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. 2025 May 12;5(5):487–501. doi: 10.1007/s43657-024-00198-2

Reduced Usage of GLS Intronic Polyadenylation Promotes Cellular Senescence

Xueping Li 1,#, Yuxin Li 1,#, Dong Ding 1, Baiyun Xie 1, Jun Yao 1, Yu Chen 1, Zhaozhao Zhao 1,2, Peng Yu 1, Shengwei Zhang 1, Fu Yang 3, Ting Ni 1,4,✉, Gang Wei 1,✉
PMCID: PMC12881237  PMID: 41659737

Abstract

Alternative polyadenylation (APA) of mRNA is a widespread transcriptomic mechanism which fine-tunes gene expression and plays important roles in diverse biological processes. Of the different APA types, intronic polyadenylation is a prevalent but not well understood one. Although the upstream regulation of intronic polyadenylation is extensively investigated, the understanding of its biological function largely lags behind. Here, we discovered that intronic polyadenylation of glutaminase-encoding gene Glutaminase (GLS) generated an isoform termed Glutaminase C (GAC, encoding glutaminase C), which played regulatory roles in cellular senescence, the basis of individual aging and also an important anti-cancer mechanism. Reduced usage of GLS intronic polyadenylation (pA) site was consistent in both human and mouse senescence models. GAC protein mainly located in mitochondria, and downregulation of GAC induced cellular senescence in both human and mouse cells. Increased reactive oxygen species (ROS) level and decreased ATP synthesis explained the senescence-associated phenotypes in GAC deficient cells. Polyadenylation factor CPSF6 bound to the last exon of GAC to regulate its expression. Rescue experiment confirmed that CPSF6-GAC signal axis did play important roles in regulating cellular senescence. Together, the present study demonstrated for the first time that intronic polyadenylation could regulate cellular senescence, largely extending our understanding of the biological roles of intronic polyadenylation.

Supplementary Information

The online version contains supplementary material available at 10.1007/s43657-024-00198-2.

Keywords: Alternative Polyadenylation (APA), Intronic Polyadenylation, Glutaminase, Mitochondria, Cellular Senescence

Introduction

Alternative polyadenylation (APA) of mRNA is a widespread phenomenon in diverse species, serving as an important contributor to transcriptome diversity (Shepard et al. 2011). Based on the relative position that poly(A) was added in gene body, APA can be divided into two major types: CR-APA and UTR-APA. The alternative polyadenylation (pA) site locates at the gene coding region (CR) is called CR-APA, which can generate protein isoforms with truncated and even distinct C-terminal domains. UTR-APA refers to the alternate pA site locates at 3' untranslated region (3' UTR), which generates transcripts with 3' UTR of different lengths that may lead to difference in RNA stability, RNA localization, translation efficiency, and even protein localization (Mayr 2016; Mitschka and Mayr 2022; Tian and Manley 2017). CR-APA can be further divided into three groups, including intronic polyadenylation (or intronic APA), alternative terminal exon APA, and exonic APA (Elkon et al. 2013). While UTR-APA mediated global changes in 3' UTR length has been extensively investigated in various biological processes such as development, cell proliferation, tumorigenesis and senescence (Chen et al. 2018; Lai et al. 2015), the function of CR-APA, especially intronic APA, is far from fully understood. Some sequences evolutionarily introduced into intron regions, such as transposon with poly(A) signal or RNA binding protein (RBP) recognition sequences, giving rise to intronic polyadenylation (Wu et al. 2014). Oppositely, trans-acting factors including splicing factors and polyadenylation factors can coordinate to repress the usage of these intronic pA sites to guarantee the generation of full-length mRNAs in most of the time (Berg et al. 2012; Kamieniarz-Gdula and Proudfoot 2019). However, some intronic pA sites can also evolve to be functional in certain biological processes. One example is the intronic polyadenylation of PDGFRα (the gene encoding platelet-derived growth factor receptor α) generates a protein with truncated kinase domain, which acts as a decoy to suppress PDGF signaling and ultimately prevents fibrosis (Mueller et al. 2016). UV-induced intronic pA site usage in ASCC3 generates a non-coding RNA that functions oppositely to the regular products of the same gene (Williamson et al. 2017). Despite a few cases connecting intronic APA to biological functions, the widespread nature and functional roles of intronic polyadenylation is still far from full understanding, especially in biological processes that closely connected to human health such as cellular senescence or aging.

Cellular senescence is a process that results from a variety of stresses, leading to a state of irreversible growth arrest. Senescence can be either harmful or beneficial to individuals. On one hand, senescent cells accumulated during aging have been implicated in promoting a variety of age-related diseases (Zheng et al. 2024). Elimination of senescent cells delays age-related pathologies in a mouse model of aging (Baker et al. 2011). On the other hand, cellular senescence is an important anti-cancer mechanism inhibiting proliferation of damaged or premalignant cells (Campisi 2013). A recent study demonstrated that induction of cancer cells to enter into senescent state could activate the immune cells (such as natural killer cells) to clear tumor cells in a KRAS-mutant lung cancer mouse model (Ruscetti et al. 2018). Thus, discovery of new genes or new layer of gene regulation that induce senescence of cancer cells could help to uncover potential novel targets for cancer immune therapy. It is well known that cellular senescence can be induced by multiple factors, such as excessive replication, telomere attrition, irradiation, oncogene activation, and oxidative damage (Campisi and d' Adda di Fagagna 2007; Hayflick and Moorhead 1961). Cellular senescence can also be induced by the perturbation in mitochondrial homeostasis, which may accelerate age-related phenotypes (Ghosh-Choudhary et al. 2021; Sahin and Depinho 2010). While several studies showed that mitochondrial defects can promote cellular senescence (Lee et al. 2024; Passos et al. 2007; Velarde et al. 2012), the underlying mechanisms are still far from clear understanding.

We were therefore curious about whether intronic polyadenylation of mitochondrial related genes played a causal role in regulating cellular senescence. By surveying the dynamic changes of intronic APA in replicative senescence models, we discovered Glutaminase (GLS), which encodes mitochondrial related enzyme glutaminase, had reduced intronic pA site usage in both human and mouse senescent cells. The senescence-accompanying altered intronic pA site usage in GLS in senescence gives us a good opportunity to look into the functional and mechanistic roles of intronic polyadenylation in senescence process. Moreover, this also renders us an opportunity to explore how alternative polyadenylation interacts with mitochondria to regulate important biological processes such as senescence. In this study, we provided evidence showing that reduced intronic polyadenylation of GLS led to increased reactive oxygen species (ROS) level and decreased ATP generation, which in turn resulted in senescence phenotypes. The polyadenylation factor CPSF6 mediated this alteration in intronic pA site usage and regulated cellular senescence. The present study largely extends our knowledge regarding the biological significance of intronic APA.

Materials and Methods

Cell Culture, Transfection and Selection of Stable Cell Lines

All cell lines (293T, Human umbilical vein endothelial cells (HUVEC), A549, Human foreskin fibroblast (HFF), NIH3T3) used in this study were cultured in Dulbecco's modified eagle medium (DMEM) medium (Gibco, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37 °C with 5% CO2. For transient transfection, cells were seeded in 6-well plate for 60%–70% confluence one day in advance, then transfected with 1 µg constructed vectors using Lipofectamine 2000 (Invitrogen) on the second day. Cells were harvested for RNA and protein extraction 48 h (hrs) after transfection. For knockdown of Glutaminase C (GAC) and CPSF6 by lentivirus transfection, shRNA oligonucleotides for GAC and CPSF6 were annealed and then cloned into the pLKO.1 vector. Then 1 µg constructed vectors or control vector pLKO.1 plus VSVG and gag/pol encoding plasmids were transfected into the 293T cells, respectively. After cultured for 12 h, antibiotics (50 U/mL penicillin and 50 µg/mL streptomycin) was added into the culture medium. After culturing 24 h and 48 h, the virus supernatant was harvested to infect HUVEC, A549, HFF and NIH3T3 cells, respectively. Twenty four hrs post the second infection, the medium was replaced with fresh DMEM supplemented with puromycin (2 µg/mL). After one day screening, the survived cells were cultured for additional two days in medium containing puromycin (2 µg/mL). Then the survived cells were harvested for RNA and protein extraction to validate GAC and CPSF6 knockdown efficiency. All shRNA sequences are listed in Table S2.

RNA Extraction, cDNA Synthesis, qRT-PCR and Western Blot

Total RNA and protein were extracted with TRIzol reagent (Sigma) according to the manufacturer's instruction. For cDNA synthesis, 1 µg total RNA was reversely transcribed into cDNA by FastQuant RT Kit (TIANGEN) and oligo (dT) primer. Then quantitative PCR was performed using 2×SYBR mix (Vazyme) on a Bio-Rad CFX manager machine. The sequences of primers for quantitative reverse transcription followed by polymerase chain reaction (qRT-PCR) are listed in Table S2. For Western blot, the protein concentration was determined by Lowry assay. Samples were resolved on SDS-polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore). Proteins were detected using indicated antibodies [anti-GLS (Abcam, ab156876, 1:1000), anti-CPSF6 (Abcam, ab175237, 1:1000), anti-GAPDH (Abways, AB2000, 1:1000)] and visualized with Tanon Chemiluminescent Imaging System. For Western blot studies, results are representative of at least three independent experiments.

Immunofluorescence (IF)

To determine the location of GAC, we adopted transient transfection. GAC plus horseradish (HA) was cloned into the pcDNA3.1 vector. Twenty four hrs post transfection, cells were used for IF. Cells were seeded in the 6 mm plate one day in advance. After 24 h incubation, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature, and then soaked in Triton X-100 at 4 °C for 5 min. Cells were washed by phosphate buffered saline (PBS) for three times before soaked in 5% BSA in PBS and then incubated with 1: 250 diluted polyclonal antibody (anti-HA, Abmart, M20003L) at 4 °C overnight. After that cells were incubated with secondary antibody 1:100 diluted conjugated anti mouse IgG (Abways, AB0132) for 90 min at 37 °C. Cells with GAC and mitochondria double staining were observed and photographed using an Olymb BIOPHOTO fluorescence microscope.

Cell Proliferation Assay

After puromycin selection, the survived cells were trypsinized and diluted, then subcultured in a 96-well plate, with 1000 cells per well and four replicates for each time point. Cell Counting Kit-8 (CCK-8) reagent (DOJINDO) was then added according to the manufacturer's protocol to each well every 24 h post seeding, lasting for four days. After each treatment, cells were incubated for two hours at 37 °C, then the optical density (OD) at 450 nm and 630 nm were measured respectively for each well by microplate reader (Biorad).

Cell Cycle Analysis

Cells were seeded in the 6-well plate one day in advance at 60%-70% confluence. After 24 h incubation, cells were harvested and washed with PBS for one time. Then cells were incubated with Triton X-100 and Propidium Iodide (PI) in 500 µL PBS for 10 min at room temperature. The fluorescence intensity of PI was measured with a FACS vantage SE flow cytometry (Becton Dickinson, USA) equipped with Cell Quest software (Becton Dickinson, USA).

SA-β-Gal Activity Assay

Cells were seeded in the 12-well plate one or two days before staining. Briefly, we adopted Sigma SA-β-Gal staining kit according to the manufacturer's instruction. After washing cells with PBS for three times, cells were fixed with the fixation buffer and then incubated with SA-β-Gal staining solution at 37 °C separated from CO2 overnight. The results were recorded using a charge-coupled device camera attached to a phase-contrast microscope.

kFluor488-EdU Assay

The kFluor488-EdU assay is performed according to the manufacturer's protocol (KGA331-100, KeyGEN BioTECH). Cells were seeded in 6-well plate for 50% confluence. Twenty-four hrs post seeding, we added EdU into the medium and cells were incubated for 2 h at 37 °C. Then cells were fixed with 4% paraformaldehyde for 15 min and washed twice by PBS with 3% BSA. Next, cells were incubated with 0.5% Triton X-100 in PBS for 20 min and then incubated with Click-iT reaction mixture for 30 min avoiding light. After washed with PBS twice, cells were added with 5 µg/mL Hoechst33342 solution in PBS and incubated for 15 min avoiding light. Finally, cells were washed with PBS and photographed using a fluorescence microscope.

Determination and Quantification of ROS

Cells were seeded in the 6-well plate at 60% confluence. Two days later, cells were washed with 1×PBS for three times and then treated with 5 µM Dihydroethidium (DHE) assay solution (Yeasen, Cat.no. 50102ES02) in 1×PBS for 60 min at 37 °C in darkness. After 60 min incubation, cells were washed with 1×PBS for three times and then harvested with 0.05% trypsin. For each sample, cells were resuspended in 300 µL 1×PBS to quantify the intracellular ROS level. Relative fluorescence intensities were determined by FACS vantage SE flow cytometry (Becton Dickinson, USA).

ATP Bioluminescence Assay

Cells were seeded in 96-well plate at 60% confluence. Then CellTiter-Glo® assay reagent (Promaga) was added to each well 24 h post seeding. The plates were gently shaken for 2 min and incubated at room temperature for 10 min in the dark. The luminescence was measured with the spark® multimode microplate reader (Tecan). All measurements were carried out in triplicate.

RNA Immunoprecipitation (RIP) Assays

After harvesting cells, we isolated the nuclei and added 1 ml cold RIP lysis buffer (50 mM Tris-HCl pH 7.4, 250 mM NaCl, 5 mM EDTA, 1% NP40, 0.5 mM DTT, 100 U/mL RNase inhibitor, 1× protease inhibitor) to the tube. Then we sonicated the chromatin for 5 × 30 s using a Bioruptor sonication device (Diagenode). After that we performed IP experiments with the following antibodies were used for IPs: rabbit anti-CPSF6 antibody (Abcam, ab175237), rabbit IgG antibody (Abmart, B30011S). The samples were incubated with corresponding antibody for 2 h at 4 °C and then with protein G beads for 2 h. After incubation, the samples were washed with RIP buffer for three times and PBS for one time and then centrifuged at 3000 rpm, 4 °C for 3 min to collect the precipitation. After immunoprecipitation, RNA was extracted with TRIzol reagent and subjected to qPCR or high-throughput sequencing. Two pairs of primers (sequences listed in Table S2) were used for RIP-qPCR and RIP-PCR.

Results

Reduced Usage of Intronic pA Site of GLS during Cellular Senescence

To examine whether intronic APA has dynamic changes during senescence, we analyzed expression data from three replicative senescence models, including our published PA-seq data from mouse embryonic fibroblasts (MEFs) and HUVECs (Chen et al. 2018), and publicly available RNA-seq data from HFF (Marthandan et al. 2015). We focused on intronic APA this time and found 22 genes showed significant intronic pA site usage changes between senescent (population doubling 11, or PD11) and young (PD6) MEFs (Table S1). Interestingly, GLS, which encodes the K-type mitochondrial glutaminase, ranked top when considering both change in intronic pA site usage and expression abundance (Table S1). Since changes in mitochondrial related functions have been widely reported to be associated with senescence and age-related diseases (Chan 2006; Guarente 2008), we selected GLS as a candidate gene to investigate the causal relationship between intronic APA and senescence. We first confirmed the authenticity of intronic pA site in GLS utilizing rapid amplification of cDNA ends (3' RACE) (Fig S1). Visualization of PA-seq data showed a dramatic decrease in intronic pA site usage in senescent MEFs while no obvious change regarding the distal pA site (Fig. 1a). Changes in intronic pA site usage illustrated in PA-seq data was further validated by quantitative reverse transcription followed by polymerase chain reaction (qRT-PCR) in senescent MEFs comparing to younger ones (Fig. 1b-c). Moreover, human GLS gene also displayed reduced intronic pA site usage in both senescent HUVECs and HFFs (Fig S2, S3). Interestingly, the gene structure and DNA sequence upstream the intronic pA site of GLS were conserved among human, rat and mouse (Fig S4), implying the biological importance of intronic polyadenylation in GLS.

Fig. 1.

Fig. 1

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The use of intronic pA site of GLS decreases during cellular senescence. (a) The track of GLS gene during MEFs' senescence from young state PD6 to senescent state PD11 analyzed from PA-seq uploaded to UCSC browser. (b) Schematic diagram of primer design to detect the expression of truncated isoform termed GAC generated by intronic pA site of GLS, and the longisoform termed KGA using the UTR's pA site. (c) The relative expression level of GAC and KGA were detected via qRT-PCR using the primers illustrated in panel (b). *** indicates p < 0.001 of t-test and ns (non-significant) indicates p > 0.05 by t-test with three qPCR reactions

Downregulation of GAC Generated by Intronic pA Site Induces Cellular Senescence

Previous biochemistry studies showed that transcript using intronic pA site of GLS generates the protein glutaminase C (GAC) while transcript using distal pA site encodes the protein KGA (the full-lenth kidney isoform) (Szeliga and Albrecht 2015). We thus named GLS transcript using intronic pA site as GAC. The reduced intronic pA site usage during senescence resulted in decreased expression of GAC transcript. To mimic the reduced usage of intronic polyadenylation in GLS, we used two different short hairpin RNAs (shRNAs) specifically targeting the last exon of GAC to specifically silence GAC expression. As induction of tumor cell to senescence has the potential to enhance immunotherapy in lung cancer (Lin et al. 2017), we tested whether specific GAC knockdown (KD) could lead to senescence in A549, a human lung cancer cell line. The knockdown of GAC in A549 cells was confirmed at both mRNA and protein levels (Fig. 2a). Interestingly, senescence-associated β-galactosidase (SA-β-Gal), a classical senescence marker (Dimri et al. 1995), showed higher staining percentage in GAC-KD cells (Fig. 2b). Other senescence associated phenotypes, such as decreased DNA replication ability, reduced cell proliferation rate and cell cycle arrest were also observed in GAC-KD cells (Fig. 2c-e). Besides, we also detected several other senescence-associated molecular markers, including decreased expression levels of cell cycle-related genes like CCND2, CCNA, and cell proliferation marker MIK67 and increased expression levels of CDKN1A and TP53 (Fig. 2f). As senescent HFF also showed a reduced usage ratio of intronic pA site in GLS (Fig S3), we knocked down GAC by two different shRNAs and found that downregulation of GAC could also lead to senescence in primary HFF cells (Fig S5a-f). Besides, such GAC-KD induced senescence-associated phenotypes remained true in mouse embryonic fibroblast cell line NIH3T3 (Fig S6a-f). These results above suggest that downregulation of GAC caused by intronic polyadenylation selection induces senescence in both human and mouse cells.

Fig. 2.

Fig. 2

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Knockdown of GAC induces senescence in human A549 cells. (a) qRT-PCR and Western blot to validate GAC knockdown efficiency in human A549 cells transfected with two different shRNAs (sh1 and sh2). GAPDH served as the internal control in both qRT-PCR and Western blot. (b) SA-β-Gal staining before and after GAC-KD via sh1 and sh2 compared to the control in A549 cells. (c) Decreased DNA replication rate in GAC-KD A549 cells evaluated by EdU incorporation assay compared to control cells. Blue: DAPI stains nuclear DNA. Green: EdU labeled DNA, the newly synthesized DNA. (d) Cell proliferation rate decreased in GAC-knockdown A549 cells compared to control cells assayed by CCK-8 kit. (e) Cell cycle arrested in G1 phase in GAC-knockdown A549 cells as detected by fluorescence-activated cell sorting (FACS) analysis. (f) Cellular senescence-associated biomarkers including decreased CCND2, CCNA and MKI67 expression and elevated CDKN1A (encoding P21) and TP53 (encoding P53) expression were detected by qRT-PCR after GAC-KD in A549 cells. GAPDH served as the internal control. ** and *** represent p < 0.01 and p < 0.001, respectively, based on t-test with three qPCR reactions in panel a and f or four biological replicates in panel d and three biological replicates in panel e

Knockdown of GAC Leads to Increased ROS Level and Decreased ATP Generation

GAC protein has been reported to specifically locate in mitochondria (Cassago et al. 2012). To validate whether it is still the case in A549 cells, we performed immunofluorescence (IF) staining and found that GAC exclusively located in mitochondria (Fig. 3a). Given its mitochondrial localization, we speculated that GAC-KD induced senescence may be mediated by dysfunction of this organelle. As dysregulated ROS level and insufficient energy generation of mitochondria has been reported to induce senescence in various cell types (Baker et al. 2011; Campisi 2013), we quantified these two metabolites in GAC-KD cells. A significantly higher level of ROS was detected upon GAC knockdown in human A549, HFF and mouse NIH3T3 cells (Fig. 3b-c and Fig S7 a-b, Fig S8 a-b ). To determine whether increased ROS contributed to cellular senescence, we used N-acetylcysteine (NAC), which is a direct antioxidant for some oxidant species such as NO2 and HOX, as an antioxidant treatment for the ROS elevated cells (Aldini et al. 2018). We found that NAC treatment could alleviate SA-β-Gal staining in GAC-KD cells (Fig. 3e-f), indicating cellular ROS is a casual factor of GAC-KD induced senescence. In addition, reduced ATP generation was also observed in GAC-KD human cells (A549 and HFF) (Fig. 3d and Fig S7 c) and mouse NIH3T3 cells (Fig S8 c). These results combined to suggest that GAC-KD induced senescence is mediated, at least in part, by increased level of ROS and reduced ATP generation.

Fig. 3.

Fig. 3

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Localization of GAC protein and knockdown of GAC promotes ROS level in A549 cells. (a) Blue: DAPI stained nuclear DNA. Green: GFP labeled mitochondria. Red: HA-tagged GAC protein. Merge: HA-tagged GAC protein localized in the mitochondria. (b) Cellular ROS level in GAC-KD (sh1 and sh2) A549 cells and control cells assayed by Dihydroethidium (DHE) assay and fluorescence-activated cell sorting (FACS). X axis represents the Fluorescence intensity of DHE while Y axis indicates cell counts. (c) Increased ROS level in GAC-KD A549 cells (two replicates, sh1 and sh2) compared to control cells. FlowJo, a well-known software for single cell flow cytometry analysis, was used for the quantification. (d) Cellular ATP level detected by CTG reagent in human A549 cells after GAC knockdown. (e-f) Representation (e) and quantification (f) of SA-β-Gal staining before (-NAC) and after (+ NAC) adding NAC in GAC-KD (two replicates, sh1 and sh2) cells compared to control. *,** and *** indicate p < 0.05, p < 0.01 and p < 0.001, respectively, based on t-test with three technical replicates in panel c and d, and three independent countings in panel f

Polyadenylation Factor CPSF6 Binds to the Last Exon of GAC to Regulate the Intronic pA Site Selection

To explore the possible mechanism by which the intronic polyadenylation of GLS is regulated, we screened for RNA binding proteins (RBPs) that potentially bound near the intronic pA site of GLS using POSTAR, a comprehensive database archiving all RBP-RNA interacting events acquired by diverse CLIP (Cross-Linking and Immunoprecipitation) sequencing protocols (Hu et al. 2017). We focused on the last 200 nucleotides (nt) in 3' UTR region of GAC where polyadenylation factors likely bind to and found 10 top-ranked candidate RBPs (Fig S9). To investigate which RBP plays the potential role in regulating GAC expression, we knocked down these 10 RBPs respectively (Fig S10 a). Intriguingly, four RBPs resulted in reduced expression of GAC upon knockdown, with CPSF6 had the most significant effect (Fig S10 b). To further verify the regulatory function of CPSF6 on GAC, we performed rescue experiment by overexpressing CPSF6 in lentivirus-mediated CPSF6 knockdown cells. Strikingly, the GAC expression was reversed upon overexpression of CPSF6 via Western blot detection (Fig. 4a). These results demonstrate that CPSF6 is a key regulator of GLS intronic polyadenylation. As a member of CFIm family, CPSF6 (also known as CFIm68) has been reported to bind to the ‘UGUA' motif (Shi and Manley 2015). We thus performed RIP-PCR (RNA immunoprecipitation followed by polymerase chain reaction) using two primer pairs spanning the ‘UGUA' motif close to the intronic pA site of GLS to examine whether CPSF6 actually binds to the 3' UTR of GAC (Fig. 4b). The result showed that GAC transcript had enriched CPSF6 binding signal when compared to non-specific IgG control (Fig. 4c), which was further validated by RIP-qPCR (Fig. 4d). Moreover, a consistent higher binding signal was detected when comparing CPSF6 antibody to IgG control in both A549 and HUVEC cells using RIP-seq (Fig. 4e). In addition, publicly available CPSF6 CLIP-seq data in MEFs (GEO Accession: GSM1481005) also supported our conclusion that CPSF6 bound to the region near GLS intronic pA site (Fig S11). All these results indicate that CPSF6 regulates intronic polyadenylation of GLS to generate GAC.

Fig. 4.

Fig. 4

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CPSF6 binds to the 3′ process region of GAC and regulates the generation of GAC isoform in A549 cells. (a) The protein levels of CPSF6 and GAC were detected by Western blot before and after CPSF6 overexpression (rescue) in CPSF6-KD A549 cells. GAPDH served as the internal control. (b) The diagram of primer design to detect the binding of CPSF6 in the 3′ UTR of GAC. (c) CPSF6 binding to GAC was validated by RIP-PCR via two pairs of primers in (b). IgG antibody served as non-specific binding control. (d) RIP-qPCR to further validate the fold enrichment of CPSF6 bound to GAC. (e) The normalized tracks analyzed from RIP-seq of CPSF6 in A549 and HUVEC cells showed higher binding signal of CPSF6 antibody compared to IgG contorl in the 3' UTR of GAC. *** in panel d represents p < 0.001 based on t-test with three technical replicates of RIP-qPCR

CPSF6 Deficiency Induces Cellular Senescence

Given decreased expression of GAC induces senescence and CPSF6-KD suppresses the intronic pA site usage of GLS, one would expect CPSF6 knockdown could also promote senescence if CPSF6-GAC signal axis plays a major role. Interestingly, CPSF6 showed decreased expression in multiple senescence models including human and mouse (Fig S12), indicating its potential roles in senescence. To examine whether downregulation of CPSF6 also induces senescence, we knocked down CPSF6 (CPSF6-KD) in human A549, HFF and mouse NIH3T3 cells. Intriguingly, CPSF6-KD cells showed multiple senescence-associated phenotypes, including higher percentage of SA-β-Gal staining, reduced cell growth rate, decreased DNA replication ability and cell cycle arrest (Fig. 5a-e and Fig S13 a-f, S14 a-d). Moreover, we also detected increased ROS level in CPSF6-KD cells comparing to control cells (Fig. 5f-g and Fig S13 g-h, S14 e-f). These results were very similar to those induced by GAC knockdown, further strengthening the CPSF6-GAC signal axis in regulating senescence-associated phenotypes.

Fig. 5.

Fig. 5

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Knockdown of CPSF6 induces cellular senescence in A549 cells. (a) qRT-PCR and Western blot to validate CPSF6 knockdown efficiency in human A549 cells transfected with two different shRNAs (sh1 and sh2). GAPDH served as the internal control. (b) SA-β-Gal staining before and after CPSF6 knockdown in A549 cells. (c) Decreased DNA replication rate in CPSF6-KD A549 cells evaluated by EdU incorporation assay compared to control cells. Blue: DAPI stains nuclear DNA. Green: EdU labeled DNA, the newly synthesized DNA. (d) Cell proliferation rate decreased in CPSF6-KD A549 cells compared to control cells assayed by CCK-8 kit. (e) Cell cycle arrested in G1 phase in CPSF6-knockdown A549 cells as detected by fluorescence-activated cell sorting (FACS) analysis. (f-g) Representation (f) and quantification (g) of ROS level in CPSF6-KD (sh1 and sh2) cells and control cells assayed by Dihydroethidium (DHE) assay and FACS. X axis represents the fluorescence intensity of DHE while Y axis indicates cell counts. ** and *** represent p < 0.01 and p < 0.001, respectively, based on t-test with three qPCR reactions in panel a or four biological replicates in panel d and three biological replicates in panel e and three technical replicates in panel g

Overexpression of GAC Rescues Senescence in CPSF6-KD Cells

To further determine whether downregulation of GAC contributes mainly to CPSF6-KD induced senescence, we overexpressed GAC in CPSF6-KD cells. Strikingly, the senescence-associated phenotypes were largely reversed including SA-β-Gal staining, cell growth rate, cell cycle arrest, and DNA replication ability in A549 cells (Fig. 6a-d). Importantly, the ROS level was also reversed upon overexpressing GAC in CPSF6-KD cells (Fig. 6e-f). These results combined to indicate that downregulation of CPSF6 promotes senescence mainly through reducing the expression of GAC, an isoform of GLS utilizing intronic polyadenylation.

Fig. 6.

Fig. 6

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Overexpressing GAC can rescue cellular senescence induced by CPSF6 knockdown. (a) Cell proliferation rate was rescued after GAC-overexpression in CPSF6-knockdown A549 cells assayed by CCK-8 kit. (b) Cell cycle arrested in G1 phase was rescued after GAC-overexpression in CPSF6-knockdown A549 cells as detected by FACS analysis. (c) Representative images (left) and percentage (right) of positive cells of SA-β-Gal staining before and after GAC overexpression in CPSF6-KD cells. (d) Decreased DNA replication rate after GAC overexpression in CPSF6-KD A549 cells evaluated by EdU incorporation assay. Percentage of EdU stained cells was shown on right. Blue: DAPI stains nuclear DNA. Green: EdU labeled DNA, the newly synthesized DNA. (e-f) Representation (e) and quantification (f) of ROS level after GAC overexpression in CPSF6-KD cells and control cells assayed by DHE assay and FACS. X axis represents the fluorescence intensity of DHE while Y axis indicates cell counts in panel e. ** and *** represent p < 0.01 and p < 0.001, respectively, based on t-test with four biological replicates in panel a and three biological replicates in panel b or three independent countings in panel c and d and three technical replicates in panel f

Discussion

Our group first demonstrated that alternative polyadenylation (APA) mediated 3′ UTR length change is a hidden post-transcriptional contributor to cellular senescence (Chen et al. 2018; Shen et al. 2019), however, the function of intronic polyadenylation during cellular senescence is still unknown. In the present study, we revealed for the first time that intronic polyadenylation played regulatory roles in cellular senescence, as exemplified by a mitochondrial related gene GLS (Fig. 7). This finding extended the functional importance of intronic APA to the aging field and also provided a novel perspective for fully understanding the regulatory mechanism underlying cellular senescence. Intronic APA can generate proteins with distinct C-terminal domains, which may differ at various aspects (such as functional domains, cellular localization and other functional properties) with the regular transcripts of the same gene. As for GLS, distal and intronic pA sites generate KGA and GAC proteins, respectively. GAC localizes in mitochondria while KGA distributes in both mitochondria and cytoplasm (Cassago et al. 2012). Although increasing investigations have revealed that GLS is involved in many biological processes (Feng et al. 2024; Johnson et al. 2018; Yu et al. 2019), this study for the first time links GAC with senescence via changing mitochondrial related functions. Given the importance of senescence to individual aging and cancer, the causal role of intronic polyadenylation to senescence will provide potential novel targets for protecting human health.

Fig. 7.

Fig. 7

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Working model for CPSF6 mediates reduced usage of intronic polyadenylation of GLS to promote cellular senescence. Here, we determined CPSF6 is a critical factor in the cleavage and intronic polyadenylation of GLS, which generates GAC via its intronic pA site. CPSF6 is downregulated in senescent cells, and then lead to reduced generation of GAC transcript and therefore producing less mitochondria-localized GAC protein. Reduced GAC protein in mitochondria leads to higher ROS level and lower ATP level, which ultimately promote senescence-associated phenotypes

Cellular senescence is also a well-known cancer prevention mechanism, we are therefore curious about if reduced intronic polyadenylation of GLS during senescence is opposite in cancer cells. Interestingly, GAC transcript derived from intronic APA of GLS was upregulated in the majority of liver tumor samples compared to matched normal tissues (eight out of 10 samples) (Fig. S15), which was in line with a previous finding that GAC was upregulated in lung cancer cells (van den Heuvel et al. 2012). Of note, GLS was found a strong shift from KGA in normal tissue to GAC in both lung and kidney cancers (LUAD, LUSC and KIRC), and lower GAC percentage was highly correlated with better survival rate in kidney cancer (Xia et al. 2014). Interestingly, we found an apparent shift from GAC in young cells to KGA in senescent cells of three additional senescence models, including two replicative senescence models (WI-38 and IMR90 fibroblasts) and one oncogene-induced senescence model (Ras-induced MIR90) (Fig. S16). These results imply that intronic APA in GLS could link senescence to cancer, though deserving further investigation.

Increased cellular ROS level and decreased ATP production possibly explain GAC-KD induced senescence. In a previous study, mice null for superoxide dismutase showed a profound decrease in lifespan and several accelerated aging phenotypes (Zhang et al. 2017). In our study, we discovered an intronic APA of GLS functioned in producing ROS (Fig. 3b-c) which linked with mitochondria homeostasis (Jezek and Hlavata 2005). ROS reduction induced by the antioxidant N-Acetyl Cysteine (NAC) alleviates SA-β-Gal staining in GAC-KD cells (Fig. 3e-f), suggesting increased ROS could mediate GAC-KD induced senescence. In addition, UTR-APA in PRELID1 (another mitochondrion-located nuclear gene) can act as a regulator of mitochondrial ROS signal in cancer cells (Gillen et al. 2017). This conclusion aligns with ours to show that APA may be a neglected and emerging mechanism in regulating mitochondria function. ATP is the main energy source for cellular metabolism and cancer cell migration (Zanotelli et al. 2018). As GAC-KD resulted in less ATP production and senescence-associated phenotypes in cancer cell line A549, targeting GAC may be one potential anti-tumor therapy via inducing cancer cells to senescence, though warrant further study.

CPSF6 is a member of the CFIm family and usually functions in conjunction with two other members of the family, CPSF5 and CPSF7 (Shi and Manley 2015). Given knockdown of CPSF6 reduced GAC expression, we examined whether knockdown of CPSF5 and CPSF7 could also regulate GAC expression. Interestingly, we found CPSF5-KD reduced GAC expression while CPSF7-KD did not affect GAC expression (Fig. S17 a-c), consistent with a previous study which showed CPSF5 regulated GLS alternative exon definition and GAC was downregulated after knockdown of CPSF5 (Masamha et al. 2016). Noteworthy, CPSF6 showed decreased expression in multiple cellular senescence models (including that of human and mouse) (Fig. S12). However, no significant changes of CPSF5 was observed in MEF senescence (Fig. S18). These above results suggest that CPSF6 changes more prevalently than its other two family members and may contribute more in regulating GAC expression and senescence. Intriguingly, a recent study reported that CPSF6 insufficiency altered protein dosage and caused a developmental syndrome by shifting poly(A) site usage between the 3’ UTR and internal sites in a pathway-specfic manner (de Prisco et al. 2023). Of note, as a widely-used polyadenylation regulator, CPSF6 could probably affect senescence through multiple regulatory axis. Overexpressing GAC partially rescues senescence-associated phenotypes induced by CPSF6 knockdown (Fig. 6), further supporting the idea that GAC is only one of the contributors.

Note worthy, GAC and KGA both function as glutaminase 1, and have very similar (or the same) function to initiate glutamine catabolism by catalyzing the conversion of glutamine into glutamate and ammonia. Their difference is that GAC located in mitochondria and KGA located in both mitochondria and cytoplasm, and GAC is reported having higher catalytic activity than KGA (Cassago et al. 2012). Interestingly, Johmura et al. reported that KGA can be involved in senescent cell survival (Johmura et al. 2021). In the present study, we also validated that KGA overexpression could rescue CPSF6-KD induced senescence in A549 cells (Fig. S19), very similar to that GAC-overexpressed CPSF6-KD A549 cells. This result coincides with the finding by Johmura and colleagues and is consistent with the fact that KGA and GAC both are glutaminase and KGA's location in both mitochondria and cytoplasm.

In summary, our results illustrated that GAC, generated from the intronic polyadenylation of GLS, played important roles in mitochondrial metabolism and protecting cells against oxidative stress. The reduced intronic pA site usage led to less GAC protein production, which in turn causes higher ROS levels and lower ATP generation, and ultimately resulted in senescence-associated phenotypes. In the last two decades, many studies have proposed several possible mechanisms underlying cellular senescence or aging regulated by mitochondria dysfunction, including changes in gene expression, energy utilization, and global metabolic shift (Butler et al. 2010; Cristina et al. 2009; Falk et al. 2008). Here, we provide evidence that GAC, one variant of GLS, with the help of the polyadenylation factor CPSF6, is essential for the metabolism in mitochondria, whose dysregulation in turn led to senescence. Our study demonstrated for the first time that intronic APA in a mitochondrion-located nuclear gene is a new contributor to cellular senescence.

Conclusion

The present study uncovered that intronic polyadenylation of glutaminase-encoding gene GLS generated an isoform termed GAC (encoding glutaminase C), which mainly located in mitochondria and played regulatory roles in cellular senescence. Downregulation of GAC induced cellular senescence in both human and mouse cells. Increased ROS level and decreased ATP synthesis explained the senescence-associated phenotypes in GAC deficient cells. The polyadenylation factor CPSF6 bound to the last exon of GAC to regulate its expression. Overall, the present study demonstrated for the first time that intronic polyadenylation could regulate cellular senescence, largely extending our understanding of the biological roles of intronic polyadenylation.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (4.1MB, docx)

Acknowledgements

All the involved person have been listed as co-authors and no additional special acknowledgment.

Abbreviations

APA

Alternative polyadenylation

CR-APA

APA located at coding region

UTR-APA

APA located at 3' untranslated region (3' UTR)

RBP

RNA binding protein

GLS

Glutaminase

GAC

Glutaminase C

KGA

The full-length kidney GLS isoform

HA

Horseradish

ROS

Reactive oxygen species

FBS

Fetal bovine serum

HUVEC

Human umbilical vein endothelial cells

MEF

Mouse embryonic fibroblast

HFF

Human foreskin fibroblast

DMEM

Dulbecco's Modified Eagle Medium

PBS

Phosphate Buffered Saline

DHE

Dihydroethidium

NAC

N-acetyl cysteine

SA-β-Gal

Senescence-associated β-galactosidase

PD

Population doubling

RACE

Rapid amplification of cDNA ends

qRT-PCR

Quantitative reverse transcription followed by polymerase chain reaction

shRNA

Short hairpin RNA

Authors’ Contributions

TN and GW conceived the overall project and supervised its execution. XL, YL and DD designed and performed the experiments, BX, YC, ZZ, PY and SZ participated in some experiments. JY performed statistical and bioinformatics analyses. XL, DD, GW and TN wrote and revised the manuscript. FY provided human samples. All authors reviewed the results and approved the manuscript.

Funding

This work was financially supported by the National Natural Science Foundation of China (92249302, 32370592), the National Key Research and Development Program of China (2023YFC3603300, 2021YFA0909300) and the Natural Science Foundation Project of Shanghai (21ZR1407000).

Data Availability

The public RNA-seq datasets used in the present study can be accessed through the NCBI GEO number GSE63577 and GSE61130.

Declarations

Ethics Approval

Not applicable.

Consent to Participate

Not applicable.

Consent for Publication

Not applicable.

Competing interests

None declared.

Footnotes

Xueping Li and Yuxin Li contributed equally to this work.

Contributor Information

Ting Ni, Email: tingni@fudan.edu.cn.

Gang Wei, Email: gwei@fudan.edu.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (4.1MB, docx)

Data Availability Statement

The public RNA-seq datasets used in the present study can be accessed through the NCBI GEO number GSE63577 and GSE61130.

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