Journal: Cell Reports

Article Title: Cohesin Disrupts Polycomb-Dependent Chromosome Interactions in Embryonic Stem Cells

doi: 10.1016/j.celrep.2019.12.057

Figure Lengend Snippet:

Article Snippet: RNA-seq libraries were then prepared from 200 ng of RNA using the NEBNext Ultra II Directional RNA-seq kit (NEB).

Techniques: Plasmid Preparation, Recombinant, Capture-C, Hybridization, Multiplex Assay, Ligation, Software

Journal: Molecular cell

Article Title: Xist deletional analysis reveals a co-dependency between Xist RNA and Polycomb complexes for spreading along the inactive X

doi: 10.1016/j.molcel.2019.01.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Input and ChIP-seq libraries were prepared in two biological replicates using NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (New England BioLabs) as per manufacturer’s instructions.

Techniques: Recombinant, Isolation, Multiplex Assay, Transfection, In Situ, Transgenic Assay, Mutagenesis, Southern Blot, Expressing, Plasmid Preparation, In Vitro, Software

Journal: Nucleic Acids Research

Article Title: Novel rapidly evolving hominid RNAs bind nuclear factor 90 and display tissue-restricted distribution

doi: 10.1093/nar/gkm668

Figure Lengend Snippet: snaRs are highly structured NF90-associated RNAs. ( A ) RNA immunoprecipitated from cell lines with anti-omni antibody was 3′-end labeled and resolved in a 5% acrylamide/7 M urea gel . Cell lines contained omni-tagged NF90a or NF90b (lanes 2 and 3) or empty vector (lane 1). Note that the cell extracts contained more NF90b than NF90a. ( B ) The most stable snaR-A and -B structures predicted by MFOLD . Base-pairing is represented by dots. snaR-B bases altered or deleted (open triangle) in snaR-C are circled. ( C ) Northern blot of supernatant ×10 −3 (Sup) and immunoprecipitated (IP) RNA from the cell lines probed with Probe-A (Figure S2A). 3′-pCp-labeled RNA precipitated with NF90a served as a marker (M). ( D ) RNA immunoprecipitated with NF90b was 3′-end labeled, digested with RNase H in the presence of sense (S) or antisense (AS) oligonucleotides corresponding to snaR (probe H, B) or 5S rRNA and resolved in a 5% acrylamide/7 M urea gel. Asterisk marks snaR-A RNase H digestion product, bromophenol blue (bpb) migration is denoted. ( E ) In vitro binding assay of T7 RNA polymerase transcribed snaR-A to equal amounts of GST fusion proteins (Figure S1) in the presence of 2000-fold molar excess yeast tRNA. NF90b [A458P,A588P] mutant is denoted by ‘GST-Mut’ and 20% input was loaded.

Article Snippet: RNA was incubated with RNase H (5 U, New England BioLabs) in the supplied reaction buffer at 30°C for 3 h. Digestion was stopped with 16 μl 2× formamide loading buffer (95% formamide, 10 mM Tris-HCl, pH 8, 20 mM EDTA) and heating at 70°C for 5 min.

Techniques: Immunoprecipitation, Labeling, Plasmid Preparation, Northern Blot, Marker, Migration, In Vitro, Binding Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Novel rapidly evolving hominid RNAs bind nuclear factor 90 and display tissue-restricted distribution

doi: 10.1093/nar/gkm668

Figure Lengend Snippet: snaR RT-PCR clones. ( A ) Clones of snaR identified from sequencing of RT-PCR products can be grouped into two subsets. Homology within each subset is denoted by an asterisk and non-homologous nucleotides are in bold font. Differences between subsets are denoted by gray shading. Consensus sequences are given below each set of clones. The majority of clones are derived from asynchronous NF90b cell line extract. Clones with ‘m’ or ‘a’ appended to their name were immunoprecipitated from NF90b G2/M phase extract or from NF90a extract, respectively. ( B ) The genomic sequence of snaR-A and -B. Genomic nucleotides matching consensus sequence are highlighted in yellow, those differing from consensus sequence are in green. 3′-Oligo(A) and oligo(T) tracts are denoted in red and blue, respectively. Dashed line denotes sequence complementary to probe H, solid line denotes predicted RNase H digestion product. ( C ) Sequence alignment of snaR-A with two potential piRNAs (bold). Alu RNA homology is denoted in yellow. Sequence homologous to the PolIII B box motif is underlined.

Article Snippet: RNA was incubated with RNase H (5 U, New England BioLabs) in the supplied reaction buffer at 30°C for 3 h. Digestion was stopped with 16 μl 2× formamide loading buffer (95% formamide, 10 mM Tris-HCl, pH 8, 20 mM EDTA) and heating at 70°C for 5 min.

Techniques: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Sequencing, Derivative Assay, Immunoprecipitation

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: ( A ) Predicted hairpins involving the sequestration of the SD sequence by the aSD1 motif. Prediction of these secondary structures were carried out on the aapA3 -Tr transcript (see for visualizing the whole structure) using the same software as in , but with no additional constraint. The A28C mutation (dark orange) generates an extra G-C base-pair; in the A40T mutant (purple), two additional A-U pairs stabilize the hairpin; the A40T antagonist mutation A33T is shown in red; the SD and aSD1 sequences are shown in turquoise and yellow, respectively. ( B ) (left) PCR constructs used to assess the SD sequestering structure by transformation assay. (right) For each transformation with the indicated PCR constructs, the number of Str R obtained per total number of transformed cells was calculated and plotted on a log scale. Error bars represent s.d; n = 3 biological replicates. (***p<0.0001; *p=0.001 according to unpaired t -test). ( C ) Graph representing the RNase H assays. The position of the oligonucleotides (FA644 for WT and A40T; FA651 for A33T/A40T; and FA652 for A28C, see ) used in the RNase H protection assay is indicated by a black arrow along the first 45 nucleotides of the aapA3 mRNA . 30 fmol of internally labeled WT and mutated aapA3 -Tr transcripts were incubated with 0 to 100 pmoles of each specific DNA oligonucleotide and subjected to digestion by E. coli RNase H1. Digestion products were analyzed on an 8% PAA denaturing gel . Substrate consumption was quantified as relative substrate band intensity, 100% corresponding to the intensity obtained in absence of oligonucleotide. Error bars represent the s.d; n = 2 technical replicates. 10.7554/eLife.47549.018 Figure 5—source data 1. Raw data to determine the transformation efficiency in . 10.7554/eLife.47549.019 Figure 5—source data 2. Numerical values of the graph shown in in .

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Sequencing, Software, Mutagenesis, Construct, Transformation Assay, Labeling, Incubation

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: 30 fmol of internally labeled RNA were used. DNA oligonucleotides were used to a final concentration of 0 to 10 μM. Reactions were incubated for 30 min at 30°C in the presence or absence (C, control) of 0.25 U E. coli RNase H1. Reactions were stopped with 10 μl of 2X Loading Buffer and products were analyzed in an 8% denaturing PAA gel. See for relative band quantification. P1 and P2 indicate the two RNase H-oligonucleotide-mediated RNA cleavage products.

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Labeling, Concentration Assay, Incubation, Control

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: ( A ) The cell lysate of the T78C strain was subjected to ultracentrifugation through a sucrose gradient. RNA was analyzed as in . The different transcripts aapA3 -FL, aapA3 -Tr, and 5S rRNA (loading control) are indicated. M+D, monosomes + disomes. ( B ) Prediction of the secondary structure involving the second aSD sequence (aSD2). Prediction was carried out on the biologically relevant mRNA ( aapA3 -Tr) (see ). No additional constrains were used. The location of the oligonucleotide used in this assay (FA633) is shown by a black arrow in . The T78C mutation is shown in dark blue, SD sequence in turquoise, anti-SD sequence in yellow and start codon in green. ( C ) A typical RNase H protection assay is shown. A total of 30 fmol of internally labeled aapA3- FL and aapA3 -Tr RNA (WT or T78C) were incubated in presence of 0 to 100 pmoles of DNA oligonucleotide (FA633) and subjected to digestion by E. coli RNase H1. Lane C contains only the labeled substrate in absence of the enzyme. Two digestion products, P1 and P2, are indicated. ( D ) Substrate consumption was quantified as the relative substrate band intensity and plotted as a function of DNA oligonucleotide concentration. Error bars represent the s.d; n = 2 technical replicates. 10.7554/eLife.47549.024 Figure 6—source data 1. Numerical values of the graph shown in in .

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Control, Sequencing, Mutagenesis, Labeling, Incubation, Concentration Assay

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: 2D structure was predicted using the RNAfold Web Server with no additional constraints. VARNA was used for drawing. Translation start and stop codons are highlighted in green and red, respectively. Shine-Dalgarno (SD) and anti-SD (aSD) sequences are shown in turquoise and yellow, respectively. The suppressor mutation T78C is highlighted in dark blue. The predicted delta G value (kcal/mol) is shown under the structure. The small black arrow indicates the binding site of the oligonucleotide used in the RNase H assay shown in .

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Mutagenesis, Binding Assay, Rnase H Assay

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet:

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Plasmid Preparation, Bacteria, DNA Extraction, Recombinant, Sequencing, Software

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: 5′ UTR regulated HBoV1 capsid mRNA abundance and protein translation. (A) Diagram of HBoV1 capsid expression constructs with the T7 promoter for the in vitro assay. (B) In vitro coupled transcription/translation assay. In vitro assays were performed according to the manufacturer's instructions. Expressed proteins were run on a 15% SDS-PAGE gel, and the signal was detected with a Cyclone Plus system (PerkinElmer) and analyzed using OptiQuant software. The ratio of VP1 to VP3 is presented at the bottom of the gel. The experiment was repeated at least three times. (C) Diagram of HBoV1 VP cDNA constructs with the cytomegalovirus (CMV) promoter. (D) Northern blot. Ten micrograms of total RNAs prepared from transfected cells were resolved on 1.5% agarose gels, transferred to Hybond-N + membranes, and hybridized with probes spanning nt 349 to 5167. The signal was detected using a ChemiDoc MP imaging system (Bio-Rad). Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. (E) Western blot (WB). The lysates of HEK293T cells transfected with the plasmids described in panel C were analyzed using an anti-Flag antibody to detect capsid expression. β-Actin served as the loading control.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Construct, In Vitro, SDS Page, Software, Northern Blot, Transfection, Imaging, Staining, Control, Western Blot

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: Identification of the cis elements in the 5′ UTR that regulated RNA abundance and translation. (A) Diagram of green fluorescent protein (GFP) reporter plasmids. Group I plasmids contained either the entire 5′ UTR or VP1 unique sequences. Group II plasmids contained both the 5′ UTR and VP1u. Group III constructs harbored VP1u with deletions in exons 1 to 4. Exon 4 in the plasmids of group IV was deleted. SV40, simian virus 40. (B) Microscopy of the fluorescent signal when reporter plasmids were transfected. (C) Western blot. The lysates of transfected HEK293T cells were analyzed using anti-GFP antibody, and β-actin served as the loading control. (D) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. The image in panel D was spliced from two different gels because the samples could not be run in one gel.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Construct, Virus, Microscopy, Transfection, Western Blot, Control, Northern Blot, Staining

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: 5′ UTR cis elements regulated HBoV1 capsid protein expression and mRNA abundance in HEK293T cells. (A) Diagram of capsid expression plasmids. Various lengths of the 5′ UTR sequence from p3XFlag-R6 were deleted to construct p3XFlag-Tru1, p3XFlag-Tru2, p3XFlag-E3 and -E4, and p3XFlag-mini. Plasmids p3XFlag-mini-T1 to p3XFlag-mini-T10 were generated by sequence deletion in exon 2 or exon 3 based on p3XFlag-mini, as indicated. (B) Western blot. The transfected HEK293T cell lysates were analyzed using anti-Flag antibody, and β-actin served as the loading control. (C) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Sequencing, Construct, Generated, Western Blot, Transfection, Control, Northern Blot, Staining

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: The alternative translation of VP1 and VP2 was IRES independent. (A) Diagram of bicistronic fluorescent reporter constructs. The 3×Flag tag, the simian virus 40 poly(A) signal, and multiple-cloning sites (MCS) are denoted. (B) Microscopy images of fluorescent signals. Images of the green fluorescent protein (GFP) fluorescent signals are on the top, and images of the mCherry fluorescent signals are on the bottom (in images of the same visual fields). (C) Western blot. The lysates of transfected HEK293T cells were analyzed using anti-GFP and anti-Flag antibodies. β-Actin served as the loading control. (D) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Construct, Virus, Cloning, Microscopy, Western Blot, Transfection, Control, Northern Blot, Staining

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: The alternative translation of HBoV1 VP proteins was based on ribosomal leaky scanning. (A) Diagram of plasmids used for in vitro or in vivo translation. (B) Western blot. The lysates of transfected HEK293T cells were analyzed using anti-Flag antibody, and β-actin served as the loading control. (C) In vitro coupled transcription/translation assay. In vitro assays were performed as described in the legend to . (D) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: In Vitro, In Vivo, Western Blot, Transfection, Control, Northern Blot, Staining

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: uATGs influenced HBoV1 capsid protein expression. (A) Diagram of HBoV1 uATG-mutated constructs. The positions of uATG1, uATG2, and uATG3 are denoted by their nucleotide sequence numbers. (B) Western blot. The transfected HEK293T cell lysates were analyzed using anti-Flag antibody, and β-actin served as the loading control. (C) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. ncRNA, noncoding RNA.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Construct, Sequencing, Western Blot, Transfection, Control, Northern Blot, Staining

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: uATG2 and uATG3 knockout mutations in an HBoV1 infectious clone decreased viral progeny production. (A) Southern blot. Hirt DNA was isolated from HEK293T cells transfected with plasmids, as indicated, at 48 h posttransfection. The DpnI-digested fragments were resolved on 1% agarose gels and transferred to a Hybond-N + membrane, followed by hybridization with an HBoV1 probe spanning from nt 1 to 5543. dRF, double replication form; mRF, monomer replication form; ssDNA, single-stranded DNA. (B) Northern blot. Total RNAs were harvested from transfected cells, and Northern blotting was carried out as described in the legend to . Ethidium bromide (EB)-stained 18S RNA bands are shown as the loading control. The images in panel B were taken from different areas of the same photo or different gels and then joined together. (C) Quantitative PCR (qPCR) for the HBoV1 genome. Cell lysates of uATG-mutated HBoV1 infectious clones were prepared by four cycles of freezing and thawing, followed by DNase I digestion. The progeny viruses were separated by cesium chloride (CsCl) density gradient centrifugation, and genomic DNA was prepared as the template for qPCR. Means and standard deviations were calculated from the results of at least three independent experiments. The pHBoV plasmid was used as a control to establish a standard curve for absolute qPCR ( E = 105.0%, R 2 = 0.999, slope = −3.208). E, efficiency. (D) Western blot. uATG-mutated infectious clones were transfected into HEK293T cells, and Western blotting was performed with anti-NS, anti-NP, and anti-VP antibodies.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Knock-Out, Southern Blot, Isolation, Transfection, Membrane, Hybridization, Northern Blot, Staining, Control, Real-time Polymerase Chain Reaction, Clone Assay, Gradient Centrifugation, Plasmid Preparation, Western Blot

Journal: Journal of Virology

Article Title: The 5′ Untranslated Region of Human Bocavirus Capsid Transcripts Regulates Viral mRNA Biogenesis and Alternative Translation

doi: 10.1128/JVI.00443-18

Figure Lengend Snippet: uATG mutations in an HBoV1 infectious clone altered viral RNA processing. RNase protection assay (RPA) with analysis of viral RNA polyadenylation at the (pA)p site. Ten micrograms of total RNAs prepared from HEK293T cells transfected with plasmids, as indicated, was protected by the (pA)p site-specific probe. RT, read through RNAs. The ratios of RNA polyadenylated at the (pA)p site versus read-through RNA are indicated as the mean and standard deviation. The numbers on the left are molecular size markers.

Article Snippet: The human embryonic kidney cell line HEK293T (ATCC, CRL-11268) was maintained in Dulbecco's modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Rnase Protection Assay, Transfection, Standard Deviation

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: Inducible RNase H1 transgene expression in vitro. A) Schematic of hiPSC differentiation from fibroblasts to NPCs and neurons. Left panel: Immunofluorescent staining of the neuronal marker MAP2 (green) in hiPSC-neurons (top) and the NPC markers SOX2 (green) and Nestin (red) in hiPSC-NPCs (bottom); scale bar: 100 mm. Right panel: Immunofluorescent staining of S9.6 (red) and DAPI (blue) in each respective cell type; scale bar: 100 mm. B) Schematic of the effect of RNase H1-mediated knockdown of R-loops, or D145N mutant that is binding- competent but catalytically inactive. C) Overview of the lentiviral constructs used in this study, including the rtTA transactivator, RH1ΔMLS, RH1ΔMLS D145N catalytically-inactive mutant, and BFP-expressing lentiviral backbone control. D) (Top) Representative Western Blot of hiPSC-neurons expressing either BFP, RH1ΔMLS, or RH1ΔMLS D145N lentiviruses. Gradient triangles indicate concentration of virus. RNase H1 protein is 5.1-fold increased in RH1ΔMLS cells relative to BFP controls (normalized to β-actin loading control). (Bottom) Immunocytochemistry for S9.6 (red) and RNase H1 (green) in hiPSC-neurons expressing BFP, RH1ΔMLS D145N , or RH1ΔMLS transgenes. RNase H1 protein is robustly increased in the cell nucleus (DAPI, blue) in RH1ΔMLS and RH1ΔMLS D145N cells relative to BFP controls, indicating successful nuclear expression of the transgene-encoded protein; scale bars = 20 mm. E) Experimental timeline. NPCs are transduced with RH1ΔMLS or a control (RH1ΔMLS D145N or BFP backbone) lentivirus. After 4 days in vitro , puromycin (1:1,000) is added to the culture media to select for transgene-expressing cells. After 6 days in culture, puromycin concentration is dropped to 1:5,000. On day 0 (indicating transition from NPC to neuron differentiation), cells are replated and fed every 2 days with neuron differentiation medium containing 1:1,000 doxycycline to activate the transgene. Every week from week 2, electrophysiological recordings are captured by multielectrode array (MEA). After 6 weeks in culture, cells are harvested for DRIP-seq, bulk- and scRNA-seq. F) (top to bottom) DRIP-seq tracks for hiPSC-derived neurons transduced with BFP (blue tracks, BFP Neuron cultures 1-3) or RH1ΔMLS (bright red tracks, cultures 1-3) constructs over a well-defined, conserved R-loop hotspot region , demonstrating robustness of our DRIP-seq experimental and computational processing. Note that y-axis scaling 0-90 for BFP Neuron DRIP-seq is wider than RH1 Neuron scale, 0-10. RNA alone processed by the DRIP-seq protocol (RNA-only DRIP, magenta track) shows no discernable peaks, indicating that our S9.6 immunoprecipitation is specific for DNA/RNA hybrids and does not represent single-stranded or double-stranded RNA. Orange track: in silico HindIII/EcoRI/XbaI/ SSPI restriction enzyme digest showing expected cutting sites of the restriction enzyme cocktail used for DRIP-seq. G) Left: Volcano plot displaying the 6,380 differential promoter-bound R-loop peaks (DRIP-seq) between hiPSC-differentiated cultures expressing RH1ΔMLS (n = 3) or BFP (n = 3) (FDR < 0.05, pink dots) after 6 weeks in culture. Of these, 4,805 DRIP-seq peaks were enriched in BFP controls (log 2 fold change > 1) and 474 DRIP-seq peaks were enriched RH1ΔMLS cells (log 2 fold change < -1), indicating successful knockdown of R-loops in the latter. Right: Volcano plot displaying the 1,753 differential promoter-bound R-loop peaks between RH1ΔMLS (n = 3) and RH1ΔMLS D145N (n = 2) (FDR < 0.1, pink dots) cells after 6 weeks in culture. Of these, 1,709 DRIP-seq peaks were enriched in RH1ΔMLS D145N controls (log 2 fold change > 1) and 35 DRIP-seq peaks were enriched in RH1ΔMLS cells (log 2 fold change < -1), again indicating successful knockdown of R-loops in the latter. H) Representative DRIP-seq tracks over two neuronal genes for BFP-, RH1ΔMLS D145N -, and RH1ΔMLS- expressing hiPSC-neurons after 6 weeks in culture. Pink highlighted region and inset displays magnified promoter region.

Article Snippet: As mitochondrial R-loops are essential for cell survival, we first used the QuikChange II Site-directed mutagenesis kit (Agilent, cat. no. #200523) according to the manufacturer’s instructions to remove the 78 bp mitochondrial localization signal (MLS) from RNase H1 cDNA (AddGene; pFRT-TODestGFP_RNAseH1; Plasmid #65784), using the primer sequences in Supplementary Table 24, RH1ΔMLS.

Techniques: Expressing, In Vitro, Staining, Marker, Knockdown, Mutagenesis, Binding Assay, Construct, Control, Western Blot, Concentration Assay, Virus, Immunocytochemistry, Transduction, Derivative Assay, Immunoprecipitation, In Silico

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: Cell specifications and transcriptomic alterations in RH1 transgenic cultures. A, B) Gene set enrichment analysis of scRNA-seq data from RH1ΔMLS and BFP control hiPSC-differentiated cultures. (A) Cnet plot and (B) dot plot showing significant gene ontologies up- (left) and down-regulated (right) following RH1ΔMLS transgene induction. Dot color indicates fold change (cnet plot) or adjusted p- value (dot plot), dot size indicates number of genes in category. n = 2 replicates from 2 cell lines/group. C) Combination of clusters into broad cell type classes, including glia (green), neurons (purple), and NPCs (blue). Gray clusters did not show canonical markers of these major neural cell types. D) RH1ΔMLS cells have significantly (p < 0.0001, Wilcoxin Rank Sum test) higher RNASEH1 expression than BFP controls (mean normalized expression: RH1ΔMLS = 2.45, BFP = 0.94). Cells from each condition were split into two groups according to RNASEH1 normalized expression value (> 1.25 = hi; ≤ 1.25 = lo) for downstream analyses. E) Bar charts showing difference in cell proportions across BFP or RH1ΔMLS transduced cells, stratified by RNASEH1 expression level. There is a significant reduction in the proportion of glia (p = 0.015) and an increased proportion of neurons (p = 0.036) in RH1ΔMLS- RNASEH1 high relative to BFP- RNASEH1 low groups (Two-way ANOVA with Dunnett’s Multiple Comparison test; n = 2 replicates from 2 cell donors per group; error bars denote S.E.M.; n.s. = not significant.) F, G) Volcano plots showing differentially expressed genes in scRNA-seq ( F ) and bulk RNA-seq ( G ) from RH1ΔMLS hiPSC-differentiated cultures and BFP controls. Green dots indicate genes more highly expressed in control cells (FDR < 0.05, log 2 fold change < 0) and purple/magenta dots indicate genes more highly expressed in RH1ΔMLS cells (FDR < 0.05, log 2 fold change > 0). H) Venn diagram showing overlap of upregulated genes in RH1ΔMLS hiPSC-differentiated cultures from scRNA and bulk RNA-seq, and promoter-bound R-loops that are knocked down following RNase H1-transgene expression. RF = Representation factor, which signifies more overlap of the two gene sets than expected based on a background list of genes if > 1. P-values calculated via hypergeometric test. I) Gene ontology of genes with upregulated expression following promoter-bound R-loop loss in RH1ΔMLS hiPSC-differentiated cultures. Numbers within gene ontology images indicate number of genes in the enriched set that occur within the ontology class, legend denotes -log 10 enrichment FDR. J) Venn diagram showing overlap of genes that have upregulated gene expression following promoter-bound R-loop loss in RH1ΔMLS hiPSC-differentiated cultures and R-loop “primed” genes in germinal matrix . RF = Representation factor, p-values calculated via hypergeometric test. K) Heatmap indicating overlap of genes that have upregulated gene expression following promoter-bound R- loop loss in RH1ΔMLS hiPSC-differentiated cultures and disease risk genes. p-values calculated via hypergeometric test. L) Optical intensity from immunofluorescence images of the R-loop-specific antibody S9.6, and the synaptic proteins PDS-95 and synapsin, in RH1ΔMLS and RH1ΔMLS D145N hiPSC-differentiated cultures. P-values calculated via two-tailed student’s t test; Error bars denote S.E.M.; n = 25 images per cell line / group. M) Representative immunofluorescence images of the synaptic protein synapsin (green) in RH1ΔMLS hiPSC- differentiated cultures and RH1ΔMLS D145N controls, along with the nuclear marker DAPI (blue) and the neuronal marker MAP2 (magenta). Scale bar: 20 mm.

Article Snippet: As mitochondrial R-loops are essential for cell survival, we first used the QuikChange II Site-directed mutagenesis kit (Agilent, cat. no. #200523) according to the manufacturer’s instructions to remove the 78 bp mitochondrial localization signal (MLS) from RNase H1 cDNA (AddGene; pFRT-TODestGFP_RNAseH1; Plasmid #65784), using the primer sequences in Supplementary Table 24, RH1ΔMLS.

Techniques: Transgenic Assay, Control, Expressing, Comparison, RNA Sequencing, Gene Expression, Immunofluorescence, Two Tailed Test, Marker

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: RNase H1-mediated R-loop loss during neuronal differentiation in vitro results in electrophysiological deficits. A) Top left: Electrophysiological recordings were taken with an Axion multielectrode array (MEA) system to assess population-wide neuronal activity. Schematic of the 48-well MEA plate used for electrophysiological recordings. Bottom Left: Schematic of an individual well within the MEA plate, containing 16 recording electrodes. Right: Screen shot of the MEA recording on week 8, with BFP-expressing hiPSC-neurons plated on the left half of the plate and RNase H1-overexpressing neurons on the right. B) Representative spike raster plots over the 10-minute recording at week 8 for BFP-expressing (left) and RNase H1-overexpressing (right) hiPSC-neurons. Each row denotes a single recording electrode within a single well of the 48-well MEA plate. C-E) The number of spontaneous spikes ( C ), weighted mean firing rate (wMFR) in Hertz ( D ), and number of bursts ( E ) were recorded weekly from BFP-controls and RNase H1 transgene expressing hiPSC-neurons from two donor lines. Data from the two donor lines were combined and a two-way ANOVA was performed with Sidak’s multiple comparisons test to test significant differences in each metric between weeks 2-8. Each condition contained at least 14-20 viable well replicates after removing wells that had less than 10 active recording electrodes. F) Statistics were additionally calculated for each electrophysiological metric at week 8 (unpaired two-tailed student’s t test) between RNase H1-transgene hiPSC-neurons and BFP controls. Individual donors represented with either filled in or hollow shapes.

Article Snippet: As mitochondrial R-loops are essential for cell survival, we first used the QuikChange II Site-directed mutagenesis kit (Agilent, cat. no. #200523) according to the manufacturer’s instructions to remove the 78 bp mitochondrial localization signal (MLS) from RNase H1 cDNA (AddGene; pFRT-TODestGFP_RNAseH1; Plasmid #65784), using the primer sequences in Supplementary Table 24, RH1ΔMLS.

Techniques: In Vitro, Activity Assay, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: R-loop landscapes in the developing human brain are linked to neural differentiation and cell-type specific transcription

doi: 10.1101/2023.07.18.549494

Figure Lengend Snippet: RNase H1 overexpression leads to reduced dendritic complexity and spine density. Expression of the RNASEH1 ΔMLS transgene, delivered via in utero electroporation into neural precursor cells of the ventricular zone in E15 mouse embryos, affects dendrite and spine morphology of cortical projection neurons. A) Expression plasmids of GFP combined with tdTomato are used as controls, while GFP combined with RNASEH1 ΔMLS are used to destroy neural R-loops in mice during prenatal development. B) Overview of the in utero electroporation technique. TdTomato or RH1ΔMLS along with GFP reporters are transfected and electroporated unilaterally in E15 mouse ventricular zone. In adult mouse, P28, strong unilateral GFP expression is observed. Scale bar = 1 mm. C) In utero electroporation experimental timeline. D) Expression of exogenous (human) RNASEH1 in RH1ΔMLS mice was confirmed in FAC-sorted GFP+ neurons, whereas no difference was observed for endogenous expression of (mouse) Rnaseh1 between groups via qPCR (Normalized to Gapdh expression). Student’s two-tailed t test; n.s. = not significant. E) Expression of human RH1ΔMLS reduces S9.6 immunoreactivity in the nucleus of mouse neural cells at P0. F) FlSH images showing RNA expression of RNASEH1 (red) in the P28 cortex of tdTomato controls and RH1ΔMLS mice, and colocalization of GFP (green) and RNASEH1 in RH1ΔMLS cells. Scale bar = 100 mm. G) Representative images of (left) control and (right) RH1ΔMLS-expressing frontal pyramidal neuron dendritic trees at P28. H) Sholl analysis, showing reduced dendritic complexity in (left) control and (right) RH1ΔMLS-expressing pyramidal neurons at P28 (tdTomato control n = 3, RH1ΔMLS n = 4). I) Representative images of (left) control and (right) RH1ΔMLS frontal pyramidal neuron dendritic spines at P28. J) Apical and basal spine densities, assessed by Imaris tracing + FOCM (see text), are significantly decreased in RH1ΔMLS-expressing prefrontal pyramidal neurons. N = 3/group. *P < 0.05, **P < 0.01 determined by Two-Tailed Student’s t-test.

Article Snippet: As mitochondrial R-loops are essential for cell survival, we first used the QuikChange II Site-directed mutagenesis kit (Agilent, cat. no. #200523) according to the manufacturer’s instructions to remove the 78 bp mitochondrial localization signal (MLS) from RNase H1 cDNA (AddGene; pFRT-TODestGFP_RNAseH1; Plasmid #65784), using the primer sequences in Supplementary Table 24, RH1ΔMLS.

Techniques: Over Expression, Expressing, In Utero, Electroporation, Transfection, Two Tailed Test, RNA Expression, Control