Aging is an irreversible physiological process that progresses with age, leading to structural disorders and dysfunctions of organs, thereby increasing the risk of chronic diseases such as neurodegenerative diseases, diabetes, hypertension, and cancer. Both organismal and cellular aging are accompanied by the accumulation of damaged organelles and macromolecules, which not only disrupt the metabolic homeostasis of the organism but also trigger the immune response required for physiological repair. Therefore, metabolic remodeling or chronic inflammation induced by damaged tissues, cells, or biomolecules is considered a critical biological factor in the organismal aging process. Notably, mitochondria are essential bioenergetic organelles that regulate both catabolism and anabolism and can respond to specific energy demands and growth repair needs. Additionally, mitochondrial components and metabolites can regulate cellular processes through damage-associated molecular patterns (DAMPs) and participate in inflammatory responses. Furthermore, the accumulation of prolonged, low-grade chronic inflammation can induce immune cell senescence and disrupt immune system function, thereby establishing a vicious cycle of mitochondrial dysfunction, inflammation, and senescence. In this review, we first outline the basic structure of mitochondria and their essential biological functions in cells. We then focus on the effects of mitochondrial metabolites, metabolic remodeling, chronic inflammation, and immune responsesthat are regulated by mitochondrial stress signaling in cellular senescence. Finally, we analyze the various inflammatory responses, metabolites, and the senescence-associated secretory phenotypes (SASP) mediated by mitochondrial dysfunction and their role in senescence-related diseases. Additionally, we analyze the crosstalk between mitochondrial dysfunction-mediated inflammation, metabolites, the SASP, and cellular senescence in age-related diseases. Finally, we propose potential strategies for targeting mitochondria to regulate metabolic remodeling or chronic inflammation through interventions such as dietary restriction or exercise, with the aim of delaying senescence. This reviewprovide a theoretical foundation for organismal antiaging strategies.

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.

Human pluripotent stem cells (hPSCs) represent a unique opportunity for understanding the molecular mechanisms underlying complex traits and diseases. CRISPR/Cas9 is a powerful tool to introduce genetic mutations into the hPSCs for loss-of-function studies. Here, we developed an episomal vector-based CRISPR/Cas9 system, which we called epiCRISPR, for highly efficient gene knockout in hPSCs. The epiCRISPR system enables generation of up to 100% Insertion/Deletion (indel) rates. In addition, the epiCRISPR system enables efficient double-gene knockout and genomic deletion. To minimize off-target cleavage, we combined the episomal vector technology with double-nicking strategy and recent developed high fidelity Cas9. Thus the epiCRISPR system offers a highly efficient platform for genetic analysis in hPSCs.

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.

T cell activation is a well-established model for studying cellular responses to exogenous stimulation. Using strand-specific RNA-seq, we observed that intron retention is prevalent in polyadenylated transcripts in resting CD4⁺ T cells and is significantly reduced upon T cell activation. Several lines of evidence suggest that intron-retained transcripts are less stable than fully spliced transcripts. Strikingly, the decrease in intron retention (IR) levels correlate with the increase in steady-state mRNA levels. Further, the majority of the genes upregulated in activated T cells are accompanied by a significant reduction in IR. Of these 1583 genes, 185 genes are predominantly regulated at the IR level, and highly enriched in the proteasome pathway, which is essential for proper T cell proliferation and cytokine release. These observations were corroborated in both human and mouse CD4⁺ T cells. Our study revealed a novel post-transcriptional regulatory mechanism that may potentially contribute to coordinated and/or quick cellular responses to extracellular stimuli such as an acute infection.