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• Research Paper Volume 11, Issue 5 pp 1356-1388

  Alternative polyadenylation dependent function of splicing factor SRSF3 contributes to cellular senescence

  Relevance score: 10.114929

  Ting Shen, Huan Li, Yifang Song, Li Li, Jinzhong Lin, Gang Wei, Ting Ni

  Keywords: senescence, alternative polyadenylation, SRSF3, PTEN, 3′UTR

  Published in Aging on March 4, 2019

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  Down-regulated splicing factor SRSF3 is known to promote cellular senescence, an important biological process in preventing cancer and contributing to individual aging, via its alternative splicing dependent function in human cells. Here we discovered alternative polyadenylation (APA) dependent function of SRSF3 as a novel mechanism explaining SRSF3 downregulation induced cellular senescence. Knockdown of SRSF3 resulted in preference usage of proximal poly(A) sites and thus global shortening of 3′ untranslated regions (3′ UTRs) of mRNAs. SRSF3-depletion also induced senescence-related phenotypes in both human and mouse cells. These 3′ UTR shortened genes were enriched in senescence-associated pathways. Shortened 3′ UTRs tended to produce more proteins than the longer ones. Simulating the effects of 3′ UTR shortening by overexpression of three candidate genes (PTEN, PIAS1 and DNMT3A) all led to senescence-associated phenotypes. Mechanistically, SRSF3 has higher binding density near proximal poly(A) site than distal one in 3′ UTR shortened genes. Further, upregulation of PTEN by either ectopic overexpression or SRSF3-knockdown induction both led to reduced phosphorylation of AKT and ultimately senescence-associated phenotypes. We revealed for the first time that reduced SRSF3 expression could promote cellular senescence through its APA-dependent function, largely extending our mechanistic understanding in splicing factor regulated cellular senescence.

  

  SRSF3 downregulation leads to global shortening of 3′ UTR in human cells. (A) Western blot confirmed lentivirus-mediated RNA interference in both human 293T and HUVEC cells. GAPDH served as internal loading control. (B) Genomic distribution of pA sites identified in 293T cells based on PA-seq method. (C) Box plot of log₂-transformed eUTR based on PA-seq in control and SRSF3-KD 293T cells. The P value of t-test is shown. (D) Histogram of gene numbers with 3′ UTR shortening or lengthening upon SRSF3 KD at different cutoffs and overlapped genes with shortened 3′ UTR (with the cutoff of |ΔeUTR| > 50) between two biological replicates in 293T cells. |ΔeUTR| > 50, 100, 200 and 400 represent the absolute difference of eUTR between SRSF3-KD and control 293T cells, respectively. Number of shared genes (labelled Shared) between two biological replicates were also shown. (E) Box plot of RUD in 293T and HUVEC cells upon knockdown of SRSF3. (F) Histogram of gene numbers with 3′ UTR shortening or lengthening upon SRSF3 KD at different ΔRUD cutoffs. |ΔRUD| > 0.05, 0.1, 0.2 and 0.3 each represents a threshold of absolute difference of RUD between SRSF3-KD and control human cells. (G) Venn diagram of genes with shortened 3′ UTR based on different methods (eUTR and RUD), different shRNAs (sh1 and sh2) and different cells (293T and HUVEV) (ΔRUD ≤ -0.05) upon knockdown of SRSF3. (H) RNA-seq tracks of four representative genes in two human cell types upon SRSF3 KD. The transcription direction is shown at the bottom. The vertical red and blue arrows represent the proximal and distal pA sites, respectively. Y axis denotes the normalized read coverage. (I, J) qRT-PCR validation of the usage of longer 3′ UTR in the total expression (L/T) in both control and SRSF3-KD 293T cells of two biological replicates (rep1 in I and rep2 in J). Rep1 and rep2 represent two biological replicates, and sh1 and sh2 denote two different shRNAs. ** and *** mean P value less than 0.01 and 0.001 (t-test), respectively.

  
  
  

  

  SRSF3 favors binding proximal pA sites and modulates APA at transcriptional level. (A-B) Box plots of public available human and mouse CLIP-seq data of SRSF3 in 3′ UTR shortened genes. Y axis represents the normalized tag intensity (reflected by RPM, reads per million) within 200 nt (A) or 100 nt (B) around the proximal and distal pA sites. Rep1 and rep2 means two biological replicates of HUVEC cells. (C) A combinated UCSC genome view near the 3′ UTR of mouse Pten containing PA-seq track, TargetScan predicted microRNA binding sties (TS miRNA sites), Mammal conservation score, SRSF3 iCLIP track (three replicates), RNA-seq track of control (Ctrl) and SRSF3-KD cells . cUTR and aUTR represent the common and alternative 3′ UTR of Pten, respectively. (D) Semi-quantitative RIP-PCR using cUTR or aUTR specific primer for PTEN on immunoprecipitated products separately harvested by anti-flag (SRSF3_flag) and IgG (Ctrl). IgG served as background binding (Ctrl). Left panel denotes the gel image while right panel represents the relative gray value quantified by imageJ software. (E) qRT-PCR quantifying the nascent RNA isolated by using the Click-iT kit in SRSF3-KD (sh1 and sh2) and control (Ctrl) 293T cells. PTEN_T, PTEN_L and PTEN_L/T represent total expression (short plus long 3′ UTR), expression of long 3′ UTR and relative expression ratio of long 3′ UTR compared to total expression, respectively. *, ** and *** mean P value less than 0.05, 0.01 and 0.001 (t-test), respectively.

  
  
  

  

  GO and KEGG enrichment analyses for 3′ UTR shortened genes shared by MEFs, 293T and HUVEC cells when knocking down SRSF3. (A) Venn diagram of 3′ UTR shortened genes (RUD-based) upon SRSF3-KD in different cells. Both the numbers and percentages were indicated. (B-C) GO term (B) and KEGG pathway (C) enrichment analysis for genes with 3′ UTR shortening shared by SRSF3-KD MEFs, 293T and HUVEC cells (221 genes in panel A). Red fonts represent functional categories related to senescence or aging.

  
  
  

  

  SRSF3 downregulation leads to senescence-associated phenotypes in both human and mouse cells. (A) SA-β-Gal staining before and after knockdown with two shRNAs (sh1 and sh2). (B) CCK-8 assay for cells with or without knockdown of SRSF3 in human and mouse cells. (C) Cell cycle analysis before and after knockdown of SRSF3 in 293T and MEF cells. (D) qRT-PCR revealed that knockdown of SRSF3 led to decreased expression of cell proliferation markers (MKI67, CDK1) and increased expression of senescence marker (CDKN1A or CDKN1B) in both human (293T and HUVEC) and mouse (MEFs and NIH3T3) cells. *, ** and *** mean P value less than 0.05, 0.01 and 0.001 (t-test), respectively.

  
  
  

  

  SRSF3-KD induced 3′ UTR shortening of PTEN produces more protein and contributes to senescence-associated phenotypes. (A) Dual luciferase assay for shorter (S) and longer (L) 3′ UTRs of PTEN in 293T (left) and HUVEC (right) cells. (B) RNA degradation curve of actD-inhibited transcription cells detected by qRT-PCR using primers specific for longer 3′ UTR (L) or targeting common region shared by longer and shorter 3′ UTR (T) of PTEN. (C, G) qRT-PCR indicated overexpression of PTEN (PTEN_OE and Pten_OE) led to decreased expression of cell proliferation marker MKI67 in 293T (C) and MEF (G) cells. (D, H) SA-β-Gal staining for 293T (D) and MEF (H) cells when overexpressing PTEN. (E, F, I, J) CCK-8 assay (E, I) and cell cycle analysis (F, J) for cells with or without overexpression (OE) of PTEN in 293T (E, F) and MEF (I, J) cells. *** and * mean P value less than 0.001 and 0.05, respectively (t-test).

  
  
  

  

  A working model for SRSF3-mediated 3′ UTR shortening contributes to cellular senescence. SRSF3 prefers proximal pA sites binding and represses nearby pA sites usage in target genes such as PTEN, PIAS1 and DNMT3A in normal conditions. Upon SRSF3 knockdown, the repression effect reduced, which in turn leads to the higher usage of corresponding pA sites and 3′ UTR shortening of target genes. Transcripts with shortened 3′ UTR generate more protein, possibly by escaping the miRNA targeting, and finally lead to senescence-associated phenotypes.