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Decreased SRSF7 mediates MDM2 variant generation in cellular senescence.
Jiwon Hong 1,2,3 , Seongki Min 1,2,3 , Gyesoon Yoon 1,2,3 and Su Bin Lim 1,2,3
1 Department of Biochemistry, Ajou University School of Medicine, Suwon, 16499, Korea
2 Inflamm-aging Translational Research Center, Ajou University Medical Center, Suwon, 16499, Korea
3 Department of Biomedical Sciences, Ajou University School of Medicine, Suwon, 16499, Korea
E-mail: sblim@ajou.ac.kr
ABSTRACT INTRODUCTION
Cellular senescence is a state of irreversible cellular growth arrest, which contributes to organismal • Growing evidence documents that more than 50% of all age-associated alterations in
aging and aging-associated diseases. Several molecular causes are defined and the change in alternative splicing are due to changes in the expression of splicing factors.
splicing pattern has been recently reported as an emerging contributor to cellular senescence. In • SR proteins enhance or repress the production of specific splice variants implicated in
our previous study, deregulation of several spliceosomal genes was involved in replicative age-related phenotype and contribute to senescence.
senescence and splicing variants of MDM2 were formed during senescence. However, it is still • It has been recently reported that juvenile-expressed splicing factor SRSF7 mediates
unclear how spliceosomal genes are individually involved in cellular senescence. Among those age-dependent alternative splicing.
genes, we focused on serine and arginine rich splicing factor 7 (SRSF7) and investigated whether
SRSF7 played a key role in the alternative splicing of MDM2. During cellular senescence, mRNA • In the previous study, we confirmed SRSF7 induced cellular senescence and MDM2
variants increased during replicative senescence.
and protein level of SRSF7 clearly decreased. Interestingly, when SRSF7 was suppressed, cellular
senescence was induced and several MDM2 variants were newly generated. We found a potential • MDM2 is well known as a negative regulator of p53. Stabilized p53 protein induces
variant through RNA-seq analysis and confirmed the formation of it in the absence of SRSF7. The cellular senescence. Dysfunction of MDM2 can lead to stabilized p53 and subsequently
lead to cellular senescence.
variant lacks p53 binding domain, suggesting that it is unable to degrade p53. These results
indicate that SRSF7 plays an important role in the alternative splicing of MDM2 and its depletion Is SRSF7 involved in MDM2 gene splicing?
triggers p53-mediated cellular senescence, implying its proper regulation is essential to prevent the If so, how can SRSF7 modulate it and induce cellular senescence?
entry into cellular senescence.
Fig 2. SRSF7 depletion-mediated senescence is linked to expression
Fig 1. SRSF7 depletion induces cellular senescence of MDM2 variants
(A-B) Expression of SRSF7 and senescence marker p21 in replicative senescence model.
(A) Western blot analysis.
(B) mRNA level of SRSF7 using qRT-PCR. (**, p<0.01 vs DT2 by student t-test.)
(C-E) Expression of SRSF7 and senescence markers (p21 and SA-β-gal activity) in oxidative stress induced senescence model.
(C) Western blot analysis.
(D) mRNA level of SRSF7 using qRT-PCR. (**, p<0.01 vs Control by student t-test.) (A-D) Expression of MDM2 variants and p53 in two senescence models (RS and OSIS).
(E) Senescence-associated β galactosidase (SA-β-gal) assay quantification. (**, p<0.01 vs Control by student t-test.) (A) mRNA level of MDM2 using qRT-PCR in RS. (**, p<0.01 vs DT2 by student t-test.)
(F-G) HDFs (DT2) were transfected with siRNA against SRSF7. Expression of SRSF7 and senescence markers. (B) Western blot analysis of RS.
(F) Western blot analysis. (C) mRNA level of MDM2 using qRT-PCR in OSIS. (**, p<0.01 vs Control by student t-test.)
(G) mRNA level of SRSF7 and CDKN1A using qRT-PCR. (**, p<0.01 vs Control by student t-test.) (D) Western blot analysis of OSIS.
(E-F) HDFs (DT2) were transfected with siRNA against SRSF7 for 3 days.
Fig 3. SRSF7 depletion modulates MDM2 expression via gene splicing (E) mRNA level of MDM2 using qRT-PCR. (**, p<0.01 vs Negative control by student t-test.)
(F) Western blot analysis.
Fig 4. SRSF7 depletion generates specifically MDM2-C variant
(A-D) HDFs (DT2) were transfected with siRNA against either U2AF1 or SRSF7 for 3 days.
(A) Heatmap showing the expression level of U2AF1 and SRSF7.
(B) Principal component analysis.
(C) Sashimi plot representing potential MDM2 splicing variants.
(D) Heatmap showing the expression level of MDM2 transcripts and schematic of corresponding protein structure.
Conclusion
• SRSF7 expression decreased in replicative senescence and
oxidative stress-induced senescence models. Also, knockdown (A) Diagram of exon compositions of MDM2 full-length (FL) and its splice variant MDM2-C with primer positions and exon numbers
of SRSF7 induced cellular senescence. according to the updated MDM2 gene information (NM_002392.6).
(B) RT-PCR for MDM2 splicing variants in RS.
• In RS and OSIS models, MDM2 variants increased in senescent (C) RT-PCR for MDM2 splicing variants in SRSF7 knockdown samples (left) and schematic of identified variants through TA cloning (right).
(D-F) HDFs (DT2) were transfected with siRNA against various splicing factors for 3 days.
cells. Also, when SRSF7 expression was suppressed by siRNAs, (D) mRNA level of each splicing factor. (**, p<0.01 vs NC by student t-test.)
(E) RT-PCR for MDM2-C variant in each splicing factor knockdown samples.
MDM2 variants were generated, including MDM2-C isoform. (F) mRNA level of SRSF7 in each splicing factor knockdown samples. (**, p<0.01 vs NC by student t-test.)
• MDM2-C isoform lacks exon 4-8, which means that it lacks NLS, Reference
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✓ Hypothesis: SRSF7 plays an important role in the alternative splicing of • iScience. 2020; 23(3):100929.
MDM2 and its depletion triggers p53-mediated cellular senescence • FASEB J. 2021; 35:e21204.
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