass Spectrometry, Software, Expressing

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Distribution of MATR3:YFP in nuclear compartments of C2C12 cells. Mouse C2C12 cells were grown on glass cover slips and transiently transfected with pEF.Bos vectors encoding MATR3:YFP variants noted on the figure. At 24 hours post-transfection, the cells were fixed and imaged. The images shown are representative of images seen in 3 independent transfections, viewing 30 to 50 cells on each cover slip (data quantified in Table ). WT MATR3 and MATR3 lacking ZnF1, ZnF2, or RRM1 sequences show a predominantly diffuse distribution within the nucleus with variable levels of small puncta. MATR3 lacking RRM2 organizes into large uniformly spherical structures. Scale bar = 100 µm.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Transfection

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Quantification of spherical structures formation by MATR3:YFP constructs.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Construct, Transfection

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Images from time-lapse video demonstrates fusion of MATR3:YFPΔRRM2 spheres in C2C12 cells. An example of the timing of fusion is illustrated in this panel of images. Two independent fusion events can clearly be seen to occur over a period of 15–30 minutes in each case. The example shown is representative of observations made in 3 separate transfection experiments tracking 20–30 cells in each field of view. Original magnification 20X.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Transfection

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Lack of WT-MATR3:YFP and WT-MATR3:YFP ΔRRM2co-localization with TDP-43:mCherry in C2C12 cells. Mouse C2C12 cells were grown on glass cover slips and transiently transfected with pEF.Bos vectors encoding MATR3:YFP variants noted on the figure. At 24 hours post-transfection, the cells were fixed and imaged. The images shown are representative of what was found in at least two separate transfection experiments analyzing 50–100 cells in each experiment (original magnification 60X with 2X digital enlargement). Each image is from a single z-plane captured on a confocal microscope (see Methods). WT-MATR3:YFP and WT-TDP-43:mCherry show limited overlap, for example TDP-43:mCherry is not seen in puncta of WT-MATR3:YFP or the spherical structures formed by MATR3:YFPΔRRM2.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Transfection, Microscopy

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Minimal co-localization of MATR3 with lamin A/C or histones. Mouse C2C12 cells were grown on glass cover slips and transiently transfected with pEF.Bos vectors encoding MATR3:YFP variants noted on the figure. At 24 hours post-transfection, the cells were fixed, immunostained with antibodies to lamin A/C (Sigma-Aldrich #SAB4200236) or histone H3 (Abcam #ab8898) and imaged. The images shown are representative of images seen in 3 independent transfections, viewing 30 to 50 cells on each cover slip. Each image is from a single z-plane captured on a confocal microscope (see Methods). Nikon elements software was used to determine the extent of interaction between MATR3 and lamin A/C or histone H3 (Supplemental Figures and ). MATR3 shows limited co-localization with either lamin A/C or histone H3.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Transfection, Microscopy, Software

Journal: Retrovirology

Article Title: Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

doi: 10.1186/1742-4690-8-60

Figure Lengend Snippet: Immunoprecipitation of HIV-1 RNA from nucleoplasmic fractions . A) Biochemical fractionation for the proteomic analysis. Nuclear extraction scheme showing the various phases of the protocol used to produce the nucleoplasmic fraction. B) Control of nuclear extraction in U2OS cells. The fractions obtained by the protocol outlined in Figure 2A were loaded on a gel for immunoblotting against α-tubulin (upper panel) that shows up only in the cytoplasmic fraction (CF) and against the nuclear protein RecQ (bottom panel) that was present only in the nucleoplasmic fraction (NF). C) Control of HIV-1 RNA associated factor Tat in the NF. Nuclear extracts from U2OS cells (mock), U2OS HIV_Exo_24 × MS2 (exo) or U2OS HIV_Intro_24 × MS2 (intro) were immunoprecipitated for HIV-1 RNA as described above, loaded on SDS-PAGE and blotted against GFP to detect the RNA-bound Tat-CFP protein (IP). Immunoblots for the nuclear extracts against GFP and flag-MS2nls (input) are shown. D) Pulldown of HIV-1 RNA and endogenous MATR3. Whole cell extracts from U2OS cells (mock), U2OS HIV_Exo_24 × MS2 (exo) or U2OS HIV_Intro_24 × MS2 (intro) were immunoprecipitated for HIV-1 RNA as described above, loaded on SDS-PAGE and blotted against MATR3 to detect the RNA-bound endogenous protein (IP). Immunoblots for the whole cell extracts against MATR3 and flag-MS2nls (input) are shown.

Article Snippet: Immunoblots were performed as described before [ ] with the following antibodies: MATR3 (Aviva Systems Biology, ARP40922_T100, 1:1000) or a gift from Yosef Shiloh and Maayan Salton (Tel Aviv University, Israel, 1:10000); p17 (NIH AIDS Reference Reagents Program, 1:1000); GFP (Roche, 11814460001, 1:1000); flag (Sigma, F1804, 1:1000); α-tubulin (Sigma, T5168, 1:10000); RecQL-1 (H-110) (Santa Cruz, sc-25547, 1:1000); β-actin-HRP (Sigma, A3854, 1:50000).

Techniques: Immunoprecipitation, Fractionation, Extraction, Control, Western Blot, SDS Page

Journal: Retrovirology

Article Title: Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

doi: 10.1186/1742-4690-8-60

Figure Lengend Snippet: MATR3 is a post-transcriptional cofactor of HIV-1 . A) MATR3 knockdown does not affect the luciferase activity. HeLa cells were transfected with the indicated siRNAs. After 48 hours siRNA-treated cells were transfected with the pNL4.3R-E-luc HIV-1 molecular clone and with pCMV-Renilla and harvested 24 hours later for luciferase assays. Relative Luc/RL expression was normalized to protein levels measured by Bradford assay. The results of three independent experiments are shown ± SD. B) MATR3 knockdown leads to decrease of the Gag expression from pNL4.3R-E-luc HIV-1 molecular clone. HeLa cells were transfected with the siRNA targeting MATR3 (siMATR3) or with a control siRNA (siCTRL). After 48 hours siRNA-treated cells were transfected with pNL4.3R-E-luc and harvested 24 hours later for immunobloting. Tubulin is the protein loading control.

Article Snippet: Immunoblots were performed as described before [ ] with the following antibodies: MATR3 (Aviva Systems Biology, ARP40922_T100, 1:1000) or a gift from Yosef Shiloh and Maayan Salton (Tel Aviv University, Israel, 1:10000); p17 (NIH AIDS Reference Reagents Program, 1:1000); GFP (Roche, 11814460001, 1:1000); flag (Sigma, F1804, 1:1000); α-tubulin (Sigma, T5168, 1:10000); RecQL-1 (H-110) (Santa Cruz, sc-25547, 1:1000); β-actin-HRP (Sigma, A3854, 1:50000).

Techniques: Knockdown, Luciferase, Activity Assay, Transfection, Expressing, Bradford Assay, Control, Western Blot

Journal: Retrovirology

Article Title: Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

doi: 10.1186/1742-4690-8-60

Figure Lengend Snippet: MATR3 knockdown impairs Rev activity . A) Knockdown of MATR3 by siRNA. 293T cells were transfected either with siRNA targeting MATR3 (siMATR3) or with a control siRNA (siCTRL) and lysed after 72 hours for western blot analysis to assess the efficiency of MATR3 knockdown. Tubulin is the protein loading control. B) RT-PCR of spliced and unspliced HIV-1 RNA levels modulated by MATR3. Spliced (S) and unspliced (US) HIV-1 RNAs were detected (lanes 1-4, upper panel) simultaneously by RT-PCR on total RNA extracted from siRNA-treated 293T cells expressing vHY-IRES-TK, Tat and Rev-EGFP as indicated. RT-PCR amplification of an unrelated RNA was not affected (β-actin mRNA) (lanes 1-4, lower panel). Reactions without RT are shown to demonstrate lack of DNA contamination (lanes 5-8). Water (mock) was used as control of DNA contamination in the reaction. C) Quantitative analysis of unspliced HIV-1 RNA levels modulated by MATR3. Unspliced (US) viral RNA expression in siRNA treated 293T cells was assayed after transfection with vHY-IRES-TK, Tat and Rev-EGFP. Unspliced RNA levels were analyzed by quantitative real-time PCR and data normalized to β-mRNA expression. Data are presented as fold change, whereby siCTRL treated cells transfected with vHY-IRES-TK and Tat in the absence of Rev were set as 1. The results of three independent experiments are shown ± SD. The inhibition was significant (p = 0.00112). D) Rev-dependent expression of HIV-1 Gag (p17*). Western blot analysis of protein extracts from siRNA-treated 293T cells expressing vHY-IRES-TK, Tat and Rev-EGFP as indicated. p17* is the product of the truncated gag gene of the vHY-IRES-TK vector. Tubulin is the protein loading control. E) Quantitative analysis of unspliced HIV-1 RNA levels modulated by MATR3 in the nucleus and the cytoplasm. Unspliced (US) viral RNA expression in siRNA treated 293T cells was assayed after transfection with vHY-IRES-TK, Tat and Rev-EGFP. Unspliced RNA levels were analyzed by quantitative real-time PCR on nuclear (NF) and cytoplasmic fractions (CF). Data were normalized to β-mRNA expression and presented as fold changes, whereby siCTRL 293T treated cells transfected with vHY-IRES-TK and Tat and Rev-EGFP were set as 1. The results of three independent experiments are shown ± SD. The inhibition was significant (p = 0.00091). F) Quantitative analysis of spliced HIV-1 RNA levels modulated by MATR3 in the nucleus and the cytoplasm. The experiment was conducted for spliced (S) HIV-1 RNA as described above (Figure 4E).

Article Snippet: Immunoblots were performed as described before [ ] with the following antibodies: MATR3 (Aviva Systems Biology, ARP40922_T100, 1:1000) or a gift from Yosef Shiloh and Maayan Salton (Tel Aviv University, Israel, 1:10000); p17 (NIH AIDS Reference Reagents Program, 1:1000); GFP (Roche, 11814460001, 1:1000); flag (Sigma, F1804, 1:1000); α-tubulin (Sigma, T5168, 1:10000); RecQL-1 (H-110) (Santa Cruz, sc-25547, 1:1000); β-actin-HRP (Sigma, A3854, 1:50000).

Techniques: Knockdown, Activity Assay, Transfection, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, RNA Expression, Real-time Polymerase Chain Reaction, Inhibition, Plasmid Preparation

Journal: Retrovirology

Article Title: Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

doi: 10.1186/1742-4690-8-60

Figure Lengend Snippet: MATR3 overexpression promotes Rev activity . A) Quantitative analysis of unspliced HIV-1 RNA levels modulated by transfected MATR3. Unspliced (US) viral RNA expression in 293T cells was assayed after transfection with Flag-MATR3, vHY-IRES-TK, Tat and Rev-EGFP. Unspliced RNA levels were analyzed by quantitative real-time PCR and data normalized to β-mRNA expression. Data are presented as fold change, whereby 293T cells transfected with vHY-IRES-TK and Tat in the absence of Rev were set as 1. The results of three independent experiments are shown ± SD. The increase was significant (p = 0.01931). B) Transfected MATR3 upregulates Rev-dependent Gag translation. Western blot analysis of protein extracts from 293T cells expressing Flag-MATR3, vHY-IRES-TK, Tat and Rev-EGFP. p17* is the product of the truncated gag gene of the vHY-IRES-TKvector. Tubulin is the protein loading control

Article Snippet: Immunoblots were performed as described before [ ] with the following antibodies: MATR3 (Aviva Systems Biology, ARP40922_T100, 1:1000) or a gift from Yosef Shiloh and Maayan Salton (Tel Aviv University, Israel, 1:10000); p17 (NIH AIDS Reference Reagents Program, 1:1000); GFP (Roche, 11814460001, 1:1000); flag (Sigma, F1804, 1:1000); α-tubulin (Sigma, T5168, 1:10000); RecQL-1 (H-110) (Santa Cruz, sc-25547, 1:1000); β-actin-HRP (Sigma, A3854, 1:50000).

Techniques: Over Expression, Activity Assay, Transfection, RNA Expression, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Control

Journal: Retrovirology

Article Title: Characterization of the HIV-1 RNA associated proteome identifies Matrin 3 as a nuclear cofactor of Rev function

doi: 10.1186/1742-4690-8-60

Figure Lengend Snippet: MATR3 interaction with Rev requires HIV-1 RNA . A) Whole cell lysates from 293T cells expressing vHY-IRES-TK and Tat with or without Rev-EGFP were subjected to immunoprecipitation with anti-MATR3 antibodies or with anti-IgG (mock). The IP were subjected to nuclease treatment and the proteins were detected by immunoblotting. B) Whole cell lysates from 293T cells expressing either vHY-IRES-TK, or v653RSN or v653SN together with Tat and Rev-EGFP were subjected to immunoprecipitation with anti-MATR3 antibodies. Immunoblots from whole cell extracts are shown on the left (input). Endogenous β-actin was used as loading control. The immunoblot for p17* shows lack of Gag expression for the RRE deficient v653SN construct (bottom panel). Immunoprecipitations are shown on the right (IP).

Article Snippet: Immunoblots were performed as described before [ ] with the following antibodies: MATR3 (Aviva Systems Biology, ARP40922_T100, 1:1000) or a gift from Yosef Shiloh and Maayan Salton (Tel Aviv University, Israel, 1:10000); p17 (NIH AIDS Reference Reagents Program, 1:1000); GFP (Roche, 11814460001, 1:1000); flag (Sigma, F1804, 1:1000); α-tubulin (Sigma, T5168, 1:10000); RecQL-1 (H-110) (Santa Cruz, sc-25547, 1:1000); β-actin-HRP (Sigma, A3854, 1:50000).

Techniques: Expressing, Immunoprecipitation, Western Blot, Control, Construct

Journal: FEBS letters

Article Title: MATR3 pathogenic variants differentially impair its cryptic splicing repression function.

doi: 10.1002/1873-3468.14806

Figure Lengend Snippet: Fig. 1. Knockdown of MATR3 results in cryptic splicing events. (A) Tabulated result of the significant cryptic splicing events found in MATR3 KD summarized by category: novel donor (ND), novel acceptor (NA), novel donor and acceptor (NDA) or novel combination of known acceptors and donors (NC). Significant events were identified using an adjusted P-value ≤0.05. (B) Sequence composition showing pyrimidine-rich sequences in the intronic region toward the 30 end of the cryptic junctions with an FDR ≤0.05 and junction difference > 0. (C, D) Sashimi plot generated using IGV genome browser illustrating the read coverage for two biological replicates of control (blue) and MATR3 knockdown (pink) for a region of UQCRC2 and UHRF2 transcribed from left to right. The bar graph denotes read depth, and the arcs represent the number of reads connecting each splice junction, with the junction coverage minimum set to 10. The region within black dashed lines represents the cryptic junction of inter- est with the cryptic exon marked by an asterisk, and inclusion levels of the cryptic exon within this region are shown as percentages to the right of the tracks. MATR3 binding analyzed from CLIP-Seq data from Coelho et al. [24], is shown in yellow.

Article Snippet: MATR3 WT and the single domain deletion constructs were gifts from Dr. Yossi Shiloh (Addgene plasmids #32880, 32881, 32882, 32883, 32884).

Techniques: Knockdown, Sequencing, Generated, Control, Binding Assay

Journal: FEBS letters

Article Title: MATR3 pathogenic variants differentially impair its cryptic splicing repression function.

doi: 10.1002/1873-3468.14806

Figure Lengend Snippet: Fig. 2. Loss of MATR3 leads to cryptic exon inclusion in UQCRC2 and UHRF2. (A) Representative RT-PCR gels showing transcript levels of GAPDH, MATR3 UTR and coding region and UQCRC2 cryptic exon (CE) from total RNA extracted from HeLa cells doubly transfected with non-targeting control siRNA (siCTRL) or siRNA targetingMATR3 (siMATR3) and control vector or FLAG-MATR3 wildtype (WT). (B) Quantifica- tion of cryptic exon inclusion in UQCRC2 normalized to GAPDH (n = 3, bar heights depict mean SEM, with each datapoint representing a biological replicate, significance determined by Welch’s t-test, *P ≤0.05). (C, D) Relative gene expression (ΔΔCt analysis) of CE-containing UQCRC2 transcripts or total UQCRC2 mRNA in HeLa cells doubly transfected with either siCTRL or siMATR3 and FLAG-GFP or FLAG- MATR3 WT as measured by quantitative RT-PCR (n = 5, bar heights depict mean SEM, with each datapoint representing a biological repli- cate, significance determined by Welch’s t-test, ns P > 0.05, **P ≤0.01, ***P ≤0.001). (E) Representative gels showing RT-PCR of UQCRC2 CE, UQCRC2, MATR3, and GAPDH from total RNA extracted from HeLa cells transfected with siRNA and then treated with cycloheximide (CHX) or DMSO control. (F) Quantification of MATR3 transcript levels normalized to GAPDH to confirm knockdown (n = 3, bar heights depict mean SEM, with each datapoint representing a biological replicate, significance determined by Welch’s t-test, *P ≤0.05). (G) Quantification of the accumulation of UQCRC2 transcripts containing the cryptic exon normalized to GAPDH (n = 3, mean SEM are plotted, with each datapoint representing a biological replicate, significance determined by two-way ANOVA, **P ≤0.01 representing the statistical comparison between siCTRL and siMATR3, statistical comparison between DMSO and CHX treatments within each siRNA is not significant). (H, I) Rela- tive gene expression (ΔΔCt analysis) of CE-containing UHRF2 transcripts or total UHRF2 mRNA in HeLa cells doubly transfected with either siCTRL or siMATR3 and FLAG-GFP or FLAG-MATR3 WT as measured by quantitative RT-PCR (n = 5, bar heights depict mean SEM, with each datapoint representing a biological replicate, significance determined by Welch’s t-test, ns P > 0.05, *P ≤0.05, **P ≤0.01, ***P ≤0.001). (J) Representative gels showing RT-PCR of UHRF2 CE, UHRF2, MATR3, and GAPDH from total RNA extracted from HeLa cells transfected with siRNA and then treated with CHX or DMSO control. (K) Quantification of the accumulation of UHRF2 transcripts con- taining the cryptic exon normalized to GAPDH (n = 3, mean SEM are plotted, with each datapoint representing a biological replicate, signifi- cance determined by two-way ANOVA, **P ≤0.01 representing the statistical comparison between siCTRL and siMATR3, ###P ≤0.001 representing the statistical comparison between DMSO and CHX treatments within each siRNA).

Article Snippet: MATR3 WT and the single domain deletion constructs were gifts from Dr. Yossi Shiloh (Addgene plasmids #32880, 32881, 32882, 32883, 32884).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Plasmid Preparation, Gene Expression, Quantitative RT-PCR, Knockdown, Comparison

Journal: FEBS letters

Article Title: MATR3 pathogenic variants differentially impair its cryptic splicing repression function.

doi: 10.1002/1873-3468.14806

Figure Lengend Snippet: Fig. 3. RT-PCR analysis shows that the RRM2 domain is required for MATR3 cryptic splicing repression in UQCRC2. (A) Schematic repre- sentation of MATR3 domains showing nuclear export signal (NES), zinc finger domains (ZF), RNA recognition motifs (RRM) and nuclear localization signal (NLS). (B) Representative gels showing RT-PCR for GAPDH, MATR3 UTR and coding region, and UQCRC2 cryptic exon (CE) from total RNA extracted from HeLa cells transfected with siRNA and MATR3 vectors. (C) Quantification of UQCRC2 cryptic exon inclu- sion normalized to GAPDH (n = 4, bar heights depict mean SEM, with each datapoint representing a biological replicate, with a total of 4 independent experiments performed, significance determined by Welch’s t-test, *P ≤0.05, **P ≤0.01, ****P ≤0.0001 ns = not significant). (D) Representative gels showing RT-PCR for GAPDH, MATR3 UTR and coding region and UQCRC2 cryptic exon (CE) from total RNA extracted from HeLa cells transfected with siRNA and MATR3 vectors. (E) Quantification of UQCRC2 cryptic exon inclusion normalized to GAPDH (n = 3, bar heights depict mean SEM, with each datapoint representing a biological replicate, with a total of 3 independent experi- ments performed, significance determined by Welch’s t-test, *P ≤0.05, ns = not significant).

Article Snippet: MATR3 WT and the single domain deletion constructs were gifts from Dr. Yossi Shiloh (Addgene plasmids #32880, 32881, 32882, 32883, 32884).

Techniques: Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: FEBS letters

Article Title: MATR3 pathogenic variants differentially impair its cryptic splicing repression function.

doi: 10.1002/1873-3468.14806

Figure Lengend Snippet: Fig. 4. RNA immunoprecipitation analysis shows enrichment of UQCRC2 and CACNB2 mRNA in MATR3 WT pulldown. (A) Western blot showing immunoprecipitation (IP) of MATR3 WT and MATR3 ΔRRM2. (B) RT-PCR of UQCRC2 and CACNB2 from cDNA of input and IP samples. (C) Quantification of binding ratio of UQCRC2 to MATR3 (n = 3, bar heights depict mean SEM, with each datapoint representing a biological replicate, with a total of 3 independent experiments performed, significance determined by Welch’s t-test, *P ≤0.05). (D) Quanti- fication of binding ratio of CACNB2 to MATR3 (n = 3, bar heights depict mean SEM, with each datapoint representing a biological replicate, with a total of 3 independent experiments performed, significance determined by Welch’s t-test, *P ≤0.05).

Article Snippet: MATR3 WT and the single domain deletion constructs were gifts from Dr. Yossi Shiloh (Addgene plasmids #32880, 32881, 32882, 32883, 32884).

Techniques: RNA Immunoprecipitation, Western Blot, Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Binding Assay

Journal: FEBS letters

Article Title: MATR3 pathogenic variants differentially impair its cryptic splicing repression function.

doi: 10.1002/1873-3468.14806

Figure Lengend Snippet: Fig. 6. MATR3 M548T is less soluble than MATR3 WT and does not rescue cryptic exon inclusion in UQCRC2 upon loss of MATR3. (A) Western blot showing Triton-X soluble (soluble fraction) protein levels of MATR3 WT and MATR3 M548T. (B) Western blot showing urea solu- ble (insoluble fraction) protein levels of MATR3 WT and MATR3 M548T. (C) Quantification of soluble MATR3 levels normalized to GAPDH (n = 3, bar heights depict mean SEM, with each datapoint representing a biological replicate, with a total of 3 independent experiments per- formed, significance determined by Welch’s t-test, **P ≤0.01). (D) Quantification of insoluble MATR3 levels (n = 3, bar heights depict mean SEM, with each datapoint representing a biological replicate, a total of 3 independent experiments was performed, significance deter- mined by Welch’s t-test, **P ≤0.01). (E) Representative gels showing RT-PCR for GAPDH, MATR3 UTR and coding region, and UQCRC2 cryp- tic exon (CE) from total RNA extracted from HeLa cells transfected with siRNA and MATR3 vectors. (F) Quantification of cryptic exon inclusion in UQCRC2 normalized to GAPDH (n = 3, bar graph heights depict mean SEM, with each datapoint representing a biological replicate, with a total of 3 independent experiments performed, significance determined by Welch’s t-test, *P ≤0.05, ****P ≤0.0001).

Article Snippet: MATR3 WT and the single domain deletion constructs were gifts from Dr. Yossi Shiloh (Addgene plasmids #32880, 32881, 32882, 32883, 32884).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Clinical features, cardiac phenotype and translocation breakpoint structure in DGAP105. ( A ) Facial features at 4 years of age included mild hypertelorism, bilateral epicanthal folds, downslanting palpebral fissures, strabismus and a broad nose with a smooth philtrum and thin vermillion border. ( B ) Ideograms depicting 46,XY,t(1;5)(p36.11;q31.2)dn . Arrows mark locations of AHDC1 and MATR3 breakpoints. ( C and D ) Echocardiograms at age of 2 days. (C) Ductal view showing distal aortic arch, CoA just distal to the left subclavian artery (LSCA), accompanied by a prominent posterior unfolding (‘posterior shelf’) and PDA. (D) Aortic arch view showing ascending aorta (5.7-mm diameter), hypoplastic aortic arch (4.4-mm diameter) and CoA posterior shelf. ( E ) Summary of the 1p36.11 and 5q31.2 breakpoints in DGAP105. The 1p36.11 breakpoint disrupts AHDC1 intron 1, whereas the 5q31.2 breakpoint disrupts MATR3 exon 15 in the 3′ UTR. BACs used in FISH analyses are indicated.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Translocation Assay

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Analysis of human MATR3 transcripts and protein expression in control and DGAP105 lymphoblasts. ( A ) Schematic of the MATR3 exon 13–15 region with the chromosomal translocation breakpoint in patient DGAP105 marked by dotted line. The proximal AAUAAA polyadenylation signal and the distal AAUAAA polyadenylation signal site are shown flanking the breakpoint in the 3′ UTR. TAA denotes the stop codon in exon 15. ( B ) MATR3 3′ RACE products in human control (Lane 1) and DGAP105 lymphoblast (Lanes 2, 3), and control human fetal heart tissue (Lanes 4, 5). RT ‘+’ or ‘−’ denote inclusion or omission of reverse transcriptase in the cDNA synthesis. The large product (1589 bp, arrow) uses the distal polyadenylation signal and predominates in control human lymphoblasts (Lane 1). In contrast, the short product (963 bp, arrow) predominates in DGAP105 lymphoblasts (Lane 2) and in control human fetal heart (Lane 4) and represents MATR3 transcripts that use the proximal polyadenylation signal. ( C ) Northern blot analysis of adult human tissues shows MATR3 transcripts of ∼3.5 and ∼2.9 kb. In heart and skeletal muscle, the 2.9-kb transcript predominates and likely corresponds to the 3′ RACE product using the proximal polyadenylation signal. In brain and other tissues, the 3.5-kb transcript predominates and corresponds to the 3′ RACE product using the distal polyadenylation signal. ( D ) Western blot analysis of protein isolated from DGAP105 and three control lymphoblast lines, showing up-regulation of Matrin 3 in DGAP105 compared with controls. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars represent the mean fold expression of four independent experiments ± SEM, corrected for loading, and normalized to Control 2; * P < 0.05 between DGAP105 and mean of the three control lines via paired Student's t test.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Expressing, Control, Translocation Assay, Reverse Transcription, cDNA Synthesis, Northern Blot, Western Blot, Isolation

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Analysis of mouse Matr3 and Ahdc1 transcripts expression in developing heart. ( A ) RT–PCR analyses of Matr3 and Ahdc1 . ( a ) Semi-quantitative RT–PCR analyses show strong Matr 3 expression in developing mouse heart, limb and brain at E11.5, 16.5 stages, ( b ) with down-regulation at the newborn (NB) and adult stages. In contrast, Ahdc1 expression is only weakly detected in limb and brain at E11.5 and 16.5. ( B ) Section in situ hybridizations at E11.5 for mouse Matr3 and Ahdc1. Matr3 is expressed in CNS, pharyngeal arches, limb buds and in the developing heart (enlarged section), whereas Ahdc1 expression was undetectable in heart (enlarged section). Sense controls (not shown) showed no expression.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, In Situ

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Analysis of the Matr3 Gt-ex13 gene trap allele. ( A ) Structure of mouse Matr3 wild-type and Gt-ex13 gene trap mutant alleles. Wild-type mouse Matr3 encodes an 846-amino acid protein. Intron 12 (2749 bp) and exon 13 (223 bp) are shown. Matr3 Gt-ex-13 gene trap allele inserts a β-Geo gene trap vector at position 118 bp in exon 13, which will generate Matr3-β-geo fusion transcripts. PCR genotyping primers depict WT-F1 (in exon 13) and WT-R1 (in intron 13) for the wild-type allele, and Mu-F1 (in exon 13) and Mu-R1 (in gene trap vector) for the mutant allele. Primers used in 3′ RACE are summarized on Materials and Methods. ( B ) E3.5 PCR genotyping shows 394-bp wild-type and 492-bp mutant alleles for wild-type (+/+), heterozygous (+/−) and homozygous (−/−) embryos. ( C ) Matr3 GT-ex13 3′ RACE analysis of mouse E14.5 brain and heart tissues detects a novel Matr3-β-Geo fusion transcript (∼4 kb) in heterozygotes. The long Matr3 3′ RACE product (1647 bp), the only form detected in brain, is reduced in heterozygous brain. Both long and short Matr3 3′ RACE products (1647 and 1025 bp) are reduced in heterozygous heart. ( D ) Western blot analysis of Matrin 3 protein isolated from wild-type and heterozygous mouse E15.5 brain and heart tissues. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars are fold ± SEM expression level from mean of three independent experiments, corrected for loading, and normalized to a value of 1.0 for wild-type heart. The small increase in expression in Matr3 Gt-ex13/+ heart is not statistically significant.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Mutagenesis, Plasmid Preparation, Western Blot, Isolation, Control, Expressing

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Early embryonic lethality in Matr3 GT-ex13 homozygous mouse embryos

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques:

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: X-Gal staining of Matr3 Gt-ex13 heterozygotes. ( A ) X-Gal staining of primitive heart (arrow) in E10.5 Matr3 heterozygote, and in CNS (brain, spinal cord), pharyngeal arches and limb bud. ( B ) X-Gal staining in primitive heart of E8.5 Matr3 heterozygote. Arrow depicts dorsal mesocardium; BC, bulbus cordis; CVC, common ventricular chamber. ( C ) Negative control wild-type E9.5 embryo with eosin counterstain. ( D ) X-Gal staining in wall (arrow) of atrial chamber (AC), bulbus cordis (BC) and ( E ) myocardium and endocardium, and ( F ) interventricular septum (IVS) of newborn (NB) Matr3 GT-ex13 heterozygote heart (arrows).

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Staining, Negative Control

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Matrin 3 protein cardiovascular expression in newborn mice. ( A ) Matrin 3 expression in wild-type newborn heart. ( B ) Pulmonary valve (PV) from (A) shows Matrin 3 in interstitial and endocardial cells. ( C ) Matrin 3 in cardiomyocyte nuclei (arrow). ( D–F ) Immunostaining in arterial vascular smooth muscle and endothelial cells for Matrin 3 (D), PECAM (E) and merged (F). Matrin 3 is expressed in both arterial smooth muscle cell nuclei (green) external to PECAM-1 endothelial cell membrane staining (red) and internal to the PECAM-1 domain, in endothelial cell nuclei. This is better seen in ( G–I ) with Matrin 3 and PECAM in small venules, which are largely devoid of vascular smooth muscle. Arrows in (I) denote Matrin 3 in endothelial cell nuclei. Scale bar: 40 μm (D–F); 5 μm (G–I).

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Expressing, Immunostaining, Membrane, Staining

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Subaortic VSD and DORV phenotypes in Matr3 Gt-ex13 heterozygotes. Transverse serial sections through the hearts of E18.5 wild type ( A and B ), and two different representative Matr3 Gt-ex13 heterozygotes (embryo 1 in C and D and embryo 2 in E and F ), each sectioned at a cranial and caudal level, illustrating the DORV with subaortic VSD phenotype in Matr3 Gt-ex13 heterozygotes (see Table ). (A and B) Wild-type sections show left and right ventricles separated by the interventricular septum, the two atrioventricular (tricuspid, mitral) valves and the aortic valve (pulmonic valve not seen in this view). Heterozygous sections do not closely resemble wild-type sections in overall cardiac configuration because, in addition to specific cardiac anomalies, affected newborn Matr3 GT-ex13 heterozygote hearts are frequently maloriented and exhibit an abnormal ‘boot shape’, with the cardiac apex pointing horizontally to the animal's left (see insets, A and C). (C) Subaortic VSD is directly inferior to and aligned with the aortic valve. (D) The aortic valve significantly overrides the right ventricle, which together with the normal communication of right ventricle to pulmonary artery (data not shown), establishes DORV. (E) In this specimen, an unusually close continuity exists between the tricuspid and aortic valves. (F) Subaortic VSD and DORV are shown. AoV, aortic valve; LA and LV, left atrium and ventricle; MV, mitral valve; RA and RV, right atrium and ventricle; TV, tricuspid valve; VSD, ventricular septal defect. Scale bar: 500 μm.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques:

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Congenital heart defects in Matr3 GT-ex13 embryonic and newborn heterozygotes

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Laser Capture Microdissection

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Semilunar heart valve defects in Matr 3 Gt-ex-13 heterozygotes. ( A ) Diagram of ascending aorta, aortic arch and descending aorta, showing plane of section for aortic valve analysis. Note left and right coronary ostia (openings) below the fibrous annulus (ring) that demarcates the valve [Cleveland Clinic Foundation (CCF), with permission]. ( B ) Wild-type newborn aortic valve (AoV) showing tri-leaflet (*) morphology in open configuration. Commissural attachments to the annulus are marked (arrows). ( C ) Matr3 GT-ex13 heterozygote newborn bicuspid AoV (BAV) showing two leaflets in closed configuration. ( D ) Wild-type newborn pulmonic valve (PuV) showing tri-leaflet morphology in open configuration. ( E ) Matr3 GT-ex13 heterozygote newborn bicuspid PuV (BPV) showing two leaflets in closed configuration.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques:

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Aortic arch abnormalities in Matr3 Gt-ex13 heterozygotes. ( A and B ) Newborn wild-type aortic arch vasculature, showing pre- (A) and post-corrosion (B) cast analysis. ( C–L ) Matr3 heterozygous newborns with various outflow tract defects. (C and D) Tubular hypoplasia and CoA. The deformed aortic arch is uniformly narrowed (segment between arrowheads), and a CoA (arrow) lies distal to the LSA near a closed DAo. (E and F) CoA (arrow) just distal to the LSA and at the level of the closed DAo also called a ‘juxaductal CoA’. (G and H) Interrupted aortic arch just distal to the LSA, with a strand of tissue joining the two segments (‘atretic aortic arch’; arrow). A VSD with left to right shunting is also present, as evident by red polymer in both ventricles. A large PDA (arrowhead) is the sole source of blood to the lower half of the body. (I and J) A wide PDA (arrowhead) and VSD are present. Following LV injection, both ventricles and the PT contain red polymer; the PT is connected to the PDA that joins the DAo. (K and L) Dual-color corrosion casting shows admixture of red (injected into LV) and blue polymers (injected into RV) in both ventricles, confirming the presence of a VSD (arrow, K). Both polymers are also present in the pulmonary trunk and aorta. A small PDA is present (arrowheads, K and L). AAo, ascending aorta; BA, brachiocephalic artery; DAo, descending aorta; IAA, interrupted aortic arch; LV, left ventricle; PT, pulmonary trunk; RCC/LCC, right/left common carotid arteries; RSA/LSA, right/left subclavian arteries; RV, right ventricle; VSD, ventricular septal defect.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Polymer, Injection

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Clinical features, cardiac phenotype and translocation breakpoint structure in DGAP105. ( A ) Facial features at 4 years of age included mild hypertelorism, bilateral epicanthal folds, downslanting palpebral fissures, strabismus and a broad nose with a smooth philtrum and thin vermillion border. ( B ) Ideograms depicting 46,XY,t(1;5)(p36.11;q31.2)dn . Arrows mark locations of AHDC1 and MATR3 breakpoints. ( C and D ) Echocardiograms at age of 2 days. (C) Ductal view showing distal aortic arch, CoA just distal to the left subclavian artery (LSCA), accompanied by a prominent posterior unfolding (‘posterior shelf’) and PDA. (D) Aortic arch view showing ascending aorta (5.7-mm diameter), hypoplastic aortic arch (4.4-mm diameter) and CoA posterior shelf. ( E ) Summary of the 1p36.11 and 5q31.2 breakpoints in DGAP105. The 1p36.11 breakpoint disrupts AHDC1 intron 1, whereas the 5q31.2 breakpoint disrupts MATR3 exon 15 in the 3′ UTR. BACs used in FISH analyses are indicated.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Translocation Assay

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Analysis of human MATR3 transcripts and protein expression in control and DGAP105 lymphoblasts. ( A ) Schematic of the MATR3 exon 13–15 region with the chromosomal translocation breakpoint in patient DGAP105 marked by dotted line. The proximal AAUAAA polyadenylation signal and the distal AAUAAA polyadenylation signal site are shown flanking the breakpoint in the 3′ UTR. TAA denotes the stop codon in exon 15. ( B ) MATR3 3′ RACE products in human control (Lane 1) and DGAP105 lymphoblast (Lanes 2, 3), and control human fetal heart tissue (Lanes 4, 5). RT ‘+’ or ‘−’ denote inclusion or omission of reverse transcriptase in the cDNA synthesis. The large product (1589 bp, arrow) uses the distal polyadenylation signal and predominates in control human lymphoblasts (Lane 1). In contrast, the short product (963 bp, arrow) predominates in DGAP105 lymphoblasts (Lane 2) and in control human fetal heart (Lane 4) and represents MATR3 transcripts that use the proximal polyadenylation signal. ( C ) Northern blot analysis of adult human tissues shows MATR3 transcripts of ∼3.5 and ∼2.9 kb. In heart and skeletal muscle, the 2.9-kb transcript predominates and likely corresponds to the 3′ RACE product using the proximal polyadenylation signal. In brain and other tissues, the 3.5-kb transcript predominates and corresponds to the 3′ RACE product using the distal polyadenylation signal. ( D ) Western blot analysis of protein isolated from DGAP105 and three control lymphoblast lines, showing up-regulation of Matrin 3 in DGAP105 compared with controls. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars represent the mean fold expression of four independent experiments ± SEM, corrected for loading, and normalized to Control 2; * P < 0.05 between DGAP105 and mean of the three control lines via paired Student's t test.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Expressing, Control, Translocation Assay, Reverse Transcription, cDNA Synthesis, Northern Blot, Western Blot, Isolation

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Analysis of mouse Matr3 and Ahdc1 transcripts expression in developing heart. ( A ) RT–PCR analyses of Matr3 and Ahdc1 . ( a ) Semi-quantitative RT–PCR analyses show strong Matr 3 expression in developing mouse heart, limb and brain at E11.5, 16.5 stages, ( b ) with down-regulation at the newborn (NB) and adult stages. In contrast, Ahdc1 expression is only weakly detected in limb and brain at E11.5 and 16.5. ( B ) Section in situ hybridizations at E11.5 for mouse Matr3 and Ahdc1. Matr3 is expressed in CNS, pharyngeal arches, limb buds and in the developing heart (enlarged section), whereas Ahdc1 expression was undetectable in heart (enlarged section). Sense controls (not shown) showed no expression.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, In Situ

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Analysis of the Matr3 Gt-ex13 gene trap allele. ( A ) Structure of mouse Matr3 wild-type and Gt-ex13 gene trap mutant alleles. Wild-type mouse Matr3 encodes an 846-amino acid protein. Intron 12 (2749 bp) and exon 13 (223 bp) are shown. Matr3 Gt-ex-13 gene trap allele inserts a β-Geo gene trap vector at position 118 bp in exon 13, which will generate Matr3-β-geo fusion transcripts. PCR genotyping primers depict WT-F1 (in exon 13) and WT-R1 (in intron 13) for the wild-type allele, and Mu-F1 (in exon 13) and Mu-R1 (in gene trap vector) for the mutant allele. Primers used in 3′ RACE are summarized on Materials and Methods. ( B ) E3.5 PCR genotyping shows 394-bp wild-type and 492-bp mutant alleles for wild-type (+/+), heterozygous (+/−) and homozygous (−/−) embryos. ( C ) Matr3 GT-ex13 3′ RACE analysis of mouse E14.5 brain and heart tissues detects a novel Matr3-β-Geo fusion transcript (∼4 kb) in heterozygotes. The long Matr3 3′ RACE product (1647 bp), the only form detected in brain, is reduced in heterozygous brain. Both long and short Matr3 3′ RACE products (1647 and 1025 bp) are reduced in heterozygous heart. ( D ) Western blot analysis of Matrin 3 protein isolated from wild-type and heterozygous mouse E15.5 brain and heart tissues. Gapdh was used as loading control. ( E ) Quantification of Matrin 3 protein expression in D. Bars are fold ± SEM expression level from mean of three independent experiments, corrected for loading, and normalized to a value of 1.0 for wild-type heart. The small increase in expression in Matr3 Gt-ex13/+ heart is not statistically significant.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Mutagenesis, Plasmid Preparation, Western Blot, Isolation, Control, Expressing

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Early embryonic lethality in Matr3 GT-ex13 homozygous mouse embryos

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques:

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: X-Gal staining of Matr3 Gt-ex13 heterozygotes. ( A ) X-Gal staining of primitive heart (arrow) in E10.5 Matr3 heterozygote, and in CNS (brain, spinal cord), pharyngeal arches and limb bud. ( B ) X-Gal staining in primitive heart of E8.5 Matr3 heterozygote. Arrow depicts dorsal mesocardium; BC, bulbus cordis; CVC, common ventricular chamber. ( C ) Negative control wild-type E9.5 embryo with eosin counterstain. ( D ) X-Gal staining in wall (arrow) of atrial chamber (AC), bulbus cordis (BC) and ( E ) myocardium and endocardium, and ( F ) interventricular septum (IVS) of newborn (NB) Matr3 GT-ex13 heterozygote heart (arrows).

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Staining, Negative Control

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Matrin 3 protein cardiovascular expression in newborn mice. ( A ) Matrin 3 expression in wild-type newborn heart. ( B ) Pulmonary valve (PV) from (A) shows Matrin 3 in interstitial and endocardial cells. ( C ) Matrin 3 in cardiomyocyte nuclei (arrow). ( D–F ) Immunostaining in arterial vascular smooth muscle and endothelial cells for Matrin 3 (D), PECAM (E) and merged (F). Matrin 3 is expressed in both arterial smooth muscle cell nuclei (green) external to PECAM-1 endothelial cell membrane staining (red) and internal to the PECAM-1 domain, in endothelial cell nuclei. This is better seen in ( G–I ) with Matrin 3 and PECAM in small venules, which are largely devoid of vascular smooth muscle. Arrows in (I) denote Matrin 3 in endothelial cell nuclei. Scale bar: 40 μm (D–F); 5 μm (G–I).

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Expressing, Immunostaining, Membrane, Staining

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Subaortic VSD and DORV phenotypes in Matr3 Gt-ex13 heterozygotes. Transverse serial sections through the hearts of E18.5 wild type ( A and B ), and two different representative Matr3 Gt-ex13 heterozygotes (embryo 1 in C and D and embryo 2 in E and F ), each sectioned at a cranial and caudal level, illustrating the DORV with subaortic VSD phenotype in Matr3 Gt-ex13 heterozygotes (see Table ). (A and B) Wild-type sections show left and right ventricles separated by the interventricular septum, the two atrioventricular (tricuspid, mitral) valves and the aortic valve (pulmonic valve not seen in this view). Heterozygous sections do not closely resemble wild-type sections in overall cardiac configuration because, in addition to specific cardiac anomalies, affected newborn Matr3 GT-ex13 heterozygote hearts are frequently maloriented and exhibit an abnormal ‘boot shape’, with the cardiac apex pointing horizontally to the animal's left (see insets, A and C). (C) Subaortic VSD is directly inferior to and aligned with the aortic valve. (D) The aortic valve significantly overrides the right ventricle, which together with the normal communication of right ventricle to pulmonary artery (data not shown), establishes DORV. (E) In this specimen, an unusually close continuity exists between the tricuspid and aortic valves. (F) Subaortic VSD and DORV are shown. AoV, aortic valve; LA and LV, left atrium and ventricle; MV, mitral valve; RA and RV, right atrium and ventricle; TV, tricuspid valve; VSD, ventricular septal defect. Scale bar: 500 μm.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques:

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Congenital heart defects in Matr3 GT-ex13 embryonic and newborn heterozygotes

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Laser Capture Microdissection

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Semilunar heart valve defects in Matr 3 Gt-ex-13 heterozygotes. ( A ) Diagram of ascending aorta, aortic arch and descending aorta, showing plane of section for aortic valve analysis. Note left and right coronary ostia (openings) below the fibrous annulus (ring) that demarcates the valve [Cleveland Clinic Foundation (CCF), with permission]. ( B ) Wild-type newborn aortic valve (AoV) showing tri-leaflet (*) morphology in open configuration. Commissural attachments to the annulus are marked (arrows). ( C ) Matr3 GT-ex13 heterozygote newborn bicuspid AoV (BAV) showing two leaflets in closed configuration. ( D ) Wild-type newborn pulmonic valve (PuV) showing tri-leaflet morphology in open configuration. ( E ) Matr3 GT-ex13 heterozygote newborn bicuspid PuV (BPV) showing two leaflets in closed configuration.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques:

Journal: Human Molecular Genetics

Article Title: MATR3 disruption in human and mouse associated with bicuspid aortic valve, aortic coarctation and patent ductus arteriosus

doi: 10.1093/hmg/ddv004

Figure Lengend Snippet: Aortic arch abnormalities in Matr3 Gt-ex13 heterozygotes. ( A and B ) Newborn wild-type aortic arch vasculature, showing pre- (A) and post-corrosion (B) cast analysis. ( C–L ) Matr3 heterozygous newborns with various outflow tract defects. (C and D) Tubular hypoplasia and CoA. The deformed aortic arch is uniformly narrowed (segment between arrowheads), and a CoA (arrow) lies distal to the LSA near a closed DAo. (E and F) CoA (arrow) just distal to the LSA and at the level of the closed DAo also called a ‘juxaductal CoA’. (G and H) Interrupted aortic arch just distal to the LSA, with a strand of tissue joining the two segments (‘atretic aortic arch’; arrow). A VSD with left to right shunting is also present, as evident by red polymer in both ventricles. A large PDA (arrowhead) is the sole source of blood to the lower half of the body. (I and J) A wide PDA (arrowhead) and VSD are present. Following LV injection, both ventricles and the PT contain red polymer; the PT is connected to the PDA that joins the DAo. (K and L) Dual-color corrosion casting shows admixture of red (injected into LV) and blue polymers (injected into RV) in both ventricles, confirming the presence of a VSD (arrow, K). Both polymers are also present in the pulmonary trunk and aorta. A small PDA is present (arrowheads, K and L). AAo, ascending aorta; BA, brachiocephalic artery; DAo, descending aorta; IAA, interrupted aortic arch; LV, left ventricle; PT, pulmonary trunk; RCC/LCC, right/left common carotid arteries; RSA/LSA, right/left subclavian arteries; RV, right ventricle; VSD, ventricular septal defect.

Article Snippet: Each cDNA sample was then subjected to Taqman real-time PCR analysis on a 7500 FAST real-time PCR system (Life Technologies): Human MATR3 TaqMan assay (Assay ID:Hs00251579_m1), Mouse Matr3 TaqMan assay (Assay ID: Mm00726619_s1, in exon 2) and Mouse Matr3 TaqMan assay (Assay ID: Mm01704913_g1 in exon 15).

Techniques: Polymer, Injection

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Genes influenced by over-expression of MATR3:YFP (no change in expression by YFP alone).

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Expressing, Over Expression

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Summary of spectral count data for the top 50 interacting proteins ranked by fold enrichment for MATR3:AviTag relative to controls.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques:

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: A graphic representation of the interactome for WT-MATR3 in human HEK293 cells. Proteomic mass spectrometry data from Supplemental Table and the software program Cytoscape (version 3.5.1) were used to generate a graphic representation of MATR3 interaction. The intensity of shading of the oval containing the gene name is linked to the spectral counting data in Supplemental Table ; the lighter the shading the higher the fold increase in spectral counts for a given protein relative to controls (untransfected cells; no expression of Avi-tagged MATR3). Proteins marked with an asterisk are interactors also identified by other studies (see Supplemental Table ). The localization or function of the identified proteins was determined by three sources { www.uniprot.org } and , . Using quantification by label-free spectral counting, HNRNPM and numerous RNA processing factors including ADAR, PDBP3, SFRS14, and PTBP1 appear to be the strongest interactors with MATR3.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Mass Spectrometry, Software, Expressing

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Distribution of MATR3:YFP in nuclear compartments of C2C12 cells. Mouse C2C12 cells were grown on glass cover slips and transiently transfected with pEF.Bos vectors encoding MATR3:YFP variants noted on the figure. At 24 hours post-transfection, the cells were fixed and imaged. The images shown are representative of images seen in 3 independent transfections, viewing 30 to 50 cells on each cover slip (data quantified in Table ). WT MATR3 and MATR3 lacking ZnF1, ZnF2, or RRM1 sequences show a predominantly diffuse distribution within the nucleus with variable levels of small puncta. MATR3 lacking RRM2 organizes into large uniformly spherical structures. Scale bar = 100 µm.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Transfection

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Quantification of spherical structures formation by MATR3:YFP constructs.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Construct, Transfection

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Images from time-lapse video demonstrates fusion of MATR3:YFPΔRRM2 spheres in C2C12 cells. An example of the timing of fusion is illustrated in this panel of images. Two independent fusion events can clearly be seen to occur over a period of 15–30 minutes in each case. The example shown is representative of observations made in 3 separate transfection experiments tracking 20–30 cells in each field of view. Original magnification 20X.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Transfection

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Lack of WT-MATR3:YFP and WT-MATR3:YFP ΔRRM2co-localization with TDP-43:mCherry in C2C12 cells. Mouse C2C12 cells were grown on glass cover slips and transiently transfected with pEF.Bos vectors encoding MATR3:YFP variants noted on the figure. At 24 hours post-transfection, the cells were fixed and imaged. The images shown are representative of what was found in at least two separate transfection experiments analyzing 50–100 cells in each experiment (original magnification 60X with 2X digital enlargement). Each image is from a single z-plane captured on a confocal microscope (see Methods). WT-MATR3:YFP and WT-TDP-43:mCherry show limited overlap, for example TDP-43:mCherry is not seen in puncta of WT-MATR3:YFP or the spherical structures formed by MATR3:YFPΔRRM2.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Transfection, Microscopy

Journal: Scientific Reports

Article Title: Characterization of gene regulation and protein interaction networks for Matrin 3 encoding mutations linked to amyotrophic lateral sclerosis and myopathy

doi: 10.1038/s41598-018-21371-4

Figure Lengend Snippet: Minimal co-localization of MATR3 with lamin A/C or histones. Mouse C2C12 cells were grown on glass cover slips and transiently transfected with pEF.Bos vectors encoding MATR3:YFP variants noted on the figure. At 24 hours post-transfection, the cells were fixed, immunostained with antibodies to lamin A/C (Sigma-Aldrich #SAB4200236) or histone H3 (Abcam #ab8898) and imaged. The images shown are representative of images seen in 3 independent transfections, viewing 30 to 50 cells on each cover slip. Each image is from a single z-plane captured on a confocal microscope (see Methods). Nikon elements software was used to determine the extent of interaction between MATR3 and lamin A/C or histone H3 (Supplemental Figures and ). MATR3 shows limited co-localization with either lamin A/C or histone H3.

Article Snippet: Flag- MATR3 delZnF1 (plasmid #32884), Flag- MATR3 delZnF1 (plasmid #32883), Flag- MATR3 del RRM1 (plasmid #32881), Flag- MATR3 delRRM2 (plasmid #32882) vectors was obtained commercially from Addgene (Cambridge, MA, USA). cDNAs inserts from these plasmids amplified by PCR, excluding the Flag tag, and were cloned into a pEF-BOS.MATR3:YFP vector by swapping out MATR3 cDNA sequences, using the Infusion HD kit.

Techniques: Transfection, Microscopy, Software