pre-mRNA Splicing Research Articles

Control of 3′ splice site selection by the yeast splicing factor Fyv6

Pre-mRNA splicing is catalyzed in two steps: 5ʹ splice site (SS) cleavage and exon ligation. A number of proteins transiently associate with spliceosomes to specifically impact these steps (first and second step factors). We recently identified Fyv6 (FAM192A in humans) as a second step factor in Saccharomyces cerevisiae; however, we did not determine how widespread Fyv6’s impact is on the transcriptome. To answer this question, we have used RNA sequencing (RNA-seq) to analyze changes in splicing. These results show that loss of Fyv6 results in activation of non-consensus, branch point (BP) proximal 3ʹ SS transcriptome-wide. To identify the molecular basis of these observations, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of a yeast product complex spliceosome containing Fyv6 at 2.3 Å. The structure reveals that Fyv6 is the only second step factor that contacts the Prp22 ATPase and that Fyv6 binding is mutually exclusive with that of the first step factor Yju2. We then use this structure to dissect Fyv6 functional domains and interpret results of a genetic screen for fyv6Δ suppressor mutations. The combined transcriptomic, structural, and genetic studies allow us to propose a model in which Yju2/Fyv6 exchange facilitates exon ligation and Fyv6 promotes usage of consensus, BP distal 3ʹ SS.

Targeting CA9 restricts pancreatic cancer progression through pH regulation and ROS production.

Lactate is a key metabolite produced by glycolytic metabolism, yet it also serves as an energy source for cancer cells. Lactate accumulation in the tumor microenvironment (TME) has been demonstrated to correlate with immunosuppressive TME and tumor progression. As a highly glycolytic tumor, it is crucial to decipher the underlying mechanism in pancreatic ductal adenocarcinoma (PDAC). Bioinformation analysis was used to identify lactate mediated carbonic anhydrase IX (CA9) upregulation. CCK-8, colony formation and mouse xenograft assay were utilized to study the effect of CA9 in PDAC. ECAR, OCR and pHi measurement confirmed the impacts of CA9 in Warburg phenotype. Using confocal microscopy, flow cytometry, qRT-PCR, co-IP, we validated the signaling pathways in PDAC to regulate reactive oxygen species (ROS) production. We confirmed that CA9 is highly expressed in PDAC and positively regulated by lactate levels. CA9 can enhance the proliferative and migratory capabilities of PDAC cells. Pharmacologic inhibition or knockdown of CA9 significantly reduce pHi, increase the intracellular lactate and reverse the Warburg phenotype. The intracellular lactate accumulation caused by CA9 knockdown upregulates ROS and mitochondrial dysfunction. Furthermore, it was discovered that the competitive binding of CA9 with FUS inhibits the facilitation of FUS on NOX4 pre-mRNA splicing. Collectively, our data illustrate that CA9 has a direct regulatory role in pHi homeostasis and ROS production, providing a potential therapeutic target for PDAC treatment.

The antisense CircRNA VvcircABH controls salt tolerance and the brassinosteroid signaling response by suppressing cognate mRNA splicing in grape.

Soil salinization is a major factor limiting the sustainable development of the grape industry. Circular RNAs (circRNAs) are more stable than linear mRNAs and are involved in stress responses. However, the biological functions and molecular mechanisms underlying antisense circRNAs in plants remain unclear. We identified the antisense circRNA VvcircABH through high-throughput sequencing. Using genetic transformation methods and molecular biological techniques, we analyzed the effects of VvcircABH on the response to salt stress and the mechanisms underlying its effects. VvcircABH was located in the nucleus and upregulated by salt stress, while the expression level of its cognate gene VvABH (alpha/beta-hydrolase) was downregulated. VvcircABH overexpression or VvABH silencing greatly enhanced grape salt tolerance. VvcircABH could bind to the overlapping region and inhibits VvABH pre-mRNA splicing, thereby decreasing the expression level of VvABH. Additionally, VvcircABH repressed the additive effect of VvABH on the interaction between VvBRI1 (brassinosteroid-insensitive 1) and VvBKI1 (BRI1 kinase inhibitor 1), thus influencing the plant's response to BR, which plays important roles in plant salt tolerance. We conclude that VvcircABH and VvABH play distinct roles in the salt tolerance and brassinosteroid signaling response, and VvcircABH could govern the expression of VvABH by inhibiting its splicing.

Effects of the therapeutic correction of U1 snRNP complex on Alzheimer’s disease

The U1 snRNP complex recognizes pre-mRNA splicing sites in the early stages of spliceosome assembly and suppresses premature cleavage and polyadenylation. Its dysfunction may precede Alzheimer’s disease (AD) hallmarks. Here we evaluated the effects of a synthetic single-stranded cDNA (APT20TTMG) that interacts with U1 snRNP, in iPSC-derived neurons from a donor diagnosed with AD and in the SAMP8 mouse model. APT20TTMG effectively binds to U1 snRNP, specifically decreasing TAU in AD neurons, without changing mitochondrial activity or glutamate. Treatment enhanced neuronal electrical activity, promoted an enrichment of differentially expressed genes related to key processes affected by AD. In SAMP8 mice, APT20TTMG reduced insoluble pTAU in the hippocampus, amyloid-beta and GFAP in the cortex, and U1-70 K in both brain regions, without cognitive changes. This study highlights the correction of the U1 snRNP complex as a new target for AD.

Sex-specific mRNA alternative splicing patterns and Dmrt1 isoforms contribute to sex determination and differentiation of oyster.

Alternative splicing (AS) of pre-mRNA is a crucial mechanism that regulates the expression of genes involved in sex determination and differentiation. Despite its importance, AS has been rarely characterized in molluscs. In this study, PacBio Iso-Seq was employed to obtain full-length transcriptome and unveil AS patterns of gonads in the Pacific oyster Crassostrea gigas. A total of 24,783 AS events were identified across 6259 genes, with many enriched in phosphorylation-related processes. Splicing factors were found to drive a high frequency of AS events in gonads. Significant sex-based differences in isoform abundance and the incidence of AS events were observed. Comparative analysis of mature female and male gonads revealed a subset of overlapping differential alternative splicing genes and differentially expressed genes enriched in processes related to microtubule function and cell motility. In addition, the expression levels of sex-biased genes were found correlated with their isoform number in both female and male gonads. A novel isoform of Dmrt1 was identified with male specific expression in mature gonads. This study provides the first comprehensive understanding of full-length transcriptome and AS patterns in molluscan gonads, shedding light on the post-transcriptional regulatory mechanisms underlying sex determination and differentiation in molluscs and potentially across other animals.

Correction of aberrant splicing of ELP1 pre-mRNA by kinetin derivatives – a structure activity relationship study

Correction of aberrant splicing of ELP1 pre-mRNA by kinetin derivatives – a structure activity relationship study

Investigation of RNA-binding protein NOVA1 in silico: Comparison of the modern human V197 with the archaic I197 variant present in Neanderthals

By comparing the high-coverage archaic genome sequences to those of modern humans, specific genetic differences have been identified. For example, a human-specific substitution has been found in neuro-oncological ventral antigen 1 (NOVA1) – an RNA-binding protein that regulates the alternative splicing of neuronal pre-mRNA. The amino acid substitution results in an isoleucine-to-valine change at position 197 in NOVA1 (archaic: I197, modern human: V197). Previous studies have utilised gene editing technology to compare the archaic and modern human forms of NOVA1 in cortical organoids, however, the structural and molecular details require further investigation. Using an in silico approach, the modern human (WT) and archaic (V197I) structures of NOVA1 were generated. Moreover, the structure of NOVA1 containing a glycine-to-valine substitution at position 68 (G68V), which occurs at the RNA-binding interface, was examined for comparison. Protein-RNA docking was subsequently performed to model the interaction of NOVA1 variants with RNA and the complexes were evaluated further using classical molecular dynamics (MD) simulations. Based on the MM-PBSA analysis, the binding free energies were similar between the WT (−956.8 ± 32.6 kcal/mol), V197I (−975.4 ± 65.6 kcal/mol), and G68V (−946.7 ± 34.3 kcal/mol) complexes. The findings highlight the binding and stability of protein-RNA complexes with only modest structural changes observed in the archaic and G68V variants compared to the WT NOVA1 protein. Further clarification is required to enhance our understanding of the impact of NOVA1 mutations on alternative splicing and disease development. In particular, delineating the effect of multiple mutations in the NOVA1 gene is of importance.

S-allylmercaptocysteine inhibits TLR4-mediated inflammation through enhanced formation of inhibitory MyD88 splice variant in mammary epithelial cells

Mastitis is an inflammatory disease affecting mammary tissues caused by bacterial infection that negatively affects milk quality and quantity. S-Allylmercaptocysteine (SAMC), a sulfur compound in aged garlic extract (AGE), suppresses lipopolysaccharide (LPS)-induced inflammation in mouse models and cell cultures. However, the mechanisms underlying this anti-inflammatory effect remain unclear. In this study, we demonstrated that oral administration of AGE suppressed the LPS-induced immune response in a mastitis mouse model and that SAMC inhibited LPS-induced interleukin-6 production and nuclear factor κB p65 subunit activation in HC11 mammary epithelial cells. Global phosphoproteomic analysis revealed that SAMC treatment downregulated 910 of the 1,304 phosphorylation sites upregulated by LPS stimulation in mammary cells, including those associated with toll-like receptor 4 (TLR4) signaling. Additionally, SAMC decreased the phosphorylation of 26 proteins involved in pre-mRNA splicing, particularly the U2 small nuclear ribonucleoprotein complex. Furthermore, we found that SAMC increased the production of the myeloid differentiation factor 88 short form (MyD88-S), an alternatively spliced form of MyD88 that negatively regulates TLR4 signaling. These findings suggest that SAMC inhibits TLR4-mediated inflammation via alternative pre-mRNA splicing, thus promoting MyD88-S production in mammary epithelial cells. Therefore, SAMC may alleviate various inflammatory diseases, such as mastitis, by modulating immune responses.

A novel pathogenic DICER1 gene variant is associated with hereditary multinodular goiter in an Argentine family as evidenced by clinical, biochemical and molecular genetic analysis.

DICER1 syndrome is an autosomal-dominant disorder that results in malignant or benign tumors. A number of distinct pathogenic germline and somatic variants have been identified as causing multinodular goiter (MNG). The purpose of the present study was to identify and characterize the genetic cause underlying the familial form of MNG through a whole-exome sequencing (WES) analysis in an Argentine family with three affected siblings. Clinical, biochemical and molecular genetics as well as bioinformatics analysis were performed. A novel heterozygous variant in the DICER1 gene was identified in the proband patient by WES. The variant was a single guanine deletion at nucleotide position 2,042 (NM_177438.3:c.2042del) resulting in a frameshift at amino acid 681 with a putative premature stop codon [NP_803187.1:p.Gly681ValfsTer4]. Family segregation analysis showed that his affected sister and his affected brother also were heterozygous for same variant, whereas the father was a healthy heterozygous carrier of the variant and the healthy mother harbor only wild-type alleles in the DICER1 gene. We have also observed that the frameshift variant does not interfere with the pre-mRNA splicing of the exon 13. In addition, two clinically relevant heterozygous variants, not associated with thyroid disease, were also identified in index sibling using the Franklin platform, a frameshift [NP_000234.1:p.Thr55AsnfsTer49] in the MEFV gene (familial mediterranean fever) and a missense [NP_004530.1:p.Ala422Thr] in the NARS1 gene (neurodevelopmental delay and ataxia). In conclusion, in the present study we have identified a novel frameshift variant corresponding to NP_803187.1:p.Gly681ValfsTer4 in the DUF 283 domain of DICER1. The results were in accordance with previous observations confirming the genetic heterogeneity of DICER1 syndrome. Moreover, the identification of this variant in the unaffected father substantiates the hypothesis of incomplete/reduced penetrance.

Structure-Activity Relationship Study of Splicing Modulators on Hsh155/SF3B1 through Chemical Synthesis and Yeast Genetics.

Meayamycins are synthetic analogs of the natural product FR901464 and exhibit potent anticancer activity against human cancers. They bind SF3B1 and PHF5A, components of the human spliceosome, and they alter pre-mRNA splicing. Detailed analysis of the active site led us to investigate a narrow pocket within the binding site that surrounds the α,β-unsaturated amide portion of meayamycin. We describe the synthesis and biological activity of two new analogs bearing a methyl substituent on the α or β position of the amide. With these analogs, we investigated the discrete interactions within the narrow region of SF3B1 using a human/yeast chimeric SF3B1 protein and found that the V1078 residue of SF3B1 affects compound binding at the amide moiety.

RNPS1 in PSAP complex controls periodic pre-mRNA splicing over the cell cycle

RNPS1 in PSAP complex controls periodic pre-mRNA splicing over the cell cycle

RNA binding protein CUGBP2/ETR-3 regulates STAT3 alternative splicing

Signal transducer and activator of transcription 3 (STAT3) is a multifactorial regulator involved in many biological responses. Alternative splicing of STAT3 pre-mRNA leads to an internal 50-nucleotide deletion of exon 23 selecting an alternative 3’ acceptor site, resulting in the generation of two splicing isoforms, STAT3α and STAT3β. STAT3β lacks 55 amino acid-residue transactivation domain at the C-terminal of STAT3α replacing seven unique amino acids. Although STAT3β was originally thought to be a dominant negative isoform of STAT3α, accumulating evidence have shown that STAT3β possesses both its unique functions and those that overlap with STAT3α in fundamental cellular processes. However, much remains unknown about STAT3 pre-mRNA alternative splicing in determining the balance between STAT3 isoforms. In this study, we identified cis-regulatory elements and CUGBP2/ETR-3 as a novel trans-acting factor that regulates STAT3 alternative splicing. Our findings demonstrate that STAT3 splicing can be modulated by CUGBP2 via association with UG-rich elements of intron 22, providing a novel insight into the mechanism of STAT3 alternative splicing. CUGBP2 would be a crucial molecule regulating the balance of STAT3 isoform expression, thus targeting CUGBP2 and its recognition sequences in intron 22 of STAT3 might impact on various biological processes regulated by STAT3 signaling pathway.

LncRNA MALAT1 promotes Erastin-induced ferroptosis in the HBV-infected diffuse large B-cell lymphoma

In a retrospective analysis of clinical data from 587 DLBCL (diffuse large B-cell lymphoma) patients in China, 13.8% of cases were associated with HBV (hepatitis B virus) infection, leading to distinct clinical features and poorer prognosis. Moreover, HBV infection has a more pronounced impact on the survival of the GCB (germinal center B-cell-like) type DLBCL patients compared to the ABC (activated B-cell-like) type. In this study, we found that the expression of LncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was downregulated in the HBV-infected GCB-type DLBCL patients, and the HBV core protein (HBX) directly inhibited the MALAT1 expression in DLBCL cells. Notably, the overexpression of HBX could attenuate the Erastin-induced ferroptosis in the GCB-type DLBCLs, while MALAT1 re-expression restored sensitivity in the HBX-overexpressing DLBCLs in vitro and in vivo. Mechanistically, MALAT1 competitively hindered SFPQ (splicing factor proline and glutamine-rich) from effectively splicing the pre-mRNA of SLC7A11 (solute carrier family 7 member 11), due to a shared TTGGTCT motif, which impeded the SLC7A11 pre-mRNA maturation and hence diminished its negative regulation on ferroptosis. Together, our study identified HBX’s role in inhibiting MALAT1 expression, promoting SFPQ-mediated splicing of SLC7A11 pre-mRNA, and reducing the GCB-type DLBCL sensitivity to Erastin-induced ferroptosis. Combined with the recent studies that ferroptosis may be involved in the occurrence and development of DLBCL, these findings explain our clinical data analysis that DLBCL patients with low expression of MALAT1 have poorer prognosis and shorter overall survival, and provide a valuable therapeutic target for the HBV-infected GCB-type DLBCL patients.

AtDGCR14L contributes to salt-stress tolerance via regulating pre-mRNA splicing in Arabidopsis.

In plants, pre-mRNA alternative splicing has been demonstrated to be a crucial tier that regulates gene expression in response to salt stress. However, the underlying mechanisms remain elusive. Here, we studied the roles of DIGEORGE-SYNDROME CRITICAL REGION 14-like (AtDGCR14L) in regulating pre-mRNA splicing and salt stress tolerance. We discovered that Arabidopsis AtDGCR14L is required for maintaining plant salt stress tolerance and the constitutively spliced and active isoforms of important stress- and/or abscisic acid (ABA)-responsive genes. We also identified the interaction between AtDGCR14L and splicing factor U1-70k, which needs a highly conserved three amino acid (TWG) motif in DGCR14. Different from wild-type AtDGCR14L, the overexpression of the TWG-substituted AtDGCR14L mutant did not change salt stress tolerance or pre-mRNA splicing of stress/ABA-responsive genes. Additionally, SWITCH3A (SWI3A) is a core subunit of the SWI/SUCROSE NONFERMENTING (SWI/SNF) chromatin-remodeling complexes. We found that SWI3A, whose splicing depends on AtDGCR14L, actively enhances salt stress tolerance. These results revealed that AtDGCR14L may play an essential role in crosstalk between plant salt-stress response and pre-mRNA splicing mechanisms. We also unveiled the potential role of SWI3A in controlling salt stress tolerance. The TWG motif in the intrinsically disordered region of AtDGCR14L is highly conserved and crucial for DGCR14 functions.

The Intricate Functional Networks of Pre-mRNA Alternative Splicing in Mammalian Spermatogenesis.

Spermatogenesis is a highly coordinated process that requires the precise expression of specific subsets of genes in different types of germ cells, controlled both temporally and spatially. Among these genes, those that can exert an indispensable influence in spermatogenesis via participating in alternative splicing make up the overwhelming majority. mRNA alternative-splicing (AS) events can generate various isoforms with distinct functions from a single DNA sequence, based on specific AS codes. In addition to enhancing the finite diversity of the genome, AS can also regulate the transcription and translation of certain genes by directly binding to their cis-elements or by recruiting trans-elements that interact with consensus motifs. The testis, being one of the most complex tissue transcriptomes, undergoes unparalleled transcriptional and translational activity, supporting the dramatic and dynamic transitions that occur during spermatogenesis. Consequently, AS plays a vital role in producing an extensive array of transcripts and coordinating significant changes throughout this process. In this review, we summarize the intricate functional network of alternative splicing in spermatogenesis based on the integration of current research findings.

Bichromatic exon-reporters reveal voltage-gated Ca 2+ -channel splice-isoform diversity across Drosophila neurons in vivo.

Every neuron contains the same genomic information but its complement of proteins is the product of countless neuron-specific steps including pre-mRNA splicing. Despite advances in RNA sequencing techniques, pre-mRNA splicing biases that favor one isoform over another are largely inscrutable in live neurons in situ . Here, in Drosophila , we developed bichromatic fluorescent reporters to investigate alternate splicing of cacophony - a gene that codes the pore-forming α 1 -subunit of the primary neuronal voltage-gated Ca 2+ channel (VGCC). These reporters reveal a neuron-specific pattern of exon biases, including stereotypical differences between neurons of the same neurotransmitter type and ostensibly the same function. Information about exon splicing biases of individual neurons in vivo provides clues to the role of VGCC motifs and the role of those neurons in the context of local circuits. The application of this technology to a large gene such as cacophony provides a precedence for effective exon-reporter design for other Drosophila genes.

Nuclear-Cytoplasmic Shuttling of the Usher Syndrome 1G Protein SANS Differs from Its Paralog ANKS4B.

The USH1G protein SANS is a small multifunctional scaffold protein. It is involved in several different cellular processes, such as intracellular transport, in the cytoplasm, or splicing of pre-mRNA, in the cell nucleus. Here, we aimed to gain insight into the regulation of the subcellular localization and the nuclear-cytoplasmic shuttling of SANS and its paralog ANKS4B, not yet reported in the nucleus. We identified karyopherins mediating the nuclear import and export by screening the nuclear interactome of SANS. Sequence analyses predicted in silico evolutionarily conserved nuclear localization sequences (NLSs) and nuclear export sequences (NESs) in SANS, but only NESs in ANKS4B, which are suitable for karyopherin binding. Quantifying the nuclear-cytoplasmic localization of wild-type SANS and NLS/NES mutants, we experimentally confirmed in silico predicted NLS and NES functioning in the nuclear-cytoplasmic shuttling in situ in cells. The comparison of SANS and its paralog ANKS4B revealed substantial differences in the interaction with the nuclear splicing protein PRPF31 and in their nuclear localization. Finally, our results on pathogenic USH1G/SANS mutants suggest that the loss of NLSs and NESs and thereby the ability to control nuclear-cytoplasmic shuttling is disease-relevant.

Homozygous synonymous FAM111A variant underlies an autosomal recessive form of Kenny-Caffey syndrome.

FAM111A (family with sequence similarity 111 member A) is a serine protease and removes covalent DNA-protein cross-links during DNA replication. Heterozygous gain-of-function variants in FAM111A cause skeletal dysplasias, such as the perinatal lethal osteocraniostenosis and the milder Kenny-Caffey syndrome (KCS). We report two siblings born to consanguineous parents with dysmorphic craniofacial features, postnatal growth retardation, ophthalmologic manifestations, hair and nail anomalies, and skeletal abnormalities such as thickened cortex and stenosis of the medullary cavity of the long bones suggestive of KCS. Using exome sequencing, a homozygous synonymous FAM111A variant, NM_001312909.2:c.81 G > A; p.Pro27=, that affects the last base of the exon and is predicted to alter FAM111A pre-mRNA splicing, was identified in both siblings. We identified aberrantly spliced FAM111A transcripts, reduced FAM111A mRNA levels, and near-complete absence of FAM111A protein in fibroblasts of both patients. After treatment of patient and control fibroblasts with different concentrations of camptothecin that induces covalent DNA-protein cross-links, we observed a tendency towards a reduced proportion of metabolically active cells in patient compared to control fibroblasts. However, under these culture conditions, we did not find consistent and statistically significant differences in cell cycle progression and apoptotic cell death between patient and control cells. Our findings show that FAM111A deficiency underlies an autosomal recessive form of FAM111A-related KCS. Based on our results and published data, we hypothesize that loss of FAM111A and FAM111A protease hyperactivity, as observed for gain-of-function patient-variant proteins, may converge on a similar pathomechanism underlying skeletal dysplasias.

Nuclear translocation of SIRT4 mediates deacetylation of U2AF2 to modulate renal fibrosis through alternative splicing-mediated upregulation of CCN2.

TGF-β stimulates CCN2 expression which in turn amplifies TGF-β signaling. This process promotes extracellular matrix production and accelerates the pathological progression of fibrotic diseases. Alternative splicing plays an important role in multiple disease development, while U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an essential factor in the early steps of pre-mRNA splicing. However, the molecular mechanism underlying abnormal CCN2 expression upon TGF-β stimulation remains unclear. This study elucidates that SIRT4 acts as a master regulator for CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing. Analyses of renal biopsy specimens from patients with CKD and mouse fibrotic kidney tissues revealed marked nuclear accumulation of SIRT4. The tubulointerstitial fibrosis was alleviated by global deletion or tubular epithelial cell (TEC)-specific knockout of Sirt4, and aggravated by adeno-associated virus-mediated SIRT4 overexpression in TECs. Furthermore, SIRT4 was found to translocate from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, which further promoted its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Importantly, exosomes containing anti-SIRT4 antibodies were found to effectively mitigate the UUO-induced kidney fibrosis in mice. Collectively, these findings indicated that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via the pre-mRNA splicing.

Nuclear translocation of SIRT4 mediates deacetylation of U2AF2 to modulate renal fibrosis through alternative splicing-mediated upregulation of CCN2

TGF-β stimulates CCN2 expression which in turn amplifies TGF-β signaling. This process promotes extracellular matrix production and accelerates the pathological progression of fibrotic diseases. Alternative splicing plays an important role in multiple disease development, while U2 small nuclear RNA auxiliary factor 2 (U2AF2) is an essential factor in the early steps of pre-mRNA splicing. However, the molecular mechanism underlying abnormal CCN2 expression upon TGF-β stimulation remains unclear. This study elucidates that SIRT4 acts as a master regulator for CCN2 expression in response to TGF-β by modulating U2AF2-mediated alternative splicing. Analyses of renal biopsy specimens from patients with CKD and mouse fibrotic kidney tissues revealed marked nuclear accumulation of SIRT4. The tubulointerstitial fibrosis was alleviated by global deletion or tubular epithelial cell (TEC)-specific knockout of Sirt4, and aggravated by adeno-associated virus-mediated SIRT4 overexpression in TECs. Furthermore, SIRT4 was found to translocate from the mitochondria to the cytoplasm through the BAX/BAK pore under TGF-β stimulation. In the cytoplasm, TGF-β activated the ERK pathway and induced the phosphorylation of SIRT4 at Ser36, which further promoted its interaction with importin α1 and subsequent nuclear translocation. In the nucleus, SIRT4 was found to deacetylate U2AF2 at K413, facilitating the splicing of CCN2 pre-mRNA to promote CCN2 protein expression. Importantly, exosomes containing anti-SIRT4 antibodies were found to effectively mitigate the UUO-induced kidney fibrosis in mice. Collectively, these findings indicated that SIRT4 plays a role in kidney fibrosis by regulating CCN2 expression via the pre-mRNA splicing.