RS Domain Research Articles

Involvement of the splicing factor SART1 in the BRCA1-dependent homologous recombination repair of DNA double-strand breaks

Although previous studies have reported that pre-mRNA splicing factors (SFs) are involved in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR), their exact role in promoting HR remains poorly understood. Here, we showed that SART1, an SF upregulated in several types of cancer, promotes DSB end resection, an essential first step of HR. The resection-promoting function of SART1 requires phosphorylation at threonine 430 and 695 by ATM/ATR. SART1 is recruited to DSB sites in a manner dependent on transcription and its RS domain. SART1 is epistatic with BRCA1, a major HR factor, in the promotion of resection, especially transcription-associated resection in the G2 phase. SART1 and BRCA1 accumulate at DSB sites in an interdependent manner, and epistatically counteract the resection blockade posed by 53BP1 and RIF1. Furthermore, chromosome analysis demonstrated that SART1 and BRCA1 epistatically suppressed genomic alterations caused by DSB misrepair in the G2 phase. Collectively, these results indicate that SART1 and BRCA1 cooperatively facilitate resection of DSBs arising in transcriptionally active genomic regions in the G2 phase, thereby promoting faithful repair by HR, and suppressing genome instability.

Design of a covalent protein-protein interaction inhibitor of SRPKs to suppress angiogenesis and invasion of cancer cells

Serine–arginine (SR) proteins are splicing factors that play essential roles in both constitutive and alternative pre-mRNA splicing. Phosphorylation of their C-terminal RS domains by SR protein kinases (SRPKs) regulates their localization and diverse cellular activities. Dysregulation of phosphorylation has been implicated in many human diseases, including cancers. Here, we report the development of a covalent protein–protein interaction inhibitor, C-DBS, that targets a lysine residue within the SRPK-specific docking groove to block the interaction and phosphorylation of the prototypic SR protein SRSF1. C-DBS exhibits high specificity and conjugation efficiency both in vitro and in cellulo. This self-cell-penetrating inhibitor attenuates the phosphorylation of endogenous SR proteins and subsequently inhibits the angiogenesis, migration, and invasion of cancer cells. These findings provide a new foundation for the development of covalent SRPK inhibitors for combatting diseases such as cancer and viral infections and overcoming the resistance encountered by ATP-competitive inhibitors.

SRSF1 interactome determined by proximity labeling reveals direct interaction with spliceosomal RNA helicase DDX23

SRSF1 is the founding member of the SR protein family. It is required-interchangeably with other SR proteins-for pre-mRNA splicing in vitro, and it regulates various alternative splicing events. Dysregulation of SRSF1 expression contributes to cancer and other pathologies. Here, we characterized SRSF1's interactome using proximity labeling and mass spectrometry. This approach yielded 190 proteins enriched in the SRSF1 samples, independently of the N- or C-terminal location of the biotin-labeling domain. The detected proteins reflect established functions of SRSF1 in pre-mRNA splicing and reveal additional connections to spliceosome proteins, in addition to other recently identified functions. We validated a robust interaction with the spliceosomal RNA helicase DDX23/PRP28 using bimolecular fluorescence complementation and in vitro binding assays. The interaction is mediated by the N-terminal RS-like domain of DDX23 and both RRM1 and the RS domain of SRSF1. During pre-mRNA splicing, DDX23's ATPase activity is essential for the pre-B to B spliceosome complex transition and for release of U1 snRNP from the 5' splice site. We show that the RS-like region of DDX23's N-terminal domain is important for spliceosome incorporation, while larger deletions in this domain alter subnuclear localization. We discuss how the identified interaction of DDX23 with SRSF1 and other SR proteins may be involved in the regulation of these processes.

Generation of an RBM20-mutation-associated left-ventricular non-compaction cardiomyopathy iPSC line (UMGi255-A) into a DCM genetic background to investigate monogenetic cardiomyopathies

RBM20 mutations account for 3 % of genetic cardiomypathies and manifest with high penetrance and arrhythmogenic effects. Numerous mutations in the conserved RS domain have been described as causing dilated cardiomyopathy (DCM), whereas a particular mutation (p.R634L) drives development of a different cardiac phenotype: left-ventricular non-compaction cardiomyopathy. We generated a mutation-induced pluripotent stem cell (iPSC) line in which the RBM20-LVNC mutation p.R634L was introduced into a DCM patient line with rescued RBM20-p.R634W mutation. These DCM-634L-iPSC can be differentiated into functional cardiomyocytes to test whether this RBM20 mutation induces development of the LVNC phenotype within the genetic context of a DCM patient.

The master energy homeostasis regulator PGC-1α exhibits an mRNA nuclear export function

PGC-1α plays a central role in maintaining mitochondrial and energy metabolism homeostasis, linking external stimuli to transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and an RNA recognition motif, however the RNA-processing function(s) were poorly investigated over the past 20 years. Here, we show that the RS domain of human PGC-1α directly interacts with RNA and the nuclear RNA export receptor NXF1. Inducible depletion of PGC-1α and expression of RNAi-resistant RS-deleted PGC-1α further demonstrate that its RNA/NXF1-binding activity is required for the nuclear export of some canonical mitochondrial-related mRNAs and mitochondrial homeostasis. Genome-wide investigations reveal that the nuclear export function is not strictly linked to promoter-binding, identifying in turn novel regulatory targets of PGC-1α in non-homologous end-joining and nucleocytoplasmic transport. These findings provide new directions to further elucidate the roles of PGC-1α in gene expression, metabolic disorders, aging and neurodegeneration.

Molecular basis of RNA-binding and autoregulation by the cancer-associated splicing factor RBM39

Pharmacologic depletion of RNA-binding motif 39 (RBM39) using aryl sulfonamides represents a promising anti-cancer therapy but requires high levels of the adaptor protein DCAF15. Consequently, novel approaches to deplete RBM39 in an DCAF15-independent manner are required. Here, we uncover that RBM39 autoregulates via the inclusion of a poison exon into its own pre-mRNA and identify the cis-acting elements that govern this regulation. We also determine the NMR solution structures of RBM39’s tandem RNA recognition motifs (RRM1 and RRM2) bound to their respective RNA targets, revealing how RRM1 recognises RNA stem loops whereas RRM2 binds specifically to single-stranded N(G/U)NUUUG. Our results support a model where RRM2 selects the 3’-splice site of a poison exon and the RRM3 and RS domain stabilise the U2 snRNP at the branchpoint. Our work provides molecular insights into RBM39-dependent 3’-splice site selection and constitutes a solid basis to design alternative anti-cancer therapies.

Abstract P2041: Identification Of The Nuclear Localization Signal In RBM20 And Its Function In The Pathogenesis Of Dilated Cardiomyopathy

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop an aggressive and arrhythmogenic form of dilated cardiomyopathy (DCM). Five of over 900 genetic mutations in RBM20 have recently been validated in animal models. Four of these five mutations are located in the arginine/serine-rich (RS) domain with the fifth in the RNA recognition motif (RRM). Interestingly, only mutations in the RS domain cause severe DCM, which suggests that disruption of RS domain function is crucial for the pathogenesis of DCM. To test this hypothesis, we generated mice expressing RBM20 with an in-frame deletion of the RS domain ( Rbm20 ΔRS ). We show that Rbm20 ΔRS mice manifest DCM with RBM20 mis-localization and granule formation, similar to that in RS domain mutation knock-in (KI) animals. Conversely, mice expressing RBM20 lacking the RRM do not exhibit RBM20 nucleocytoplasmic transport or granule formation, and, similar to RRM mutation KI mice, do not develop DCM. These data suggest that the critical nuclear localization signal (NLS) in RBM20 is located within the RS domain and that disruption of this sequence leads to RBM20 mis-localization and granule formation. In vitro experiments employing sequence deletion plasmids and immunocytochemical staining revealed that the first nine amino acids in the RS domain constitute the core NLS in RBM20, which is further supported by the fact that all three validated DCM-causing mutations are located in this segment. Analysis of plasmids containing DCM-associated mutations in other regions of RBM20, as well as in the non-NLS RS domain sequence, further confirmed that only mutations in the NLS facilitate re-localization and granule formation. Phosphorylation of residues within the NLS has been shown play an important role in protein nucleocytoplasmic transport. To test this, we mutated all phosphorylatable serine residues in the NLS to unphosphorylatable alanine or phosphomimetic aspartate and provide evidence that phosphorylation is dispensable for nucleocytoplasmic transport. Collectively, our findings identify the critical NLS in RBM20 and demonstrate that mutations in this NLS cause severe DCM through disruption of RS domain-mediated nuclear localization and sarcoplasmic granule formation.

Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knock-in (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We show that Rbm20ΔRS mice manifest DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 is mis-localized to the sarcoplasm in Rbm20ΔRS mice, which led to the formation of RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif (RRM) show similar mis-splicing of RBM20 target genes, but do not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrate that only DCM-associated mutations in the RS domain facilitate RBM20 nucleocytoplasmic transport and promote granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain. Mutation analysis of phosphorylation sites in the RS domain indicate that this modification is dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain-mediated nuclear localization is crucial for severe DCM caused by NLS mutations.

Plant serine/arginine-rich proteins: versatile players in RNA processing.

Serine/arginine-rich (SR) proteins participate in RNA processing by interacting with precursor mRNAs or other splicing factors to maintain plant growth and stress responses. Alternative splicing is an important mechanism involved in mRNA processing and regulation of gene expression at the posttranscriptional level, which is the main reason for the diversity of genes and proteins. The process of alternative splicing requires the participation of many specific splicing factors. The SR protein family is a splicing factor in eukaryotes. The vast majority of SR proteins' existence is an essential survival factor. Through its RS domain and other unique domains, SR proteins can interact with specific sequences of precursor mRNA or other splicing factors and cooperate to complete the correct selection of splicing sites or promote the formation of spliceosomes. They play essential roles in the composition and alternative splicing of precursor mRNAs, providing pivotal functions to maintain growth and stress responses in animals and plants. Although SR proteins have been identified in plants for three decades, their evolutionary trajectory, molecular function, and regulatory network remain largely unknown compared to their animal counterparts. This article reviews the current understanding of this gene family in eukaryotes and proposes potential key research priorities for future functional studies.

Peptides that Mimic RS repeats modulate phase separation of SRSF1, revealing a reliance on combined stacking and electrostatic interactions.

Phase separation plays crucial roles in both sustaining cellular function and perpetuating disease states. Despite extensive studies, our understanding of this process is hindered by low solubility of phase-separating proteins. One example of this is found in SR and SR-related proteins. These proteins are characterized by domains rich in arginine and serine (RS domains), which are essential to alternative splicing and in vivo phase separation. However, they are also responsible for a low solubility that has made these proteins difficult to study for decades. Here, we solubilize the founding member of the SR family, SRSF1, by introducing a peptide mimicking RS repeats as a co-solute. We find that this RS-mimic peptide forms interactions similar to those of the protein's RS domain. Both interact with a combination of surface-exposed aromatic residues and acidic residues on SRSF1's RNA Recognition Motifs (RRMs) through electrostatic and cation-pi interactions. Analysis of RRM domains from human SR proteins indicates that these sites are conserved across the protein family. In addition to opening an avenue to previously unavailable proteins, our work provides insight into how SR proteins phase separate and participate in nuclear speckles.

Sgh1, an SR-like Protein, Is Involved in Fungal Development, Plant Infection, and Pre-mRNA Processing in Fusarium graminearum.

Serine/arginine (SR) proteins are essential pre-mRNA splicing factors in eukaryotic organisms. Our previous studies have shownthat the unique SR-specific protein kinase Srk1 is important for RNA splicing and gene transcription in Fusarium graminearum, and interacts with two SR proteins, FgSrp1 and FgSrp2. In this study, we have identified an SR-like protein called Sgh1 in F. graminearum, which is orthologous to budding yeast paralogous Gbp2 and Hrb1. Our data have shownthat the Sgh1 is involved in vegetative growth, conidiation, sexual reproduction, DON synthesis, and plant infection. Moreover, the Sgh1 is mainly localized to the nucleus. RNA-seq analysis has shownthat the expression of over 1100 genes and the splicing efficiency in over 300 introns were affected in the Δsgh1 mutant. Although the RS domain and all three of the RRM domains are important for the Sgh1 functions, only the RS domain is responsible for its nuclear localization. Finally, we verified that the Sgh1 interacts with the unique SR-specific kinase Srk1 in F. graminearum by the yeast-two hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Taken together, our results have revealed that the Sgh1 regulates the fungal development, plant infection, and the pre-mRNA processing, and the RS domain regulates the function of the Sgh1 by modulating its nucleocytoplasmic shuttling.

I536T variant of RBM20 affects splicing of cardiac structural proteins that are causative for developing dilated cardiomyopathy

RBM20 is one of the genes predisposing to dilated cardiomyopathy (DCM). Variants in the RS domain have been reported in many DCM patients, but the pathogenicity of variants within the RNA-recognition motif remains unknown. Two human patients with the I536T-RBM20 variant without an apparent DCM phenotype were identified in sudden death cohorts. A splicing reporter assay was performed, and an I538T knock-in mouse model (Rbm20I538T) was generated to determine the significance of this variant. The reporter assay demonstrated that the human I536T variant affected the TTN splicing pattern compared to wild-type. In the mouse experiments, Rbm20I538T mice showed different splicing patterns in Ttn, Ldb3, Camk2d, and Ryr2. The expressions of Casq1, Mybpc2, and Myot were upregulated in Rbm20I538T mice, but Rbm20I538T mice showed neither DCM nor cardiac dysfunction on histopathological examination and ultrasound echocardiography. The I536T-RBM20 (I538T-Rbm20) variant changes gene splicing and affects gene expression, but the splicing and expression changes in Ttn and Ca handling genes such as Casq1, Camk2d, and Ryr2 do not cause DCM morphology in the mouse model.Key messages• Two human patients with the I536T-RBM20 variant without a DCM phenotype were identified.• A splicing reporter assay demonstrated that the variant affected the TTN splicing.• Rbm20I538T mice showed neither DCM nor cardiac dysfunction.• Rbm20I538T mice showed different splicing patterns and the gene expressions.

SR Splicing Factors Promote Cancer via Multiple Regulatory Mechanisms.

Substantial emerging evidence supports that dysregulated RNA metabolism is associated with tumor initiation and development. Serine/Arginine-Rich proteins (SR) are a number of ultraconserved and structurally related proteins that contain a characteristic RS domain rich in arginine and serine residues. SR proteins perform a critical role in spliceosome assembling and conformational transformation, contributing to precise alternative RNA splicing. Moreover, SR proteins have been reported to participate in multiple other RNA-processing-related mechanisms than RNA splicing, such as genome stability, RNA export, and translation. The dysregulation of SR proteins has been reported to contribute to tumorigenesis through multiple mechanisms. Here we reviewed the different biological roles of SR proteins and strategies for functional rectification of SR proteins that may serve as potential therapeutic approaches for cancer.

SR Protein Kinases Regulate the Splicing of Cardiomyopathy-Relevant Genes via Phosphorylation of the RSRSP Stretch in RBM20.

(1) Background: RNA binding motif 20 (RBM20) regulates mRNA splicing specifically in muscle tissues. Missense mutations in the arginine/serine (RS) domain of RBM20 lead to abnormal gene splicing and have been linked to severe dilated cardiomyopathy (DCM) in human patients and animal models. Interestingly, many of the reported DCM-linked missense mutations in RBM20 are in a highly conserved RSRSP stretch within the RS domain. Recently, it was found that the two Ser residues within this stretch are constitutively phosphorylated, yet the identity of the kinase(s) responsible for phosphorylating these residues, as well as the function of RSRSP phosphorylation, remains unknown. (2) Methods: The ability of three known SR protein kinases (SRPK1, CLK1, and AKT2) to phosphorylate the RBM20 RSRSP stretch and regulate target gene splicing was evaluated by using both in vitro and in vivo approaches. (3) Results: We found that all three kinases phosphorylated S638 and S640 in the RSRSP stretch and regulated RBM20 target gene splicing. While SRPK1 and CLK1 were both capable of directly phosphorylating the RS domain in RBM20, whether AKT2-mediated control of the RS domain phosphorylation is direct or indirect could not be determined. (4) Conclusions: Our results indicate that SR protein kinases regulate the splicing of a cardiomyopathy-relevant gene by modulating phosphorylation of the RSRSP stretch in RBM20. These findings suggest that SR protein kinases may be potential targets for the treatment of RBM20 cardiomyopathy.

Expansion and functional analysis of the SR-related protein family across the domains of life.

Serine/arginine-rich (SR) proteins comprise a family of proteins that is predominantly found in eukaryotes and plays a prominent role in RNA splicing. A characteristic feature of SR proteins is the presence of an S/R-rich low-complexity domain (RS domain), often in conjunction with spatially distinct RNA recognition motifs (RRMs). To date, 52 human proteins have been classified as SR or SR-related proteins. Here, using an unbiased series of composition criteria together with enrichment for known RNA binding activity, we identified >100 putative SR-related proteins in the human proteome. This method recovers known SR and SR-related proteins with high sensitivity (∼94%), yet identifies a number of additional proteins with many of the hallmark features of true SR-related proteins. Newly identified SR-related proteins display slightly different amino acid compositions yet similar levels of post-translational modification, suggesting that these new SR-related candidates are regulated in vivo and functionally important. Furthermore, candidate SR-related proteins with known RNA-binding activity (but not currently recognized as SR-related proteins) are nevertheless strongly associated with a variety of functions related to mRNA splicing and nuclear speckles. Finally, we applied our SR search method to all available reference proteomes, and provide maps of RS domains and Pfam annotations for all putative SR-related proteins as a resource. Together, these results expand the set of SR-related proteins in humans, and identify the most common functions associated with SR-related proteins across all domains of life.

RBM20 phosphorylation and its role in nucleocytoplasmic transport and cardiac pathogenesis.

Arginine–serine (RS) domain(s) in splicing factors are critical for protein–protein interaction in pre‐mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA‐binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho‐antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle‐down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein‐RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock‐in (KI) mouse model (Rbm20S637A) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)‐like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.

Abstract 13431: Gain-of-Function Mutations in RNA Binding Motif 20 Result in Cardiac Remodelling and Pathogenesis in Mice

Gain-of-function mutations of a gene result in new molecular function. Genetic studies show that mutations in a gene named as RNA binding motif 20 (RBM20) are associated with dilated cardiomyopathy (DCM). To test whether Rbm20 mutations are responsible for cardiac remodelling and potential underlying mechanisms, we selected two mutations (S637A and S639G) located in arginine/serine-rich domain of RBM20 and generated two knock-in mouse models ( Rbm20 S637A and Rbm20 S639G ) respectively. Both mouse lines were subjected to histological and anatomical analysis as well as cardiac function assessment with echocardiography. The results revealed that heterozygous (HT) and homozygous (HM) male and female mice from both lines displayed DCM phenotype with enlarged left ventricular (LV) chamber, thinner LV walls and reduced ejection fraction. Interestingly, we observed that Rbm20 S639G mice manifested more severe phenotype than that in Rbm20 S637A mice. Rbm20 S639G HM mice exhibited higher mortality rate (~50%) than that (~34%) in Rbm20 S637A HM mice at an early age (<100 days). Differentially expressed and spliced genes from RNA-seq data are involved in arrhythmia, cardiomyopathy, and sudden death based on Human Phenotype Ontology enrichment analysis (FDR≤0.05) in both lines. Immunofluorescent analysis in tissues and single cardiomyocytes respectively showed both mutations promote RBM20 nucleocytoplasmic trafficking and protein condensates, suggesting they are gain-of-function mutations, leading to high mortality rate at younger age. Both mutations also interrupted the alternative splicing of TTN and calcium handling genes. Force measurements and Ca 2+ transient evaluation of intact myocytes showed that the reduced cellular diastolic stiffness and impaired contractility in HT and HM mice. Studies suggested that phosphorylation of RS domain is critical for protein trafficking. We then performed mass spectrometry on the in vitro expressed RBM20 and identified phosphorylation on S637 and S639. Phosphomimetic assay showed that phosphorylation was not responsible for RBM20 trafficking and condensates. Together, we reported a new mechanism by which cardiac genetic mutations causes protein trafficking and aggregation in cardiac remodelling.

Abstract 14462: RBM20 Regulation In Healthy And Diseased Cardiomyocytes: A New Opportunity For Targeted Therapeutics

Introduction: Mutations in the splicing factor RBM20 cause a severe form of dilated cardiomyopathy (DCM) characterized by early onset, fast disease progression, and high risk of sudden death. We have previously shown that modulation of RBM20 expression is a promising strategy for patient with mutations in RBM20. However, our understanding of its endogenous regulation is limited. Hypothesis: DCM-causing mutations in RBM20 interfere with phosphorylation of RBM20 RS domain and with localization of the protein. We hypothesize that modulation of RBM20 phosphorylation could be used to normalize function in the presence of mutations that impair it. We sought to uncover the pathways responsible for such regulation and test their therapeutic potential. Methods: We developed a fluorescent in vitro reporter system that allows for the quantification of RBM20 expression and localization in human cardiomyocytes. We generated an induced Pluripotent Stem cells (iPSC) line in which the endogenous RBM20 is tagged at the C-term with an in-frame mCherry (RBM20-mCh). The tag leaves the regulatory regions at the 5’ end of the gene intact. RBM20-mCh hiPSC were differentiated into cardiomyocytes (iPSC-CMs) and matured for 4 weeks. We screened 2-month-old matured iPSC-CM on Matrigel-coated 384-well plates against a library of siRNA targeting all human kinases and phosphatases. We tested the hits in a contractility assay Results: We identified 35 kinases and 16 phosphatases that alter RBM20 expression and localization. Functional studies showed that the kinases that upregulated RBM20 expression increased cardiomyocyte contractility while downregulating kinases reduced contractility. Conclusions: In conclusion, understanding the kinase regulation of RBM20 provides insight into control of mRNA splicing of proteins such as Titin and may point to new targeted therapeutic strategies for RBM20 mutant DCM.

SAM homeostasis is regulated by CFIm-mediated splicing of MAT2A.

S-adenosylmethionine (SAM) is the methyl donor for nearly all cellular methylation events. Cells regulate intracellular SAM levels through intron detention of MAT2A, the only SAM synthetase expressed in most cells. The N6-adenosine methyltransferase METTL16 promotes splicing of the MAT2A detained intron by an unknown mechanism. Using an unbiased CRISPR knock-out screen, we identified CFIm25 (NUDT21) as a regulator of MAT2A intron detention and intracellular SAM levels. CFIm25 is a component of the cleavage factor Im (CFIm) complex that regulates poly(A) site selection, but we show it promotes MAT2A splicing independent of poly(A) site selection. CFIm25-mediated MAT2A splicing induction requires the RS domains of its binding partners, CFIm68 and CFIm59 as well as binding sites in the detained intron and 3´ UTR. These studies uncover mechanisms that regulate MAT2A intron detention and reveal a previously undescribed role for CFIm in splicing and SAM metabolism.

Cleaved PGAM5 dephosphorylates nuclear serine/arginine-rich proteins during mitophagy

PGAM5 is a protein phosphatase located in the inner mitochondrial membrane through its transmembrane (TM) domain and is cleaved within the TM domain upon mitochondrial dysfunction. We found previously that cleaved PGAM5 is released from mitochondria, following proteasome-mediated rupture of the outer mitochondrial membrane during mitophagy, a selective form of autophagy specific to mitochondria. Here, we examined the role of cleaved PGAM5 outside mitochondria. Deletion mutants that mimic cleaved PGAM5 existed not only in the cytosol but also in the nucleus, and a fraction of cleaved PGAM5 translocated to the nucleus during mitophagy induced by the uncoupler CCCP. We identified serine/arginine-related nuclear matrix protein of 160 kDa (SRm160)/SRRM1, which contains a highly phosphorylated domain rich in arginine/serine dipeptides, called the RS domain, as a nuclear protein that interacts with PGAM5. PGAM5 dephosphorylated SRm160, and incubation of lysates from WT cells, but not of those from PGAM5-deficient cells, induced dephosphorylation of SRm160 and another RS domain-containing protein SRSF1, one of the most characterized serine/arginine-rich (SR) proteins. Moreover, phosphorylation of these proteins and other SR proteins, which are commonly reactive toward the 1H4 monoclonal antibody that detects phosphorylated SR proteins, decreased during mitophagy, largely because of PGAM5 activity. These results suggest that PGAM5 regulates phosphorylation of these nuclear proteins during mitophagy. Because SRm160 and SR proteins play critical roles in mRNA metabolism, PGAM5 may coordinate cellular responses to mitochondrial stress at least in part through post-transcriptional and pre-translational events.