Alternative polyadenylation (APA), a phenomenon that RNA molecules with different 3′ ends originate from distinct polyadenylation sites of a single gene, is emerging as a mechanism widely used to regulate gene expression. In the present review, we first summarized various methods prevalently adopted in APA study, mainly focused on the next-generation sequencing (NGS)-based techniques specially designed for APA identification, the related bioinformatics methods, and the strategies for APA study in single cells. Then we summarized the main findings and advances so far based on these methods, including the preferences of alternative polyA (pA) site, the biological processes involved, and the corresponding consequences. We especially categorized the APA changes discovered so far and discussed their potential functions under given conditions, along with the possible underlying molecular mechanisms. With more in-depth studies on extensive samples, more signatures and functions of APA will be revealed, and its diverse roles will gradually heave in sight.

Cellular senescence has been viewed as a tumor suppression mechanism and also as a contributor to individual aging. Widespread shortening of 3′ untranslated regions (3′ UTRs) in messenger RNAs (mRNAs) by alternative polyadenylation (APA) has recently been discovered in cancer cells. However, the role of APA in the process of cellular senescence remains elusive. Here, we found that hundreds of genes in senescent cells tended to use distal poly(A) (pA) sites, leading to a global lengthening of 3′ UTRs and reduced gene expression. Genes that harbor longer 3′ UTRs in senescent cells were enriched in senescence-related pathways. Rras2, a member of the Ras superfamily that participates in multiple signal transduction pathways, preferred longer 3′ UTR usage and exhibited decreased expression in senescent cells. Depletion of Rras2 promoted senescence, while rescue of Rras2 reversed senescence-associated phenotypes. Mechanistically, splicing factor TRA2B bound to a core “AGAA” motif located in the alternative 3′ UTR of Rras2, thereby reducing the RRAS2 protein level and causing senescence. Both proximal and distal poly(A) signals showed strong sequence conservation, highlighting the vital role of APA regulation during evolution. Our results revealed APA as a novel mechanism in regulating cellular senescence.

Intron retention (IR) is the least well‐understood alternative splicing type in animals, and its prevalence and function in physiological and pathological processes have long been underestimated. Cellular senescence contributes to individual aging and age‐related diseases and can also serve as an important cancer prevention mechanism. Dynamic IR events have been observed in senescence models and aged tissues; however, whether and how IR impacts senescence remain unclear. Through analyzing polyA⁺ RNA‐seq data from human replicative senescence models, we found IR was prevalent and dynamically regulated during senescence and IR changes negatively correlated with expression alteration of corresponding genes. We discovered that knocking down (KD) splicing factor U2AF1, which showed higher binding density to retained introns and decreased expression during senescence, led to senescence‐associated phenotypes and global IR changes. Intriguingly, U2AF1‐KD‐induced IR changes also negatively correlated with gene expression. Furthermore, we demonstrated that U2AF1‐mediated IR of specific gene (CPNE1 as an example) contributed to cellular senescence. Decreased expression of U2AF1, higher IR of CPNE1, and reduced expression of CPNE1 were also discovered in dermal fibroblasts with age. We discovered prevalent IR could fine‐tune gene expression and contribute to senescence‐associated phenotypes, largely extending the biological significance of IR.

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.

Intronic polyadenylation (IpA) usually leads to changes in the coding region of an mRNA, and its implication in diseases has been recognized, although at its very beginning status. Conveniently and accurately identifying IpA is of great importance for further evaluating its biological significance. Here, we developed IPAFinder, a bioinformatic method for the de novo identification of intronic poly(A) sites and their dynamic changes from standard RNA-seq data. Applying IPAFinder to 256 pan-cancer tumor/normal pairs across six tumor types, we discovered 490 recurrent dynamically changed IpA events, some of which are novel and derived from cancer-associated genes such asTSC1,SPERD2, andCCND2. Furthermore, IPAFinder revealed that IpA could be regulated by factors related to splicing and m⁶A modification. In summary, IPAFinder enables the global discovery and characterization of biologically regulated IpA with standard RNA-seq data and should reveal the biological significance of IpA in various processes.

Despite the manifestation and contribution of cellular senescence to aging and various diseases, accurate identification of heterogeneous senescent cells remains challenging. Current senescence evaluation methods rely mainly on limited markers or homogeneous samples, which might fail to capture universal senescence features, limiting their generalizability. Here we developed the human universal senescence index (hUSI), an accurate and robust senescence evaluation method for diverse cells and samples. Based on features learned from the most comprehensive cellular senescence-associated transcriptome data so far, hUSI demonstrated its convincing connections with senescence phenotypes and superior robustness in predicting senescence state. Using hUSI, we discovered potential senescence regulators and mapped senescent cell accumulation across cell types in coronavirus disease 2019 (COVID-19). The method also facilitates decoding heterogeneous senescence states in melanoma tumors, identifying prognosis-associated signaling pathways. Overall, hUSI demonstrates its utility in characterizing cellular senescence across biological contexts, with broad applications in aging research and clinical practice.