Journal: Acta Biochimica et Biophysica Sinica

Article Title: CARF regulates the alternative splicing and piwi/piRNA complexes during mouse spermatogenesis through PABPC1

doi: 10.3724/abbs.2024224

Figure Lengend Snippet: CARF is highly expressed in spermatocytes and spermatids of the testis (A) NCBI database analysis of the expression profile of Carf mRNA in mouse tissues. (B) qPCR analyses of Carf mRNA levels in multiple organs of mice. Data are presented as the mean ± SEM, n = 3. (C) Immunofluorescence staining analysis of CARF (green) in testis sections. γH2AX (red) was used as a marker for spermatocytes. The nuclei were stained with DAPI (blue). Spc indicates spermatocytes, Ser indicates Sertoli cells, Ley indicates Leydig cells, and Rspd indicates round sperm. Scale bar: 50 μm. (D) Immunofluorescence staining analysis of CARF (red) and PNA (green) in testis sections. The nuclei were stained with DAPI (blue), Scale bar: 50 μm. (E,F) The expression pattern of CARF in the mouse germline atlas was analyzed via a single-cell sequencing database (http://malehealthatlas.cn/ ).

Article Snippet: Anti-rabbit PABPC1 (1:100, Cat No. #53348; Cell Signaling, Beverly, USA) and CARF (1:100, Cat No. #16615-1-AP; Proteintech) primary antibodies were used for staining overnight.

Techniques: Expressing, Immunofluorescence, Staining, Marker, Sequencing

Journal: Acta Biochimica et Biophysica Sinica

Article Title: CARF regulates the alternative splicing and piwi/piRNA complexes during mouse spermatogenesis through PABPC1

doi: 10.3724/abbs.2024224

Figure Lengend Snippet: CARF interacts with PABPC1 to participate in RNA alternative splicing (A) Abnormal alternative splicing patterns, including skipped exons, alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), mutually exclusive exons (MXE) and retained intron (RI) caused by Carf defects. (B) Abnormal variable splicing of the functional gene Hira, which is related to germ cell development caused by Carf defects. It belongs to the alternative 5′ splice site (A5SS) exception mode. (C) Abnormal variable splicing of the functional gene Surf1, which is related to germ cell development caused by Carf defects. It is an alternative 3′ splice site (A3SS). (D) Abnormal variable splicing of the functional gene Usf2, which is related to germ cell development caused by Carf defects. It belongs to the mutually exclusive exons (MXE). (E) Abnormal variable splicing of the functional gene Lrmp, which is related to germ cell development caused by Carf defects. It belongs to the retained intron (RI) family. (F) Abnormal variable splicing of the functional gene Pkd2l1, which is related to germ cell development caused by Carf defects. It belongs to the retained intron (RI) family. (G) Co-IP analysis of the interaction between CARF and PABPC1. PABPC1 expression was detected in the IP products of CARF, and IgG was used as a control. GAPDH served as a loading control. (H) Co-IP analysis of the interaction between CARF and PABPC1. CARF expression was detected in the IP products of PABPC1, and IgG was used as a control. GAPDH served as a loading control. (I) Immunostaining of PABPC1 in wild-type and Carf–/– testis sections (PABPC1: green); DAPI was used to stain the dye the nuclei, scale bar: 50 μm. (J) Western blot analysis of PABPC1 protein levels in testes from wild-type and Carf–/– mice. GAPDH served as a loading control. (K) Immunohistochemical analysis of the expression of PABPC1 in testes from wild-type and Carf–/– testis sections. Scale bar: 50 μm. (L) Quantitative results of (K). n = 3, Data are presented as the mean ± SEM. ***P < 0.001.

Article Snippet: Anti-rabbit PABPC1 (1:100, Cat No. #53348; Cell Signaling, Beverly, USA) and CARF (1:100, Cat No. #16615-1-AP; Proteintech) primary antibodies were used for staining overnight.

Techniques: Alternative Splicing, Functional Assay, Co-Immunoprecipitation Assay, Expressing, Control, Immunostaining, Staining, Western Blot, Immunohistochemical staining

Journal: Nucleic Acids Research

Article Title: A Myc–microRNA network promotes exit from quiescence by suppressing the interferon response and cell-cycle arrest genes

doi: 10.1093/nar/gks1452

Figure Lengend Snippet: miR-22 targets genes mediating cell-cycle arrest. ( A ) Ago2 IP. Ago2 forms a complex with the miRNA and the target mRNA. This complex is immunoprecipitated and the mRNA levels are quantified with qRT-PCR. ( B ) Ago2 IPs identify genes mediating cell-cycle arrest and apoptosis as direct targets of miR-22. REDD1, TP53INP1, p21 and CARF transcripts show significantly higher Ago2 occupancy in miR-22–transfected HeLa cells compared with mock-transfected cells. Ago2 occupancy of the target genes transcripts was measured using qRT-PCR as described in the text. The Y -axis shows fold change in mRNA levels from Ago2 IP–isolated RNA normalized to input RNA, which served as the control. ( C ) Luciferase assays show that miR-22 directly targets the 3 ′ UTRs of the genes indicated on the X -axis. IKKg and Empty are negative controls. IKKg was not repressed by miR-22 and Empty is the luciferase vector with no UTR. The Y -axis shows relative luciferase units from miR-22-transfected cells normalized to control siRNA transfection. For the mutant 3′UTRs, 3 bp in each 6-mer miR-22 target site in the 3′UTRs were mutated. ( D ) qRT-PCR results for REDD1, TP53INP1, p21 and CARF transcripts show increased expression in quiescent fibroblasts relative to proliferating fibroblasts. Y -axis indicates fold enrichment in quiescent versus proliferating fibroblasts. ( E ) qRT-PCR shows miR-22 suppresses TP53INP1, REDD1 and p21 transcript levels in quiescent fibroblasts. Fold changes are denoted on the Y -axis relative to the control siRNA transfection. ( F ) REDD1, TP53INP1, p21 and CARF protein expression in fibroblasts was downregulated by transfection with miR-22 compared with control siRNA transfection. For B, D and E, GAPDH was used as a control for normalizing input RNA levels. For B, C, D and E, bars indicate the mean, and error bars denote ±SD, n = 3. P -values were estimated by Student’s t -test. * P < 0.05.

Article Snippet: Membranes were blocked with 5% milk in TBST and probed with corresponding primary antibodies against specific proteins [high mobility group box-1 (HMGB1): Cell Signaling Technology, IRF5: Abcam ab33478, REDD1: Abcam ab106356, TP53INP1: Abcam ab9755, p21: Abcam ab7960, CARF: ab88322, MXD4: Santa Cruz Biotechnology sc-771, MYC: Santa Cruz Biotechnology sc-764X].

Techniques: Immunoprecipitation, Quantitative RT-PCR, Transfection, Isolation, Control, Luciferase, Plasmid Preparation, Mutagenesis, Expressing

Journal: Human Molecular Genetics

Article Title: Cis-acting single nucleotide polymorphisms alter MicroRNA-mediated regulation of human brain-expressed transcripts

doi: 10.1093/hmg/ddw317

Figure Lengend Snippet: SNPs in the 3’UTR MRE modify miRNA-mediated regulation of target genes. (A) Flowchart describing steps utilized to prioritize candidate miRNA-MRE interactions for further study. Of the 24 candidates selected, 11 SNP-resident MREs are in the 3’UTR and 13 SNP-resident MREs are in the CDS of target genes. (B) Schematic describing how a SNP in a MRE could enhance or disrupt miRNA-mediated target gene regulation. (C–H) HEK293 cells were transfected with constructs containing the reference SNP (Ref), the alternative ‘candidate’ SNP (Alt), or a scrambled MRE (Scr) for the following genes ( C ) RAPGEF1, ( D ) SOD1, ( E ) CARF, ( F ) TUBB4A, ( G ) FBXO45 and ( H ) RPRD1A and analysed for RNA and protein expression. RNA and protein was harvested 48 h post-transfection. Left panel: RT-qPCR for Renilla (normalized to Firefly mRNA), presented as fold change relative to WT ( n = 9). Right panel: Relative luminescence units (RLU) normalized ( Renilla / Firefly ) and presented as fold change relative to WT ( n = 12). All panels: data presented as box & whiskers plot (min to max) and analysed by one-way ANOVA with Sidak’s multiple comparison test, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies used in this study were procured from the following sources: Flag-M2 (F3165, Sigma); GAPDH (sc32233, SantaCruz); b-tubulin (sc5274, SantaCruz); RAPGEF1 (A301-965A, Bethyl Labs); SOD1 (A303-811A, Bethyl Labs); SREBF1 (ab3259, Abcam); ABCA7 (A304-427A, Bethyl Labs); CARF (A303-861A, Bethyl Labs); PICALM (ab172962, Abcam); His (ab9108, Abcam); Argonaute 2 (ab57113, Abcam).

Techniques: Transfection, Construct, Expressing, Quantitative RT-PCR

Journal: Human Molecular Genetics

Article Title: Cis-acting single nucleotide polymorphisms alter MicroRNA-mediated regulation of human brain-expressed transcripts

doi: 10.1093/hmg/ddw317

Figure Lengend Snippet: SNPs in MREs alter miRNA-mediated regulation in iPSCs. Three iPSC lines representing the following genotypes: altSNP -/- (homozygous for reference allele), altSNP +/+ (homozygous for the alternative candidate SNP), and altSNP +/- (alternative SNP/reference allele) were selected for each gene. iPSC lines were left untreated (Ut) or transfected with the non-targeting (Nt) control or respective miRNA reagents: (A) RAPGEF1: anti-miR-124-3p; (B) SOD1: anti-miR-139-5p; (C) CARF: mimic of miR-181-5p; (D) SREBF1: anti-miR-29b-3p; (E) ABCA7: anti-miR-34a-5p; (F) PICALM: anti-miR-34a-5p + anti-miR-103a-3p. RNA and protein was harvested 48 h post-transfection. Top panel: RT-qPCR for the respective genes (normalized to HPRT), presented as fold change relative to WT ( n = 4). Bottom panel: Densitometry of immunoblots performed for each gene (shown in ) presented as fold change relative to Ut ( n = 3). iPSC lines used (reference ): RAPGEF1 ( , , , , , ); CARF ( , , , , , ); SOD1 ( , , , , , ); SREBF1 ( , , , , , ); ABCA7 ( , , , , , ); PICALM ( , , , , , ). Data are presented as box & whiskers plot (min to max) and analysed by one-way ANOVA with Sidak’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies used in this study were procured from the following sources: Flag-M2 (F3165, Sigma); GAPDH (sc32233, SantaCruz); b-tubulin (sc5274, SantaCruz); RAPGEF1 (A301-965A, Bethyl Labs); SOD1 (A303-811A, Bethyl Labs); SREBF1 (ab3259, Abcam); ABCA7 (A304-427A, Bethyl Labs); CARF (A303-861A, Bethyl Labs); PICALM (ab172962, Abcam); His (ab9108, Abcam); Argonaute 2 (ab57113, Abcam).

Techniques: Transfection, Quantitative RT-PCR, Western Blot

Journal: Human Molecular Genetics

Article Title: Cis-acting single nucleotide polymorphisms alter MicroRNA-mediated regulation of human brain-expressed transcripts

doi: 10.1093/hmg/ddw317

Figure Lengend Snippet: SNPs in the MRE alter miRNA binding in iPSCs. Three altSNP +/- iPSC lines for each of the 6 genes were left untreated (Ut) or transfected with the non-targeting (Nt) control or respective miRNA reagents (RAPGEF1:anti-miR-124-3p, SOD1:anti-miR-139-5p, CARF: mimic of miR-181-5p, SREBF1:anti-miR-29b-3p, ABCA7:anti-miR-34a-5p, PICALM: anti-miR-34a-5p + anti-miR-103a-3p). Ago2 immunoprecipitation (anti-Ago2 Ab) and associated RNA was harvested 24 h later. RT-qPCR using allele-specific primers (Ref vs Alt) for respective genes (normalized to HPRT and total gene expression Ref + Alt) was performed. iPSC lines used (reference Figure S6 ): RAPGEF1 (2,13,14); CARF ( , , ); SOD1 (4,10,12); SREBF1 (4,5,6); ABCA7 (1,7,10); PICALM ( , , ). Data are presented as the ratio of SNP/WT ( n = 12). Data are presented as box & whiskers plot (min to max) and analysed by one-way ANOVA with Sidak’s multiple comparison test, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies used in this study were procured from the following sources: Flag-M2 (F3165, Sigma); GAPDH (sc32233, SantaCruz); b-tubulin (sc5274, SantaCruz); RAPGEF1 (A301-965A, Bethyl Labs); SOD1 (A303-811A, Bethyl Labs); SREBF1 (ab3259, Abcam); ABCA7 (A304-427A, Bethyl Labs); CARF (A303-861A, Bethyl Labs); PICALM (ab172962, Abcam); His (ab9108, Abcam); Argonaute 2 (ab57113, Abcam).

Techniques: Binding Assay, Transfection, Immunoprecipitation, Quantitative RT-PCR, Expressing

Journal: PLoS ONE

Article Title: Genome-Wide Identification of Calcium-Response Factor (CaRF) Binding Sites Predicts a Role in Regulation of Neuronal Signaling Pathways

doi: 10.1371/journal.pone.0010870

Figure Lengend Snippet: a) WebLogo ( http://weblogo.berkeley.edu/ ) representation of the 10bp motif discovered by the PRIORITY motif finder in the ChIP peak sequences. The height of each letter represents the enrichment of that base at each position. If all four bases are equally represented, no base is shown at that position. b) Competition EMSA analysis of CaRF binding to the consensus chCaRE motif in the ChIP peak of the Camk2n1 gene (camCaRE). Arrow indicates the CaRF-camCaRE complex, and the right triangles indicate increasing concentrations (50 or 100 fold molar excess) of the unlabeled competitor probes. c) Competition EMSA analysis to examine the relative importance of each base across the 10bp chCaRE motif. Recombinant CaRF was incubated with radiolabeled camCaRE in the absence (-) or presence of a 50 or 100-fold molar excess of unlabeled competitor probes. The right triangle indicates increasing competitor concentrations. Competitor probes were based on the camCaRE sequence ( AAAGCGAGGC ) with the indicated changes at each position (e.g. 1G has a G rather than an A at position 1 of the motif while the rest of the motif is unchanged). Degenerate code: Y = C/T, N = A,C,G, or T, B = C,G, or T, R = A/G, H = A, C, or T, D = A, G, or T. d) Alignment of the cCaRE, mCaRE, chCaRE, and camCaRE sequences. The mCaRE, which fails to bind CaRF, differs from the CaRF binding sequences at 5 positions, which are shown in gray. Degenerate bases are as described above along with S = C/G.

Article Snippet: To characterize CaRF binding to the chCaRE sequences, we first tested the ability of CaRF to bind the specific chCaRE motif ( 5′-AAAGCGAGGC-3′ ) found in a CaRF ChIP peak from the promoter of the Camk2n1 gene (camCaRE, ).

Techniques: Binding Assay, Recombinant, Incubation, Sequencing

Journal: PLoS ONE

Article Title: Genome-Wide Identification of Calcium-Response Factor (CaRF) Binding Sites Predicts a Role in Regulation of Neuronal Signaling Pathways

doi: 10.1371/journal.pone.0010870

Figure Lengend Snippet: a) Primary data from the UCSC genome browser ( http://genome.ucsc.edu ) showing the CaRF ChIP peak overlapping exon 1 of the Carf gene. b) Position of CaRF-binding motifs in the CaRF ChIP peak from the Carf gene. Capital letters denote exon 1. The underlined sequences show the two potential CaRF-binding motifs. The more 3′ motif in intron 1 was identified by the PRIORITY motif finder. c) Competition EMSA analysis demonstrates that CaRF can bind both motifs in the Carf ChIP peak. Recombinant CaRF was bound to a radiolabeled cCaRE probe in the absence (-) or presence of a 100-fold molar excess of competitor probes. Arrow indicates the CaRF-cCaRE complex. Unlabeled probes used as competitors are listed across the top. d) Expression of Carf mRNA in a Carf exon 8 KO mouse. Cortical neurons from individual P0 WT or CaRF exon 8 deleted (KO) mice were cultured for 5 days, treated with 1µM TTX overnight, then RNA was harvested for cDNA synthesis and quantitative PCR. Carf mRNA was detected with primers against exons 11–12 distal to the deleted region in Carf . Carf mRNA expression was normalized for expression of Gapdh in the same sample to control for sample handling. e) Chromatin immunoprecipitation for RNA polymerase II on the Carf promoter. Carf promoter DNA co-precipitated with an anti-RNA polymerase II antibody or control IgG was quantitated by Q-PCR, and normalized as a percent of signal in the input DNA. Bars show the mean and error bars show SEM. * p <0.05.

Article Snippet: To characterize CaRF binding to the chCaRE sequences, we first tested the ability of CaRF to bind the specific chCaRE motif ( 5′-AAAGCGAGGC-3′ ) found in a CaRF ChIP peak from the promoter of the Camk2n1 gene (camCaRE, ).

Techniques: Binding Assay, Recombinant, Expressing, Cell Culture, cDNA Synthesis, Real-time Polymerase Chain Reaction, Control, Chromatin Immunoprecipitation