Title:
Alternative polyadenylation dependent function of splicing factor SRSF3 contributes to cellular senescence
剪接因子SRSF3的选择性多聚腺苷酸化依赖性功能有助于细胞衰老
Abstract:
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.
下调的剪接因子SRSF3通过其在人类细胞中的 选择性剪接依赖性 功能促进细胞衰老，这是预防癌症和促进个体衰老的重要生物学过程。在这里，我们发现 SRSF3的选择性多聚腺苷酸化（APA）依赖性功能 可作为解释下调表达的SRSF3诱导细胞衰老的新机制。敲除SRSF3导致优先使用近端poly（A）位点，进而导致全局mRNA的3’非翻译区（3’UTR）的缩短。 SRSF3的敲除还会在人和小鼠细胞中 诱导衰老相关的表型 。这些3’UTR缩短了的基因主要富集在衰老相关途径中。缩短的3’UTR往往比较长的UTR产生更多的蛋白质。我们通过过表达三种候选基因（PTEN，PIAS1和DNMT3A）来模拟3’UTR缩短的影响，发现其都会导致衰老相关的表型。从机制上讲，SRSF3在近端poly（A）位点附近的结合密度要高于3’UTR缩短基因中的远端位点。此外，通过异位过表达或敲除SRSF3来使得PTEN的表达上调，均会导致AKT的磷酸化降低并最终导致衰老相关的表型。我们首次发现表达减少的SRSF3可通过其APA依赖性功能来促进细胞衰老，这一发现在很大程度上拓宽了我们对剪接因子调节细胞衰老的机制理解。
Introduction:
Alternative splicing plays an important role in cellular senescence and aging [1-5]. Core splicing machinery and related splicing factors undergo dramatic changes during aging [6], accompanied with global splicing changes of downstream target genes [7-9]. Heterogeneous nuclear ribonucleoproteins (hnRNPs) and serine/arginine-rich (SR) splicing factors (SRSFs) are two groups of factors regulating alternative splicing and play important roles in numerous biological processes including aging [5, 10-12]. Known examples include that the expression changes of multiple such factors (Hnrnpa1, Hnrnpa2b1, Sf3b1, Srsf3, etc.) are associated with mice lifespan and some (HNRNPA1 and HNRNPA2B1) are even related to parental longevity in humans [13]. Notably, decreased expression of splicing factor SRSF3 (also known as SRp20) is found in multiple cellular senescence models, and depletion of SRSF3 intriguingly induces cellular senescence via its influence on the choice of TP53 splicing isoforms in human fibroblast cells [5]. Reversely, elevated SRSF3 expression level is universal in many cancers, which can promote cell growth and maintain the transformation properties of cancer cells [14]. It has also been reported that higher expression of SRSF3 and the consequent splicing dysfunction is associated with neurodegenerative diseases and cancers [15-17]. These findings suggest that splicing-dependent function of SRSF3 plays an important role in senescence and related biological processes.
选择性剪接在细胞衰老和人体衰老中起着重要的作用。核心剪接机制和相关的剪接因子会在衰老期间发生显着变化，进而伴随着下游靶基因的全局剪接变化。异质核核糖核蛋白（hnRNPs）和 富含丝氨酸/精氨酸（SR）的剪接因子（SRSFs）是调节选择性剪接的两组因子，它们在包括衰老在内的多种生物过程中发挥着重要作用。已知的实例包括：多种此类因子（Hnrnpa1，Hnrnpa2b1，Sf3b1，Srsf3等）的表达变化与小鼠寿命相关，并且一些因子（HNRNPA1和HNRNPA2B1）甚至与人类的亲本寿命相关。值得注意的是，在多个细胞衰老模型中发现剪接因子 SRSF3（也称为SRp20）的表达降低，并且SRSF3的消耗通过其对人成纤维细胞中TP53剪接同种型的选择的影响而有趣地诱导细胞衰老。相反，SRSF3的表达水平升高在许多癌症中是普遍存在的 ，这可以促进细胞生长并维持癌细胞的转化特性。据报道，SRSF3的高表达和随之而来的剪接功能障碍与神经退行性疾病和癌症有关。这些发现表明SRSF3的剪接依赖性功能在衰老和相关的生物学过程中起重要作用。
However, increasing evidence has come to highlight the biomedical importance of revealing splicing-independent function of splicing factors in fully understanding their regulation mechanism [18, 19]. As an example, splicing factor RBFox2 directly interacts with Polycomb complex 2 (PRC2) to regulate genome-wide transcription in mammals [19]. In addition, RBFox2 binding to 3′ UTR of Jph2 gene can antagonise miR34a-mediated gene suppression and plays a role in heart failure [20]. Splicing factor SRSF3 can directly bind to transcripts of histone H2a gene to facilitate their nucleus-to-cytoplasm transport [21]. Interestingly, SRSF3 can modulate the translation efficiency of a viral RNA through interacting with an RNA-binding protein PCBP2 [22]. Noteworthy, SRSF3 can also regulate the alternative poly(A) (pA) site recognition in calcitonin coding gene CALCA by affecting CSTF2 binding [23]. These above findings on splicing-independent function of SRSF3 inspire us to hypothesize that alternative polyadenylation (APA) dependent function of SRSF3 could also play a role in regulating cellular senescence. APA is a phenomenon that one gene contains multiple polyadenylation (pA) sites to produce transcript isoforms differ either at the lengths of 3′ untranslated regions (UTR-APA) or C-terminal domains (CR-APA) [24, 25]. UTR-APA is more prevalent than CR-APA at genome-wide level [25], which could lead to distinct difference in RNA stability, translation efficiency, localization of RNA and protein among isoforms with different lengths of 3′ UTR [26, 27]. The dynamic APA changes have been reported to occur in multiple physiological or pathological processes [28-32]. Global 3′ UTR shortening due to the favorite usage of the proximal pA site took place in cell proliferation and tumorigenesis, and genome-wide lengthening of 3′ UTRs occurs during development and differentiation [33]. It has been discovered that APA regulation is widespread in eukaryotes, and there are more than 70% genes in human genome undergoing APA [25, 34], further supporting the prevalence and importance of APA. As for the regulation mechanisms, the cis-acting elements and 3′ end processing factors can both affect pA site selection [24, 33, 35-37]. For example, CSTF2 is a well-known factor that participates in mRNA 3′ end processing, and its cellular concentration can affect pA site usage [38, 39]. Knockdown of CSTF2 plus its paralog CSTF2t can promote genes to preferentially use the distal pA site [40, 41]. Besides, CFIm25 and CFIm68 were another two 3′ end processing factors that have been reported to be involved in pA site selection. Favorite usage of the proximal pA site was observed when CFIm25 or CFIm68 was down-regulated [42-45]. Polyadenylation can also be coupled with splicing [46], recent studies demonstrated that multiple splicing factors (such as U1 snRNP [47, 48], HnRNP H/H’ [49] and NOVA2 [50]) could regulate APA. Additionally, factors of other aspects, such as transcription [51], chromatin state [52] and other RNA binding proteins [53-55], can also be involved in the modulation of APA.
然而，越来越多的证据强调，揭示剪接因子的剪接独立功能对于完全理解剪接因子的调控机制，具有很强的生物医学重要性。例如，剪接因子RBFox2直接与Polycomb复合物2（PRC2）相互作用，以调节哺乳动物的全基因组转录。此外，RBFox2与Jph2基因的3’UTR结合可拮抗miR34a介导的基因抑制，并在心力衰竭中发挥作用。剪接因子SRSF3可以直接结合于组蛋白H2a基因的转录本，以促进其细胞核到细胞质的转运。有趣的是，SRSF3可以通过与RNA结合蛋白PCBP2相互作用来调节病毒RNA的翻译效率。值得注意的是，SRSF3还可以通过影响CSTF2的结合来调节降钙素编码基因CALCA中的选择性poly（A）（pA）的位点识别。以上关于SRSF3的剪接独立功能的发现激发了我们去假设SRSF3的选择性多聚腺苷酸化（APA）依赖性功能也可以在调节细胞衰老中起作用。 APA是一种基因含有多个多腺苷酸化（pA）位点以产生转录物同种型的现象，其在3’非翻译区（UTR-APA）或C-末端结构域（CR-APA）的长度上不同。 从全基因组水平上来看，UTR-APA比CR-APA更普遍，这可能导致具有不同长度的3’UTR的同种型在RNA稳定性，翻译效率，RNA和蛋白质中的明显差异。据报道，动态APA变化发生在多种生理或病理过程中。全局3’UTR缩短最偏好使用近端pA位点，其主要发生在细胞增殖和肿瘤发生中；而全基因组3’UTR的延长主要发生在发育和分化期间。已经发现APA调控在真核生物中广泛存在，并且在人类基因组中有超过70％的基因正在接受APA，这进一步支持了APA的普遍性和重要性。至于调节机制，顺式作用元件和3’末端加工因子都可以影响pA位点选择。例如，CSTF2是参与mRNA 3’末端加工的一个著名的因子，其细胞浓度可影响pA位点的使用。敲除CSTF2及其旁系同源物CSTF2t可以促进基因优先使用远端pA位点。此外，据报道，CFIm25和CFIm68是参与pA位点选择的另外两个3’末端加工因子。当CFIm25或CFIm68下调时，可以观察到最偏好使用近端pA位点。多腺苷酸化还可以与剪接相结合，最近的研究表明，多个剪接因子（如U1 snRNP，HnRNP H / H和NOVA2）也可以调节APA。此外，其他方面的因素，如转录，染色质状态和其他RNA结合蛋白，也可能参与APA的调节。
To examine whether down-regulation of splicing factor SRSF3 promotes cellular senescence via its APA-dependent mechanism, we performed transcriptome-wide APA profiling on SRSF3-knockdown (SRSF3-KD) and control cells by PA-seq [56] (a 3′ end specific enrichment RNA-seq method) and strand-specific RNA-seq methods [57]. Interestingly, we observed SRSF3-KD induced global shortening of 3′ UTRs in both human and mouse cells. SRSF3 has higher binding density near proximal pA sites than distal ones in 3′ UTR shortened genes. These 3′ UTR-shortened genes were enriched in senescence-associated pathways, and shortened 3′ UTRs tended to produce more corresponding proteins. We further found that mimicking the effect of 3′ UTR shortening by overexpression of three candidate genes promoted senescence-associated phenotypes. These results combined to support the model that APA-dependent function of SRSF3 depletion can lead cellular senescence.
为了检验剪接因子SRSF3的下调是否通过其APA依赖性机制促进细胞衰老，我们通过PA-seq和链特异性RNA-seq的方法对敲除了SRSF3的细胞（SRSF3-KD）和对照细胞进行转录组范围的APA分析。 有趣的是，我们观察到SRSF3-KD可以诱导人和小鼠细胞中3’UTR的全局缩短。 SRSF3在近端pA位点附近的结合密度高于3’UTR缩短基因中的远端结合密度。 这些3’UTR缩短的基因富集在衰老相关途径，缩短的3’UTR倾向于产生更多相应的蛋白质。 我们进一步发现，通过过表达三个候选基因来模拟3’UTR缩短的作用促进了衰老相关的表型。 这些结果支撑了SRSF3敲低后产生的APA依赖性功能可导致细胞衰老的模型。
RESULTS
Down-regulation of SRSF3 leads to global shortening of 3′ UTR in human and mouse cells
SRSF3的下调导致人和小鼠细胞中3’UTR的全球缩短
To examine whether SRSF3-KD induces downstream changes other than alternative splicing, we firstly applied our published PA-seq protocol, which specifically enriched 3′ ends of mRNA by reverse transcription with modified oligo(dT) primer to capture the polyA tail and precisely identify polyadenylation site at the genome scale [56], to detect global APA changes in human 293T cells. Two biological replicates of lentivirus-mediated short hairpin RNA (shRNA) interference were performed and down-regulation of SRSF3 protein was confirmed by western blot (Fig. 1A, Fig. S1). The reliability of the identified pA sites was analyzed before comparing the dynamic changes of APA between SRSF3-KD and control cells. Known pA sites and those located at 3′ UTR regions were the top two categories of the identified pA sites (Fig. 1B), consistent with previous reports [32, 56]. Besides, 85.5% of the identified pA sites were covered by PolyA_DB3 [58] and nucleotides composition near pA sites were in line with previous reports (Fig. S2). Moreover, canonical polyA signals (AAUAAA and AUUAAA) occupy ~75% of identified pA sites (Fig. S2C). These quality control results demonstrated the satisfied quality of the identified pA sites and their reliability for further analyses. The changes of pA site usage upon SRSF3 knockdown were next analyzed. Effective 3′ UTR (eUTR), which considering both location and abundance of pA sites for genes with APA, was used to reflect the weighted length of 3′ UTR for each gene [32, 56]. Interestingly, an overall 3′ UTR shortening pattern evaluated by eUTR was observed in SRSF3-KD human 293T cells with two biological replicates (Fig. 1C), suggesting that SRSF3 downregulation favored the usage of proximal pA sites. We further examined the eUTR changes at individual gene level and found that SRSF3-KD induced more genes to use proximal pA sites in both replicates (Fig. 1D). Notably, a considerable proportion of overlapped genes between two biological replicates using PA-seq method and eUTR calculation further supported the reproducibility of such global trend (Fig. 1D).
为了检查SRSF3-KD是否可以诱导除了选择性剪接之外的下游变化，我们首先应用我们已公布的PA-seq方案，该方案通过用修饰的oligo（dT）引物，以逆转录的方式来特异性富集mRNA的3’末端以捕获polyA尾并精确鉴定全基因组范围内的多腺苷酸化位点，用于检测人的293T细胞的全局APA变化。我们使用了慢病毒介导的短发夹RNA（shRNA）干扰的两个生物学重复，并通过蛋白质印迹证实SRSF3蛋白的下调（图1A，图S1）。在比较SRSF3-KD与对照细胞之间APA的动态变化之前，我们先分析了鉴定出的pA位点的可靠性。已知的pA位点和位于3’UTR区域的pA位点是已鉴定的pA位点的前两类（图1B），与之前的报道一致。此外，85.5％的已识别的pA位点被PolyA_DB3覆盖，pA位点附近的核苷酸组成与之前的报道一致（图S2）。此外，规范的polyA信号（AAUAAA和AUUAAA）约占已鉴定的pA位点的75％（图S2C）。这些质量控制结果证明了我们所鉴定的pA位点的满意质量及其进一步分析的可靠性。接下来我们分析了SRSF3敲除后pA位点使用的变化。有效的3’UTR（eUTR），其同时考虑到了具有APA的基因的pA位点的位置和丰度，它被用于反映每个基因的3’UTR的加权长度。有趣的是，在具有两个生物学重复的SRSF3-KD人293T细胞中观察到由eUTR评估的全局3’UTR缩短的模式（图1C），这表明SRSF3的下调有利于近端pA位点的使用。我们进一步检查了个体基因水平上的eUTR变化，发现SRSF3-KD诱导了更多的基因在两次重复中使用近端pA位点（图1D）。值得注意的是，我们同时使用PA-seq方法和eUTR计算，在两个生物重复间存在着高比例的的重叠基因，这进一步支持了这种全局趋势的再现性（图1D）。
As an independent validation, we next adopted the method of RUD index [59], which reflected the relative usage of distal pA sites compared to total pA sites, to confirm the APA changes based on a separate RNA-seq data. Consistent with the results based on eUTR method, we detected a global reduction of RUD index upon SRSF3 KD in 293T cells (Fig. 1E), suggesting the favoring of proximal pA sites and shortening of 3′ UTRs. At individual gene level, we also detected more genes using shortened 3′ UTRs than lengthened ones in SRSF3-KD 293T cells (Fig. 1F). To expand this conclusion in more human cells, we applied the same RNA-seq and RUD analysis in Human Umbilical Vein Endothelial Cells (HUVECs), which is widely used as a vascular senescence model [32, 60-62]. In line with the results in human 293T cells, knockdown SRSF3 with two replicates in HUVECs both displayed a similar 3′ UTR shortening trend at both genome-wide (Fig. 1E) and individual gene level (Fig. 1F).
作为一项独立验证，我们接下来采用了RUD指数的方法，该方法反映了远端pA位点与总pA位点相比的相对使用情况，并基于单独的RNA-seq数据来确认APA变化。 与基于eUTR方法的结果一致，我们检测到293T细胞中SRSF3 KD的RUD指数的全局降低（图1E），这显示出对近端pA位点的偏好和3’UTR的缩短。 在个体基因水平上，相比于延长的3’UTR，我们在SRSF3-KD 293T细胞中使用缩短的3’UTR可以检测到更多的基因（图1F）。 为了在更多的人类细胞中扩展这一结论，我们在人脐静脉内皮细胞（HUVECs）中应用了相同的RNA-seq和RUD分析，其广泛用作血管衰老模型。 与人293T细胞中的结果一致，敲除了SRSF3的两个HUVEC重复在基因组范围内（图1E）和个体基因水平（图1F）都显示出类似的3’UTR缩短趋势。
To gain a comprehensive comparison of genes tending to use shorter 3′ UTRs upon SRSF3-KD based on different methods, biological replicates and types of cells, the interrelation of these gene sets was shown in a venn diagram (Fig. 1G). The majority (1134 genes) of SRSF3-KD induced 3′ UTR shortened genes were shared between two biological replicates of HUVECs (Fig. 1G). A considerable overlap (483 genes) between two different bioinformatical methods (eUTR and RUD) was also detected in 293T cells (Fig. 1G). Importantly, there were 355 genes showed 3′ UTR shortening in both 293T and HUVECs based on different methods and biological replicates (Fig. 1G), which were probably the common targets of SRSF3 in different cell types. SRSF3-KD induced 3′ UTR shortening in four representative genes was visualized in tracks of RNA-seq (Fig. 1H) and PA-seq (Fig. S3). Ten candidate genes were further selected for validation by reverse transcription coupled with quantitative real-time polymerase chain reaction (qRT-PCR), nine of which were confirmed to have reduced usage of distal pA sites (i.e., favor the proximal pA sites) in SRSF3-KD human cells (Fig. 1I-J). These above results indicated that downregulation of SRSF3 caused global 3′ UTR shortening in human cells.
为了全面比较基于不同方法，生物学重复和细胞类型中得出的SRSF3-KD偏好使用较短3’UTR基因的结果，我们使用维恩图显示这些基因组的相互关系（图1G）。 SRSF3-KD诱导的3’UTR缩短基因的大多数（1134个基因）在HUVEC的两个生物学重复之间共享（图1G）。在293T细胞中也检测到两种不同生物信息学方法（eUTR和RUD）之间的相当大的重叠（483个基因）（图1G）。重要的是，基于不同的方法和生物学重复，有355个基因在293T和HUVEC中显示3’UTR缩短（图1G），这些基因可能是不同细胞类型中SRSF3的共同靶标。 4个SRSF3-KD诱导3’UTR缩短的代表性基因，其在RNA-seq（图1H）和PA-seq（图S3）中的轨迹被可视化。我们通过qRT-PCR进一步选择10个候选基因进行验证，其中9个被证实在SRSF3-KD的人类细胞中减少了远端pA位点的使用（即，偏好近端pA位点）（图1I-J）。以上结果表明，SRSF3的下调会导致人类细胞中全局3’UTR的缩短。
To examine whether Srsf3 could play a similar APA regulatory role in mouse cells, we knocked down Srsf3 in mouse embryonic fibroblasts (MEFs) (Fig. S4A) and constructed RNA-seq libraries followed by RUD analysis. Consistent with the trend in human cells, knockdown of Srsf3 in MEFs also led to global shortening of 3′ UTRs (Fig. S4B) and the majority of genes with APA changes favored proximal pA sites (Fig. S4C). Both visualization of RNA-seq results and qRT-PCR validation of selected genes supported the shortening of 3′ UTR in Srsf3-KD mouse cells (Fig. S4D-F). Altogether, our results proved that downregulation of splicing factor SRSF3 resulted in global shortening of 3′ UTR in both human and mouse cells.
为了检查Srsf3是否可以在小鼠细胞中发挥类似的APA调节作用，我们在小鼠胚胎成纤维细胞（MEF）中敲除Srsf3（图S4A）并构建RNA-seq文库，然后进行RUD分析。 与人类细胞的趋势一致，MEF中Srsf3的敲除也导致3’UTR的全局缩短（图S4B），并且大多数具有APA变化的基因偏好近端pA位点（图S4C）。 RNA-seq结果的可视化和所选基因的qRT-PCR验证均支持Srsf3-KD小鼠细胞中3’UTR的缩短（图S4D-F）。 总之，我们的结果证明，剪接因子SRSF3的下调导致人和小鼠细胞中3’UTR的整体缩短。
SRSF3 favors proximal pA sites binding and transcriptionally modulates APA
SRSF3偏好与近端pA位点结合并转录调节APA
We next examined whether SRSF3 directly regulated alternative polyadenylation by integrative analysis of public SRSF3 CLIP-seq (crosslinking-immunopre-cipitation and high-throughput sequencing) data [63, 64] and our PA-seq and RNA-seq data before and after SRSF3 knockdown. As we focused on UTR-APA, CLIP signal located at 3′ UTRs was analyzed. Interestingly, the binding intensity of SRSF3 was significantly higher near proximal pA sites than distal ones for 3′ UTR shortened genes in both human and mouse cells (Fig. 2A). Since about 90% of genes had ≥ 100 nucleotides (nt) distance between proximal pA site and stop codon (Fig. S5), we analyzed CLIP signal within 100 nt around proximal or distal pA sites and observed same results (Fig. 2B). These results suggested that SRSF3 globally favors the proximal pA site binding. To confirm such result at individual gene level, three candidate genes (PTEN, a well-known tumor suppressor and related to longevity; DNMT3A, a known methyltransferase associated with aging and cancer; PIAS1, a repressor of transcription factor STAT1 that related to breast tumorigenesis), which were validated undergoing 3′ UTR shortening in SRSF3-KD cells (Fig. 1I-J, Fig. S4E-F), were visualized with their CLIP-seq and RNA-seq signal in UCSC genome browser. Noteworthy, SRSF3 had higher binding signal around the proximal pA site than the distal one for PTEN in all three biological replicates in mouse cells (see iCLIP of SRSF3 track in Fig. 2C, Fig. S6). SRSF3 iCLIP tracks of PIAS1 and DNMT3A showed similar proximal pA site preference (Fig. S7, S8). Consistent with the shortening of 3′ UTR, RNA-seq tracks of these three candidate genes showed considerable ratio change between alternative 3′ UTR (aUTR) and constitutive 3′UTR (cUTR) in SRSF3-KD samples (Fig. 2C, Fig. S7, S8). Importantly, the 3′ UTRs of PTEN, PIAS1 and DNMT3A are all evolutionarily conserved and ranked at top 14%, 13.6% and 10.6% in all human coding genes, respectively (Fig.S9), implying the importance of such regulatory role of SRSF3 in an evolutionary view.
我们接下来通对过公共SRSF3的CLIP-seq数据、SRSF3敲除前后的PA-seq和RNA-seq数据的综合分析，来检查SRSF3是否可以直接调节选择性多聚腺苷酸化。由于我们关注的是UTR-APA，所以我们分析了位于3’UTR的CLIP信号。有趣的是，对于人和小鼠细胞中3’UTR缩短的基因，SRSF3在近端pA位点附近的结合强度要显着高于远端pA位点（图2A）。由于约90％的基因在近端pA位点和终止密码子之间具有≥100个核苷酸（nt）距离（图S5），我们分析了在近端或远端pA位点周围100nt内的CLIP信号，并观察到相同的结果（图2B）。这些结果表明SRSF3在全基因组范围内有利于近端pA位点结合。为了从单个基因水平来证实这个结果，我们在UCSC基因组浏览器中，对三个可在SRSF3-KD细胞中被证明3′ UTR缩短的候选基因（PTEN; DNMT3A; PIAS1）（图1I-J，图S4E-F）进行了CLIP-seq和RNA-seq信号的可视化。值得注意的是，在小鼠细胞的所有三个生物学重复中，SRSF3在近端pA位点周围的结合信号高于PTEN的远端结合信号（参见图2C中的SRSF3轨迹的iCLIP，图S6）。 PIAS1和DNMT3A的SRSF3 iCLIP轨迹显示出类似的近端pA位点偏好（图S7，S8）。与3’UTR的缩短一致，这三种候选基因的RNA-seq轨迹在SRSF3-KD样品中的选择性3’UTR（aUTR）和组成型3’UTR（cUTR）之间显示出相当大的比例变化（图2C，图2）。 S7，S8）。重要的是，PTEN，PIAS1和DNMT3A的3’UTR均在进化上保守，并且在所有人类编码基因中分别排在前14％，13.6％和10.6％（图9），从进化的角度来看，这暗示出SRSF3调节作用的重要性。
Alternative polyadenylation contributing to different isoforms of PTEN has been reported by other research [65-67], however, SRSF3 directly binding to its 3′ UTR and regulating its APA is novel, we performed further experimental validations in human cell. RNA immunoprecipitation coupled with semi-quantitative PCR (RIP-PCR) showed higher binding signal of SRSF3 near the proximal pA site than the distal one of PTEN (Fig. 2D). These results indicated that SRSF3 regulated APA of PTEN by its binding preference to the proximal pA site. To further explore whether SRSF3 regulated PTEN’s APA at the transcriptional level, we applied analysis on nascent RNA. qRT-PCR showed that SRSF3-KD increased the usage of proximal pA site in nascent poly(A)+ RNA, indicating SRSF3 regulated APA of PTEN at transcriptional level (Fig. 2E). Together, SRSF3 favored proximal pA site binding of PTEN and regulated its APA at transcriptional level.
其他研究已经报道了对PTEN的不同亚型有贡献的选择性多聚腺苷酸化，然而，SRSF3直接与其3’UTR结合并调节其APA这一观点是非常新颖的，我们在人类细胞中进行了进一步的实验验证。 RNA免疫沉淀结合半定量PCR（RIP-PCR）显示，SRSF3在近端pA位点附近的结合信号高于PTEN的远端（图2D）。 这些结果表明，SRSF3通过其对近端pA位点的结合偏好来调节PTEN的APA。 为了进一步探索SRSF3是否在转录水平上调节PTEN的APA，我们对新生RNA进行了分析。 qRT-PCR显示SRSF3-KD增加了新生poly（A）+ RNA中近端pA位点的使用，表明SRSF3在转录水平上调节PTEN的APA（图2E）。 总之，SRSF3有利于PTEN的近端pA位点结合并在转录水平上调节其APA。
SRSF3-KD induced 3′ UTR-shortened genes enrich in senescence-associated pathways
SRSF3-KD诱导3’UTR缩短的基因在衰老相关途径中富集
To understand the functional consequence of SRSF3-KD induced 3′ UTR shortening, functional enrichment analyses were performed on those 3′ UTR shortened genes using gene ontology (GO) and KEGG pathway (Fig. 3A). We first analyzed 3′ UTR-shortened genes shared by two biological replicates of HUVEC, and discovered that four (cell division, protein ubiquitination, cell cycle and Wnt signaling pathway) out of the top ten enriched GO terms were associated with senescence (Fig. S10A) [68, 69]. And seven out of the top ten enriched KEGG pathways (Endocytosis, Protein processing in endoplasmic reticulum, Ubiquitin mediated proteolysis, Insulin signaling pathway, mTOR signaling pathway, AMPK singling pathway and FoxO signaling pathway) were associated with senescence or aging (Fig. S10B) [70-73]. Next, we analyzed SRSF3-KD induced 3′ UTR shortening genes shared by 293T and HUVEC cells, and found that they were enriched in senescence-associated GO terms (cell division, cellular response to DNA damage stimulus, cell cycle and protein ubiquitination) and senescence/aging related pathways (Protein processing in endoplasmic reticulum, Ubiquitin mediated proteolysis, Endocytosis, FoxO signaling pathway and mTOR signaling pathway) (Fig. S11) [68, 70, 71, 74]. 3′ UTR-shortened genes in MEFs were also enriched in senescence/aging related pathways (cell division, Ras signaling pathway, Wnt signaling pathway, Ubiquitin mediated proteolysis, PI3K-Akt signaling pathway and Endocytosis) (Fig. S12). Finally, we analyzed SRSF3-KD induced 3′ UTR shortening genes shared by 293T, HUVEC and MEF cells (221 genes showed in Fig. 3A), and the result showed that they were enriched in senescence-associated GO terms (cell division, cell cycle, insulin receptor signaling pathway and regulation of microtubule cytoskeleton organization) and senescence/aging related pathways (Protein processing in endoplasmic reticulum, FoxO signaling pathway, PI3K-Akt signaling pathway and AMPK signaling pathway) (Fig. 3B,C) [75-77]. These results above indicated that SRSF3-KD induced 3′ UTR-shortened genes possibly had the potential to function in senescence and aging in both human and mouse cells.
为了理解SRSF3-KD诱导的3’UTR缩短的功能性结果，我们使用GO和KEGG途径对这些3’UTR缩短的基因进行功能富集分析（图3A）。我们首先分析了在HUVEC的两个生物重复间有重叠的3个’UTR缩短的基因，并且发现前十个富集的GO术语中的四个与衰老相关（图S10A）。十大富集的KEGG途径中的七个与衰老或衰老相关（图S10B）。接下来，我们分析了在293T和HUVEC细胞中重叠的SRSF3-KD诱导的3’UTR缩短基因，并发现它们富含衰老相关的GO术语和衰老/衰老相关途径（图S11）。 MEF中3’UTR缩短的基因也富集在衰老/衰老相关途径（图S12）。最后，我们分析了在293T，HUVEC和MEF细胞三者重叠的SRSF3-KD诱导的3’UTR缩短基因（图3A中显示221个基因），结果显示它们富含衰老相关的GO术语和衰老/衰老相关通路（图3B，C）。上述结果表明，SRSF3-KD诱导3’UTR缩短的基因可能具有在人和小鼠细胞中衰老和衰老中起作用的潜力。
Decreased SRSF3 causes senescence-related phenotypes in human and mouse cells
降低的SRSF3导致人和小鼠细胞中衰老相关的表型
We next examined whether knockdown of SRSF3 could lead to senescence-associated phenotypes in human and mouse cells. RNA interferences using two shRNAs targeting SRSF3 caused increased senescence-associated β-galactosidase (SA-β-gal) staining [78] in both human (293T and HUVEC) and mouse (MEF and NIH3T3) cells (Fig. 4A). In addition, SRSF3-KD reduced cell growth rate in tested cell lines (Fig. 4B). Further investigation showed that SRSF3-KD resulted in cell cycle arrest in G2/M phase in 293T cells while arrest in G1 phase in MEFs (Fig. 4C). Notably, SRSF3-KD caused a common decrease of S phase percentage in both 293T and MEF cells (Fig. 4C). What’s more, knockdown of SRSF3 in human and mouse cells also led to the decreased expression of MKI67, a molecular marker for cell proliferation (Fig. 4D) [79]. In addition, SRSF3-KD resulted in upregulation of senescence-related marker CDKN1A (encodes p21) and/or CDKN1B (encodes p27) in human and mouse cells (Fig. 4D). These results demonstrated that knockdown of SRSF3 could induce senescence-related phenotypes in both human and mouse cells.
我们接下来检查了SRSF3的敲除是否会导致人和小鼠细胞的衰老相关表型。我们使用靶向SRSF3的两种shRNA的RNA干扰来诱使人类（293T和HUVEC）和小鼠（MEF和NIH3T3）细胞中衰老相关的β-半乳糖苷酶（SA-β-gal）染色增加（图4A）。此外，SRSF3-KD还降低了测试细胞系中的细胞生长速率（图4B）。进一步的研究表明，在293T细胞中的SRSF3-KD会致使细胞周期停滞在G2 / M期，而在MEF中则是停滞在G1期（图4C）。值得注意的是，SRSF3-KD还导致293T和MEF细胞中S期百分比的共同降低（图4C）。更重要的是，SRSF3在人和小鼠细胞中的敲除也导致MKI67的表达降低，MKI67是细胞增殖的分子标记[图4D]。此外，SRSF3-KD导致人和小鼠细胞中衰老相关标志物CDKN1A（编码p21）和/或CDKN1B（编码p27）的上调表达（图4D）。这些结果表明SRSF3的敲低可以在人和小鼠细胞中诱导衰老相关的表型。
SRSF3-KD induced 3′ UTR shortening genes promote senescence-related phenotypes via their increased protein levels
SRSF3-KD诱导的3’UTR缩短基因通过增加的蛋白质水平促进衰老相关的表型
As knockdown of SRSF3 led to global shorting of 3′ UTRs and senescence-associated phenotypes, we hypothesized that 3′ UTR shortening mediated expression change of target genes can be an alternative mechanism in explaining SRSF3-KD induced senescence. Our results already showed that SRSF3 directly regulated APA of candidate genes including PTEN, PIAS1 and DNMT3A (Fig. 2, Fig. S7, S8). These three genes belong to enriched pathways associated with senescence/aging (PTEN belongs to pathways in cancer, PI3K-Akt signaling pathway; PIAS1 and DNMAT3A belong to transcription, DNA-templated, regulation of transcription). We thus chose these genes to test the hypothesis. To examine whether SRSF3-KD induced 3′ UTR shortening affects gene expression, we carried out dual luciferase assay with different lengths of 3′ UTRs. It has been reported that protein abundance of the variable PTEN isoforms resulting from alternative polyadenylation are distinct because of miRNA effects or difference in protein translation efficiency [65, 66, 80, 81]. Our result showed that transcripts with the shorter 3′ UTR of PTEN can markedly produce more protein than those with the longer one in two different human cell types (Fig. 5A, Fig. S13). The result remained true in mouse cells (Fig. S14). In addition, shorter 3′ UTR of PIAS1 also generated more protein than the longer one (Fig. S15). Interestingly, multiple TargetScan (TS) predicted microRNA (miRNA) binding sites existed in the regions between proximal and distal pA sites for both PTEN and PIAS1 (see TS miRNA sites in Fig. 2C, Fig. S7). This result suggested that shortened 3′ UTR could enhance the protein expression through escaping from targeting by miRNAs. Further, there were more predicted miRNA binding sites within the alternative 3′ UTR of DNMT3A (see TS miRNA sites in Fig. S8), however, the dual luciferase assay cannot be performed due to the technical failure of cloning the longer 3′ UTR of DNMT3A. Next, RNA turnover rate analysis showed that transcripts with shorter 3′ UTR of PTEN was more stable than those with the longer one in HUVEC cells, though a less difference of such stability in 293T cells (Fig. 5B). These results suggested that SRSF3-KD induced shortening was likely to increase the protein production of affected genes, possibly contributed by miRNA-mediated stability control, translation efficiency or other mechanisms.
由于SRSF3的敲除会导致3’UTR的的全局缩短和衰老相关表型，我们假设由3’UTR缩短而介导的靶基因的表达变化可以作为一种解释SRSF3-KD诱导的衰老的选择性机制。我们的结果已经表明，SRSF3直接调节包括PTEN，PIAS1和DNMT3A在内的候选基因的APA（图2，图S7，S8）。这三个基因属于与衰老/衰老相关的富集途径，所以我们选择这些基因来检验这一假设。为了检查SRSF3-KD是否诱导3’UTR缩短，进而影响基因表达，我们进行了具有不同长度的3’UTR的双荧光素酶测定。据报道，由于miRNA效应或蛋白质翻译效率的差异，由选择性多聚腺苷酸化产生的选择性PTEN亚型的蛋白质丰度是不同的。我们的结果显示，相比于具有较长3’UTR的PTEN的转录本，具有较短3’UTR的PTEN的转录本可显着产生更多的蛋白质（图5A，图S13）。这一结果在小鼠细胞中仍然存在（图S14）。此外，较短的3’UTR PIAS1也比较长的3’UTR PIAS1产生更多的蛋白质（图S15）。有趣的是，多个TargetScan（TS）预测的microRNA（miRNA）结合位点存在于PTEN和PIAS1的近端和远端pA位点之间的区域中（参见图2C中的TS miRNA位点，图S7）。该结果表明，缩短的3’UTR可以通过逃避miRNA的靶向来增强蛋白质表达。此外，在DNMT3A的选择性3’UTR内有更多预测的miRNA结合位点（参见图S8中的TS miRNA位点），然而，由于克隆较长的3’UTR的技术失败，我们无法对DNMT3A进行双荧光素酶测定。之后的RNA转换率分析显示，相比于HUVEC的3’UTR较长的转录本，PTEN的3’UTR较短的转录本更稳定，尽管这一差异在293T细胞中较小（图5B）。这些结果表明，SRSF3-KD诱导的3’UTR的缩短可能会使得那些受影响基因的蛋白产物的增加，这可能是由miRNA介导的稳定性控制，翻译效率或其他机制所促成的。
We next mimicked the elevated expression of these 3′ UTR-shortened genes by overexpressing candidate genes and examined whether they could contribute to cell senescence. Overexpression of PTEN in human cells led to decreased expression of MKI67, a well-known cell proliferation marker (Fig. 5C). Upregulation of PTEN also caused senescence-related phenotypes in human cells including increased SA-β-gal activity (Fig. 5D), decreased cell growth rate (Fig. 5E) and reduced percentage of S phase cells (Fig. 5F). Additionally, cells transfected with PTEN shorter isoform grew slower than those with the longer one (Fig. S16). Importantly, upregulated Pten promoted alike senescence-associated phenotypes in mouse cells (Fig. 5G-J). Besides, we also proved that overexpression of Pias1 and Dnmt3a resulted in similar senescence-related phenotypes, including increased SA-β-gal activity, reduced cell proliferation rate and changed cell cycle (Fig. S17). All these results supported the notion that 3′ UTR shortening contributed to, at least in part, SRSF3-KD induced senescence.
我们接下来通过过表达候选基因来模拟这些3’UTR缩短基因表达的升高，并检查它们是否可以促进细胞衰老。PTEN在人细胞中的过表达导致MKI67的表达降低，MKI67是众所周知的细胞增殖标记物（图5C）。 PTEN的上调还引起人细胞中衰老相关的表型，包括增加的SA-β-gal活性（图5D），降低的细胞生长速率（图5E）和降低的S期细胞百分比（图5F）。另外，用PTEN较短的亚型转染的细胞比具有较长亚型的细胞生长得缓慢（图S16）。重要的是，上调的Pten在小鼠细胞中促进了相似的衰老相关表型（图5G-J）。此外，我们还证明Pias1和Dnmt3a的过表达均可导致类似的衰老相关表型，包括增加的SA-β-gal活性，降低的细胞增殖率和改变的细胞周期（图S17）。所有这些结果均支持3’UTR缩短至少部分促成SRSF3-KD诱导的衰老的观点。
PTEN is a well-known tumor suppressor and its overexpression extends mice lifespan through reduced PI3K activity and downstream cancer protection mechanisms [82], consistent with our observation that overexpression of PTEN induced cellular senescence (Fig. 5), an important tumor prevention mechanism [83]. Since PTEN can negatively regulate PI3K/AKT pathway through dephosphorylating phosphatidyl-inositol-3,4,5-trisphosphate (PIP3) and thus reduce phosphorylated AKT (p-AKT) abundance [84, 85], we next examined whether PTEN-induced senescence related to altered level of p-AKT. In good consistence with known reports, overexpressing PTEN in 293T and HUVEC cells reduced p-AKT abundance significantly while the AKT level did not exhibit a significant change (Fig. S18A). As knockdown of SRSF3 induced 3′ UTR shortening of PTEN and transcripts with shortened 3′ UTR of PTEN generated more protein than those with the longer one (validated by dual luciferase assay, Fig. 5A), one would expect that SRSF3-KD can increase the protein level of PTEN. Consistently, SRSF3 knockdown with two shRNAs both led to higher PTEN protein abundance in human 293T and HUVEC cells (Fig. S18B). Furthermore, SRSF3 knockdown attenuated the abundance of p-AKT but not that of the total AKT (Fig. S18B), coinciding with the result of PTEN upregulation. Together, SRSF3-KD induced senescence can be partially explained by PTEN upregulation, which at least in part contributed by 3′ UTR shortening.
PTEN是一种众所周知的肿瘤抑制因子，它的过表达会降低PI3K活性和并产生下游癌症保护机制，来延长小鼠寿命，这与我们观察到PTEN过表达会诱导细胞衰老的结果相一致（图5），而这是一种重要的肿瘤预防机制一致。由于PTEN可以通过去PIP3来负调节PI3K / AKT途径，从而减少磷酸化AKT（p-AKT）的丰度，我们接下来检查了是否PTEN诱导的衰老与改变的p-AKT水平有关。与已知的报道一致，在293T和HUVEC细胞中过表达PTEN显着降低了p-AKT的丰度，而AKT的水平没有显示出显着变化（图S18A）。由于SRSF3的敲除可以诱导PTEN的3’UTR缩短，并且PTEN的缩短的的转录本比具有较长的3’UTR的PTEN转录本能产生更多的蛋白质（通过双荧光素酶测定验证，图5A），所以我们预测，SRSF3-KD可以增加PTEN的蛋白质水平。相似地，我们用两种shRNA来敲除SRSF3，均导致了人类293T和HUVEC细胞中更高的PTEN蛋白丰度（图S18B）。此外，SRSF3的敲除还减弱了p-AKT的丰度而非总AKT的丰度（图S18B），这与PTEN上调表达产生的结果一致。总之，SRSF3-KD诱导的衰老可以部分地通过PTEN上调来解释，而部分的PTEN上调是由3’UTR的缩短贡献的。
DISCUSSION
Cellular senescence is a cancer prevention mechanism, and SRSF3 could be one of the regulators given SRSF3 is downregulated in multiple senescence models and upregulated in many cancer types [5]. What’s more, knockdown of SRSF3 induced cellular senescence while increased SRSF3 expression promoted cancer-related cellular phenotypes further highlighted its regulatory importance in both biological systems [5, 14], wherein splicing-dependent function of SRSF3 was mainly focused. However, SRSF3 can also regulate RNA export, RNA stability, alternative polyadenylation and translation [21-23]. Growing evidences have highlighted the biomedical importance of understanding the splicing-independent functions of multiple splicing factors such as RBFox2, SRSF2 and U2AF1 [20, 86]. Here, our results also showed that reduction of SRSF3 expression indeed affected splicing of multiple genes, including TP53 gene whose splicing pattern change was consistent with the previous research (Fig. S19) [5]. However, splicing-independent function of SRSF3 in cellular senescence has not been explored. In this study, we were surprised to find that knockdown of SRSF3 led to over one thousand genes favoring proximal pA site usage, resulting in the global shortening of 3′ UTRs in both human and mouse cells. 3′ UTR shortened genes were enriched in senescence-associated pathways and likely produced more protein, as demonstrated by candidate genes. Mimicking the effect of 3′ UTR shortening by overexpression of three candidate genes all caused senescence-related phenotypes. Specifically, SRSF3 regulated PTEN’s APA at transcriptional level and contributed to senescence. Thus, SRSF3-KD induced senescence can be explained, at least in part, by its APA-dependent function (Fig. 6).
细胞衰老是一种癌症预防机制，SRSF3可能是调控因子之一，这是因为SRSF3在多个衰老模型中被下调并在许多癌症类型中被上调。更重要的是，SRSF3的敲除诱导了细胞衰老，而SRSF3表达的增加则会促进癌症相关的细胞表型，这进一步凸显了其在两种生物系统中的调节的重要性，而其中SRSF3的剪接依赖性功能是最被关注的。此外，SRSF3还可以调节RNA输出，RNA稳定性，选择性多聚腺苷酸化和翻译。越来越多的证据强调了理解RBFox2，SRSF2和U2AF1等多种剪接因子的剪接独立功能在生物医学方面的重要性。在此，我们的结果还表明，SRSF3表达的减少的确影响了包括TP53基因在内的多个基因的剪接，其剪接模式地变化与先前的研究一致（图S19）。但是，关于SRSF3在细胞衰老中的剪接独立功能仍未被探索。在本项研究中，我们惊讶地发现SRSF3的敲除会导致超过一千个基因中偏好使用近端pA位点，并导致人和小鼠细胞中3’UTR的全局缩短。正如通过候选基因证明的，3’UTR缩短的基因会在衰老相关途径中富集并且可能产生更多蛋白质。我们通过过表达三个候选基因来模拟3’UTR缩短的影响，其均会导致衰老相关的表型。具体而言，SRSF3可以在转录水平上调节PTEN的APA并促进衰老。因此，SRSF3-KD诱导的衰老可以至少部分地通过其APA依赖性功能来解释（图6）。
The functional link between SRSF3-KD induced APA changes and cellular senescence was supported by multiple evidences. First, SRSF3-KD induced 3′ UTR shortened genes were enriched in senescence-associated pathways. Second, overexpression of three candidate genes (PTEN, PIAS1 and DNMT3A), emulating their effect of 3′ UTR shortening upon SRSF3 knockdown (Fig. 1,2, Fig. S4,S7,S8), promoted senescence-associated phenotypes. Specifically, PTEN, a well-known tumor suppressor and lifespan regulator [82], can be regulated at APA level by SRSF3 in both human and mouse cells. Supporting this, it was recently reported that nuclear poly(A) polymerases also regulated alternative polyadenylation of PTEN [66]. We further verified that SRSF3-KD induced PTEN’s 3′ UTR shortening generated more proteins, leading to reduced level of p-AKT and ultimately senescence-related phenotypes. Additionally, we did not find any obvious changes on the splicing pattern of PTEN compared SRSF3-KD to the control (Fig. S20). These data combined support the notion that APA-dependent function of SRSF3 contributes to cellular senescence.
SRSF3-KD诱导的APA变化与细胞衰老之间的功能联系得到了多种证据的支持。首先，SRSF3-KD诱导的3’UTR缩短的基因在衰老相关途径中富集。其次，通过对三种候选基因（PTEN，PIAS1和DNMT3A）的过表达，模拟了它们在SRSF3敲除的3’UTR缩短后所造成的影响（图1,2，图S4，S7，S8），其促进了衰老相关的表型。具体而言，PTEN，一种众所周知的肿瘤抑制因子和寿命调节因子，可在人和小鼠细胞中通过SRSF3在APA水平进行调节。最近有报道称，核poly（A）聚合酶也可以调节PTEN的选择性多聚腺苷酸化。我们进一步证实，SRSF3-KD诱导PTEN的3’UTR缩短会产生更多蛋白质，并导致p-AKT水平的降低并最终导致衰老相关表型。此外，我们没有发现PTEN的剪接模式与SRSF3-KD相比有任何明显的变化（图S20）。这些数据结合起来可以支持SRSF3的APA依赖性功能有助于细胞衰老的观点。
To profile the global APA changes in SRSF3-KD comparing to the control, we exploited two high throughput sequencing strategies (PA-seq and strand-specific RNA-seq methods) in 293T cells and HUVEC. Although we identified lots of genes undergoing 3′ UTR shortening either by eUTR or RUD analysis on PA-seq and RNA-seq, respectively, the overlap was relative small as showed in Fig. 1G. Three major differences between these two methods may underlie the phenomenon. First, the library construction step is considerable different. eUTR is calculated based on PA-seq data, which specifically enriches 3′ end sequence of polyA+ RNA. However, RUD is calculated based on RNA-seq data, which covers the full length of polyA+ RNA. Second, eUTR considers all identified pA sites located in 3′ UTR while RUD only calculates two pA sites (the most proximal pA site and the most distal pA site) within 3′ UTR. Thus eUTR and RUD will show less overlap considering this issue. Third, eUTR uses tag number in the pA cluster to reflect the usage preference and RUD reflects the relative read coverage on alternative 3′ UTR comparing to common region of 3′ UTR. Lastly, different PCR bias in different genomic region due to factors such as GC context, secondary structure may also underlie some of the difference between eUTR and RUD. Together, these above three major difference may explain the relative small overlap between eUTR and RUD. Consistent with this, overlap between two biological replicates with the same RUD calculation (52.7% for HUVEC_RUD_sh1 and HUVEC_RUD_sh2) is much higher than overlap between eUTR and RUD (22.5%).
为了分析SRSF3-KD与对照相比的全局APA变化，我们在293T细胞和HUVEC中利用了两种高通量测序方法（PA-seq和链特异性RNA-seq方法）。尽管我们分别通过对PA-seq和RNA-seq的eUTR或RUD分析鉴定了许多产生3’UTR缩短的基因，但其重叠相对较少，如图1G所示。这两种方法之间的三个主要差异可能是造成这种现象的根本原因。首先，建库的步骤有很大不同。 eUTR基于PA-seq数据计算，其特异性地富集polyA + RNA的3’末端序列。然而，RUD基于RNA-seq数据计算，其涵盖polyA + RNA的全长。其次，eUTR考虑位于3’UTR的所有鉴定的pA位点，而RUD仅计算3’UTR内的两个pA位点（最近的pA位点和最远的pA位点）。因此，考虑到这个问题，eUTR和RUD将显示较少的重叠。第三，eUTR使用pA簇中的标签号来反映使用偏好，并且RUD反映了选择性3’UTR上相对于3’UTR的公共区域的相对读取覆盖。最后，由于GC背景，二级结构等因素导致的不同基因组区域中的不同PCR偏差也可能是eUTR和RUD之间的一些差异的基础。总之，以上三个主要差异可以解释eUTR和RUD之间相对较少的重叠。与此一致，具有相同RUD计算的两个生物重复之间的重叠（对于HUVEC_RUD_sh1和HUVEC_RUD_sh2为52.7％）远高于eUTR和RUD之间的重叠（22.5％）。
Increasing evidences support the idea that splicing factors can regulate alternative polyadenylation. A well-known example was that downregulation of U1 small nuclear ribonucleoproteins (snRNP), which plays an important role in alternative splicing, led to the production of the truncated transcripts resulting from using cryptic pA sites located in introns [47, 48]. To regulate alternative polyadenylation located within 3′ UTR, SRSF3 may need to bind to nearby locations. We thus analyzed publicly available SRSF3 CLIP-seq data to confirm whether SRSF3 can bind to 3′ UTR-shortened genes and what’s the binding difference between proximal and distal pA sites. The results showed higher SRSF3 binding density near proximal pA sites than the distal ones (Fig. 2). Given the higher usage of proximal pA site upon SRSF3 knockdown, the higher SRSF3 binding nearby proximal pA sites possibly inhibited the selection of corresponding pA sites. Further investigation is definitely warranted to elucidate the detailed mechanism of such regulation.
越来越多的证据支持了剪接因子可以调节选择性多聚腺苷酸化这一观点。一个众所周知的例子是U1 snRNP的下调，其在选择性剪接中起到重要作用，会导致截短的转录物的产生，这是由于使用位于内含子中的隐蔽pA位点所导致的。为了调节位于3’UTR内的选择性多聚腺苷酸化，SRSF3可能需要结合到附近的位置。因此，我们分析了公开获得的SRSF3 CLIP-seq数据，以确认SRSF3是否可以与3’UTR缩短的基因结合，以及近端和远端pA位点之间的结合差异是什么。结果显示，近端pA位点附近的SRSF3结合密度高于远端位点（图2）。鉴于SRSF3敲除后近端pA位点的使用较高，邻近pA位点附近的SRSF3的较高结合可能抑制相应pA位点的选择。我们有必要进一步对其进行研究，以阐明这调控的详细机制。
3′ UTR shortening induced protein upregulation probably requires the expression of miRNAs targeting the alternative 3′ UTRs between proximal and distal pA sites. We thus examined whether such miRNAs were expressed by analyzing public small RNA sequencing data in human (293T, HUVEC) and mouse cells (MEFs). Interestingly, the result showed that miRNAs specifically targeting alternative 3′ UTRs of shortened genes have similar expression profiles compared with all expressed miRNAs (Fig. S21), supporting the notion that 3′ UTR shortening produced more protein through escaping of expressed miRNAs [87]. Which miRNA(s) involved in SRSF3-induced cellular senescence deserves extensive experimental validation.
3’UTR缩短诱导的蛋白质上调可能需要表达靶向近端和远端pA位点之间的选择性3’UTR的miRNA。 因此，我们通过分析人类（293T，HUVEC）和小鼠细胞（MEF）中的公共小RNA测序数据来检查是否这些miRNA表达。 有趣的是，结果显示，与所有表达的miRNA相比，特异性靶向缩短基因的选择性3’UTR的这类miRNA具有相似的表达谱（图S21），这支持了3’UTR缩短通过逃逸表达的miRNA来产生更多蛋白质这一观点。哪些miRNA参与了SRSF3诱导的细胞衰老，值得我们进行广泛的实验验证。
Our recent publication indicated that hundreds of genes underwent 3′ UTR lengthening in mouse embryonic fibroblasts (MEFs) replicative senescence model and rat Vascular Smooth Muscle Cells (rVSMCs) derived from old animal [32]. Specifically, longer 3′ UTR of Rras2 could produce less protein and induce senescence-related phenotypes [32]. However, it did not rule out the possibility that 3′ UTR shortening of certain genes could also promote senescence, given the complicated and sometimes opposite functions of genes in the same regulatory network. For example, kinase PI3K transforms PIP2 to PIP3, while phosphatase PTEN reverses PIP3 to PIP2. The two proteins act oppositely in the PI3K/AKT pathway, and thus similar 3′ UTR length change of these two genes might result in opposite consequences [88, 89]. In the present study, we showed that SRSF3-KD induced 3′ UTR shortening of PTEN could produce more protein and promote senescence-associated phenotypes. These data support a model that both 3′ UTR lengthening and shortening could regulate cellular senescence, and the function of the corresponding gene in the senescence pathway is the key. Keeping in mind that replicative senescence is a complicated biological process that multiple factors including SRSF3 are changed. Although reduced SRSF3 led to 3′ UTR shortening of target genes, changes of other regulators during replicative senescence may cause 3′ UTR lengthening of target genes. Actually, previous reports demonstrated that both 3′ UTR lengthening and shortening can be observed upon knockdown of different RNA binding proteins [53, 54]. Thus, SRSF3-KD induced global 3′ UTR shortening is not a contradiction compared to 3′ UTR lengthening in replicative senescence given the changed expression of multiple APA regulators. Understanding the downstream consequence of each APA regulators will definitely extend our understanding of the much-complicated APA changes during cellular senescence.
我们最近发表的文章表明，数百个基因在小鼠胚胎成纤维细胞（MEFs）复制衰老模型和来自老年动物的大鼠血管平滑肌细胞（rVSMCs）中出现了3’UTR延长。具体而言，Rras2较长的3’UTR可以产生较少的蛋白质并诱导衰老相关的表型。然而，鉴于同一调控网络中基因的复杂且有时相反的功能，我们不能排除某些基因的3’UTR缩短促进衰老的可能性。例如，激酶PI3K将PIP2转化为PIP3，而磷酸酶PTEN可将PIP3逆转为PIP2。这两种蛋白质在PI3K / AKT途径中起相反作用，因此这两种基因相似的3’UTR长度变化可能导致相反的后果。在本研究中，我们发现SRSF3-KD诱导PTEN的3’UTR缩短可以产生更多的蛋白质并促进衰老相关的表型。这些数据支持3’UTR延长和缩短均可调节细胞衰老的模型，并且这些基因在衰老途径中起到的功能是关键的。请记住，复制衰老是一个复杂的生物过程，包括SRSF3在内的多种因素都会发生变化。尽管SRSF3的减少会导致靶基因3’UTR缩短，但在复制衰老过程中其他调节因子的变化也可能导致靶基因3’UTR延长。实际上，之前的报道表明，在敲除不同的RNA结合蛋白后，可以观察到3’UTR延长和缩短。因此，SRSF3-KD诱导的全局3’UTR缩短与复制衰老的3’UTR延长相比并不矛盾，因为多种APA调节因子的表达发生变化。了解每个APA调控因子的下游进程肯定会扩展我们对细胞衰老过程中复杂的APA变化的理解。
