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Meiotic resumption in mammalian oocytes involves nucleus and organelle structural changes, notably chromatin configuration transitioning from non-surrounding nucleolus (NSN) to surrounding nucleolus (SN) in germinal vesicle (GV) oocytes. Our study found that nuclear speckles, a subnuclear structure mainly composed of serine-arginine (SR) proteins, changed from a diffuse spotted distribution in mouse NSN oocytes to an aggregation pattern in SN oocytes. We further discovered that SRPK1, an enzyme phosphorylating SR proteins, co-localized with NS at SN stage and NSN oocytes failed to convert into SN oocytes after inhibiting the activity of SRPK1. Furthermore, the typical structure of chromatin ring around the nucleolus in SN oocytes collapsed after inhibitor treatment. To explore the underlying mechanism, phosphorylated SR proteins were confirmed to be associated with chromatin by salt extraction experiment, and in situ DNase I assay showed that the accessibility of chromatin enhanced in SN oocytes with SRPK1 inhibited, accompanied by decreased repressive modification on histone and abnormal recurrence of transcriptional signal. In conclusion, our results indicated that SRPK1-regulated phosphorylation on SR proteins was involved in the NSN to SN transition and played an important role in maintaining the condensation nucleus of SN oocytes via interacting with chromatin.
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Alternative splicing (AS) of pre-mRNAs increases the diversity of transcriptome and proteome and plays fundamental roles in plant development and stress responses. However, the prevalent changes in AS events and the regulating mechanisms of plants in response to pathogens remain largely unknown. Here, we show that AS changes are an important mechanism conferring cotton immunity to Verticillium dahliae (Vd). GauSR45a, encoding a serine/arginine-rich RNA binding protein, was upregulated expression and underwent AS in response to Vd infection in Gossypium australe, a wild diploid cotton species highly resistant to Vd. Silencing GauSR45a substantially reduced the splicing ratio of Vd-induced immune-associated genes, including GauBAK1 (BRI1-associated kinase 1) and GauCERK1 (chitin elicitor receptor kinase 1). GauSR45a binds to the GAAGA motif that is commonly found in the pre-mRNA of genes essential for PTI, ETI, and defense. The binding between GauSR45a and the GAAGA motif in the pre-mRNA of BAK1 was enhanced by two splicing factors of GauU2AF35B and GauU1-70 K, thereby facilitating exon splicing; silencing either AtU2AF35B or AtU1-70 K decreased the resistance to Vd in transgenic GauSR45a Arabidopsis. Overexpressing the short splicing variant of BAK1GauBAK1.1 resulted in enhanced Verticillium wilt resistance rather than the long one GauBAK1.2. Vd-induced far more AS events were in G. barbadense (resistant tetraploid cotton) than those in G. hirsutum (susceptible tetraploid cotton) during Vd infection, indicating resistance divergence in immune responses at a genome-wide scale. We provided evidence showing a fundamental mechanism by which GauSR45a enhances cotton resistance to Vd through global regulation of AS of immunity genes.
Assuntos
Processamento Alternativo , Ascomicetos , Resistência à Doença , Regulação da Expressão Gênica de Plantas , Gossypium , Doenças das Plantas , Proteínas de Plantas , Gossypium/genética , Gossypium/microbiologia , Gossypium/imunologia , Processamento Alternativo/genética , Resistência à Doença/genética , Doenças das Plantas/microbiologia , Doenças das Plantas/imunologia , Doenças das Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ascomicetos/fisiologia , Imunidade Vegetal/genética , VerticilliumRESUMO
The Ser/Arg-rich splicing factors (SR proteins) constitute a crucial protein family in alternative splicing, comprising twelve members characterized by unique repetitive Arg-Ser dipeptide sequences (RS) and one to two RNA-recognition motifs (RRM). The RS regions of SR proteins undergo variable phosphorylation, resulting in unphosphorylated, partially phosphorylated, or hyper-phosphorylated states based on functional requirements. Despite the identification of the SR protein family over 30 years ago, the purification of native SR proteins in soluble form at large quantities has presented challenges due to their low solubility. This protocol delineates a method for acquiring soluble, full-length, unphosphorylated, hypo- and hyper-phosphorylated SRSF1, a prototypical SR family member. Notably, this protocol facilitates the purification of SRSF1 in ample quantities suitable for NMR, as well as various biophysical and biochemical studies. The methodologies and principles outlined herein are expected to extend beyond SRSF1 protein production and can be adapted for purifying other SR protein family members or SR-related proteins, such as snRNP70 and U2AF-35. Given the involvement of these proteins in numerous essential biological processes, this protocol will prove beneficial to researchers in related fields. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Purification of SRSF1 from E. coli Support Protocol: Purification of ULP1 Basic Protocol 2: Purification of hypo-phosphorylated SRSF1 from E. coli Basic Protocol 3: Purification of hyper-phosphorylated SRSF1 from E. coli.
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Escherichia coli , Proteínas , Escherichia coli/genética , Fosforilação , Processamento AlternativoRESUMO
Background: Serine/arginine-rich (SR) proteins regulate pre-mRNA splicing. However, structurally similar proteins often behave differently in splicing regulation and the underlying mechanisms are largely unknown. Here, using SMN1/2 minigenes we extensively analyzed four SR proteins, SRSF1/5/6/9. Methods: In this study, the effects of these proteins on SMN1/2 exon 7 splicing when tethered at either intron 6 or 7 were evaluated using an MS2-tethering assay. Deletion analysis in four SR proteins and co-overexpression analysis were performed. Results: Splicing outcomes varied among all four SR proteins, SRSF1 and SRSF5 function the same at the two sites, acting as repressor and stimulator, respectively; while SRSF6 and SRSF9 promote exon 7 inclusion at only one site. Further, the key domains of each SR proteins were investigated, which identified a potent inhibitory nonapeptide in the C-terminus of SRSF1/9 ribonucleic acid recognition motif-1 (RRM1) and a potent stimulatory heptapeptide at the N-terminus of SRSF5/6 RRM1. Conclusion: The insight of the four SR proteins and their domains in affecting SMN gene splicing brings a new perspective on the modes of action of SR proteins; and the functional peptides obtained here offers new ideas for developing splice switching-related therapies.
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RNA , Serina , RNA/metabolismo , Serina/química , Splicing de RNA/genética , Proteínas/metabolismo , Peptídeos/metabolismo , Arginina/químicaRESUMO
Triple-negative breast cancer (TNBC), although highly lethal, lacks validated therapeutic targets. Here, we report that U2 snRNP-associated SURP motif-containing protein (U2SURP), a poorly defined member of the serine/arginine rich protein family, was significantly upregulated in TNBC tissues, and its high expression was associated with poor prognosis of TNBC patients. MYC, a frequently amplified oncogene in TNBC tissues, enhanced U2SURP translation through an eIF3D (eukaryotic translation initiation factor 3 subunit D)-dependent mechanism, resulting in the accumulation of U2SURP in TNBC tissues. Functional assays revealed that U2SURP played an important role in facilitating tumorigenesis and metastasis of TNBC cells both in vitro and in vivo. Intriguingly, U2SURP had no significant effects on proliferative, migratory, and invasive potential of normal mammary epithelial cells. Furthermore, we found that U2SURP promoted alternative splicing of spermidine/spermine N1-acetyltransferase 1 (SAT1) pre-mRNA by removal of intron 3, resulting in an increase in the stability of SAT1 mRNA and subsequent protein expression levels. Importantly, spliced SAT1 promoted the oncogenic properties of TNBC cells, and re-expression of SAT1 in U2SURP-depleted cells partially rescued the impaired malignant phenotypes of TNBC cells caused by U2SURP knockdown both in vitro and in mice. Collectively, these findings reveal previously unknown functional and mechanism roles of the MYC-U2SURP-SAT1 signaling axis in TNBC progression and highlight U2SURP as a potential therapy target for TNBC.
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Acetiltransferases , Processamento Alternativo , Proteínas Proto-Oncogênicas c-myc , Ribonucleoproteínas , Neoplasias de Mama Triplo Negativas , Animais , Humanos , Camundongos , Acetiltransferases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Fator de Iniciação 3 em Eucariotos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ribonucleoproteínas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
The Androgen Receptor (AR) gene plays a key role in castration-resistant prostate cancer (CRPC). Controlling the progression of CRPC by inhibiting AR gene expression is one of the core directions for prostate cancer (Pca) drug development. A 23-amino acids retention, named exon 3a, into the DNA binding domain of the splice variant AR23 has been shown to prevent AR from entering the nucleus and restore the sensitivity of cancer cells to related therapies. In this study, we conducted a preliminary investigation of the splicing modulation of the AR gene in order to develop a splice-switching therapy for Pca by promoting exon 3a inclusion. Using mutagenesis-coupled RT-PCR with AR minigene and over-expression of certain splicing factors, we found that serine/arginine-rich (SR) proteins are key factors facilitating the recognition of the 3' splice site of exon 3a (L-3' SS), while the deletion or blocking of the polypyrimidine tract (PPT) region of the original 3' splice site of exon 3 (S-3' SS) could strongly enhance exon 3a splicing without affecting the function of any SR protein. Furthermore, we designed a series of antisense oligonucleotides (ASOs) to screen drug candidates, and ASOs targeting S-3' SS and its PPT region or the exonic region of exon 3 turned out to be most effective in rescuing exon 3a splicing. A dose-response test indicated ASO12 as the lead candidate drug significantly promoting the inclusion of exon 3a to more than 85%. MTT assay confirmed that the cell proliferation was significantly inhibited after ASO treatment. Our results provide the first glance to AR splicing regulation. With several promising therapeutic ASO candidates obtained here, further development of ASO drugs to treat CRPC is strongly encouraged.
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Neoplasias de Próstata Resistentes à Castração , Receptores Androgênicos , Masculino , Humanos , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Sítios de Splice de RNA , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Éxons , Linhagem Celular Tumoral , Proteínas/genética , Regulação Neoplásica da Expressão Gênica , Processamento AlternativoRESUMO
Alternative splicing of pre-mRNAs is crucial for plant growth and development. Serine/arginine-rich (SR) proteins are a conserved family of RNA-binding proteins that are critical for both constitutive and alternative splicing. However, how phosphorylation of SR proteins regulates gene transcription and alternative splicing during plant development is poorly understood. We found that the Arabidopsis thaliana L. SR protein-specific kinase II family proteins (SRPKIIs) play an important role in plant development, including flowering. SRPKIIs regulate the phosphorylation status of a subset of specific SR proteins, including SR45 and SC35, which subsequently mediates their subcellular localization. A phospho-dead SR45 mutant inhibits the assembly of the apoptosis-and splicing-associated protein complex and thereby upregulates the expression of FLOWERING LOCUS C (FLC) via epigenetic modification. The splicing efficiency of FLC introns was significantly increased in the shoot apex of the srpkii mutant. Transcriptomic analysis revealed that SRPKIIs regulate the alternative splicing of c. 400 genes, which largely overlap with those regulated by SR45 and SC35-SCL family proteins. In summary, we found that Arabidopsis SRPKIIs specifically affect the phosphorylation status of a subset SR proteins and regulate the expression and alternative splicing of FLC to control flowering time.
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Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Processamento Alternativo/genética , Arabidopsis/metabolismo , Fosforilação , Expressão Gênica , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
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.
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(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.
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Cardiomiopatia Dilatada , Proteínas Quinases , Proteínas de Ligação a RNA , Animais , Arginina/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Humanos , Fosforilação/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , SerinaRESUMO
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 (â¼94%), 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.
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Proteoma , Proteínas de Ligação a RNA , Animais , Arginina/metabolismo , Humanos , Proteínas Nucleares/genética , Proteoma/genética , RNA/metabolismo , Precursores de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Serina/genética , Fatores de Processamento de Serina-Arginina/genética , Fatores de Processamento de Serina-Arginina/metabolismoRESUMO
Human papillomaviruses (HPV) are epitheliotropic DNA tumor viruses that are prevalent in the human population. A subset of the HPVs termed high-risk HPVs (HR-HPVs) are causative agents of anogenital cancers and head-and-neck cancers. Cancer is the result of persistent high-risk HPV infections that have not been cleared by the immune system of the host. These infections are characterized by dysregulated HPV gene expression, in particular constitutive high expression of the HPV E6 and E7 oncogenes and absence of the highly immunogenic viral L1 and L2 capsid proteins. HPVs make extensive use of alternative mRNA splicing to express its genes and are therefore highly dependent on cellular RNA-binding proteins for proper gene expression. Levels of RNA-binding proteins are altered in HPV-containing premalignant cervical lesions and in cervical cancer. Here we review our current knowledge of RNA-binding proteins that control HPV gene expression. We focus on RNA-binding proteins that control expression of the E6 and E7 oncogenes since they initiate and drive development of cancer and on the immunogenic L1 and L2 proteins as there silencing may contribute to immune evasion during carcinogenesis. Furthermore, cellular RNA-binding proteins are essential for HPV gene expression and as such may be targets for therapy to HPV infections and HPV-driven cancers.
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Proteínas Oncogênicas Virais , Infecções por Papillomavirus , Humanos , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/genética , RNA Mensageiro/metabolismo , Carcinógenos , Proteínas de Ligação a RNA/genética , Carcinogênese/genéticaRESUMO
Serine/arginine-rich (SR) proteins are important splicing factors in plant development and abiotic/hormone-related stresses. However, evidence that SR proteins contribute to the process in woody plants has been lacking. Using phylogenetics, gene synteny, transgenic experiments, and RNA-seq analysis, we identified 24 PtSR genes and explored their evolution, expression, and function in Popolus trichocarpa. The PtSR genes were divided into six subfamilies, generated by at least two events of genome triplication and duplication. Notably, they were constitutively expressed in roots, stems, and leaves, demonstrating their fundamental role in P. trichocarpa. Additionally, most PtSR genes (~83%) responded to at least one stress (cold, drought, salt, SA, MeJA, or ABA), and, especially, cold stress induced a dramatic perturbation in the expression and/or alternative splicing (AS) of 18 PtSR genes (~75%). Evidentially, the overexpression of PtSCL30 in Arabidopsis decreased freezing tolerance, which probably resulted from AS changes of the genes (e.g., ICE2 and COR15A) critical for cold tolerance. Moreover, the transgenic plants were salt-hypersensitive at the germination stage. These indicate that PtSCL30 may act as a negative regulator under cold and salt stress. Altogether, this study sheds light on the evolution, expression, and AS of PtSR genes, and the functional mechanisms of PtSCL30 in woody plants.
Assuntos
Regulação da Expressão Gênica de Plantas , Populus/metabolismo , Fatores de Processamento de RNA/metabolismo , Estresse Fisiológico , Processamento Alternativo , Arabidopsis/genética , Especificidade de Órgãos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Populus/genética , Fatores de Processamento de RNA/genética , TemperaturaRESUMO
Pathogen sensing via pattern recognition receptors triggers massive reprogramming of macrophage gene expression. While the signaling cascades and transcription factors that activate these responses are well-known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Given their crucial role in regulating pre-mRNA splicing and other RNA processing steps, we hypothesized that members of the SR/hnRNP protein families regulate innate immune gene expression in distinct ways. We analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage-like cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We identified thousands of transcripts whose abundance is increased or decreased by SR/hnRNP knockdown in macrophages. Notably, we observed that SR and hnRNP proteins influence expression of different genes in uninfected versus Salmonella-infected macrophages, suggesting functionalization of these proteins upon pathogen sensing. Likewise, we found that knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for thousands of macrophage transcripts and that these alternative splicing changes were distinct in uninfected and Salmonella-infected macrophages. Finally, having observed a surprising degree of similarity between the differentially expressed genes (DEGs) and DIUs in hnRNP K and U knockdown macrophages, we found that hnRNP K and U knockdown macrophages are both more restrictive to Vesicular Stomatitis Virus (VSV), while hnRNP K knockdown macrophages are more permissive to Salmonella Typhimurium. Based on these findings, we conclude that many innate immune genes evolved to rely on one or more SR/hnRNPs to ensure the proper magnitude of their induction, supporting a model wherein pre-mRNA splicing is critical for regulating innate immune gene expression and controlling infection outcomes in macrophages ex vivo.
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Processamento Alternativo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Imunidade Inata/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Transcriptoma , Animais , Biomarcadores , Biologia Computacional/métodos , Ontologia Genética , Redes Reguladoras de Genes , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Masculino , Camundongos , Modelos Biológicos , Células RAW 264.7 , Infecções por Salmonella/genética , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/imunologiaRESUMO
Plasmodium falciparum, the parasite responsible for the deadliest form of human malaria, replicates within the erythrocytes of its host, where it encounters numerous pressures that cause extensive DNA damage, which must be repaired efficiently to ensure parasite survival. Malaria parasites, which have lost the non-homologous end joining (NHEJ) pathway for repairing DNA double-strand breaks, have evolved unique mechanisms that enable them to robustly maintain genome integrity under such harsh conditions. However, the nature of these adaptations is unknown. We show that a highly conserved RNA splicing factor, P. falciparum (Pf)SR1, plays an unexpected and crucial role in DNA repair in malaria parasites. Using an inducible and reversible system to manipulate PfSR1 expression, we demonstrate that this protein is recruited to foci of DNA damage. Although loss of PfSR1 does not impair parasite viability, the protein is essential for their recovery from DNA-damaging agents or exposure to artemisinin, the first-line antimalarial drug, demonstrating its necessity for DNA repair. These findings provide key insights into the evolution of DNA repair pathways in malaria parasites as well as the ability of the parasite to recover from antimalarial treatment.
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Malária Falciparum , Malária , Parasitos , Animais , Reparo do DNA/genética , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genéticaRESUMO
Serine/arginine protein kinases (SRPKs) phosphorylate Arg/Ser dipeptide-containing proteins that play crucial roles in a broad spectrum of basic cellular processes. The existence of a large internal spacer sequence that separates the bipartite kinase catalytic core and anchors the kinases in the cytoplasm is a unique structural feature of SRPKs. Here, we report that exposure of HeLa and T24 cells to DNA damage inducers triggers the nuclear translocation of SRPK1 and SRPK2. Furthermore, we show that nuclear SRPKs did not protect from, but on the contrary, mediated the cytotoxic effects of genotoxic agents, such as 5-fluorouracil (5-FU) and cisplatin. Confirming previous data showing that the kinase activity is essential for the entry of SRPKs into the nucleus, SRPIN340, a selective SRPK1/2 inhibitor, blocked the nuclear accumulation of the kinases, thus diminishing the cytotoxic effects of the drugs. ATR/ATM-dependent phosphorylation of threonine 326 and serine 408 in the spacer domain of SRPK1 was essential for the redistribution of the kinase to the nucleus. Substitution of either of these two residues to alanine or inhibition of ATR/ATM kinase activity abolished nuclear localization of SRPK1 and conferred tolerance to 5-FU treatment. These findings suggest that SRPKs may play an important role in linking cellular signaling to DNA damage in eukaryotic cells.
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Núcleo Celular/metabolismo , Cisplatino/farmacologia , Fluoruracila/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Morte Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Dano ao DNA , Células HeLa , Humanos , Modelos Biológicos , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Fosforilação/efeitos dos fármacos , Piperidinas/farmacologia , Substâncias Protetoras/farmacologia , Transporte Proteico/efeitos dos fármacosRESUMO
The evolutionarily conserved TRanscript-EXport (TREX) complex plays central roles during mRNP (messenger ribonucleoprotein) maturation and export from the nucleus to the cytoplasm. In yeast, TREX is composed of the THO sub-complex (Tho2, Hpr1, Tex1, Mft1, and Thp2), the DEAD box ATPase Sub2, and Yra1. Here we present a 3.7 Å cryo-EM structure of the yeast THOâ¢Sub2 complex. The structure reveals the intimate assembly of THO revolving around its largest subunit Tho2. THO stabilizes a semi-open conformation of the Sub2 ATPase via interactions with Tho2. We show that THO interacts with the serine-arginine (SR)-like protein Gbp2 through both the RS domain and RRM domains of Gbp2. Cross-linking mass spectrometry analysis supports the extensive interactions between THO and Gbp2, further revealing that RRM domains of Gbp2 are in close proximity to the C-terminal domain of Tho2. We propose that THO serves as a landing pad to configure Gbp2 to facilitate its loading onto mRNP.
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Família Multigênica , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Microscopia Crioeletrônica , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
The assembly and activation of the spliceosome rely upon the phosphorylation of an essential family of splicing factors known as the serine-arginine (SR) proteins. Although it has been demonstrated recently that two enzyme families, the SR protein kinases (SRPKs) and the Cdc2-like kinases (CLKs), can function as a complex to efficiently phosphorylate these SR proteins in the nucleus, the molecular features involved in such a connection are unknown. In this study, we identified a group of conserved residues in the large lobe of SRPK1 that interact with the N terminus of CLK1 stabilizing the SRPK1-CLK1 complex. Mutations in this motif not only disrupt formation of the kinase-kinase complex but also impair SRPK1-dependent release of the phospho-SR protein from CLK1. The binding motif potently up-regulates CLK1-specific phosphorylation sites, enhances SR protein diffusion from nuclear speckles, and impacts the alternative splicing of several target genes. These results indicate that CLK1 binds a conserved, electronegative surface on SRPK1, thereby controlling SR protein phosphorylation levels for enhanced subnuclear trafficking and alternative splicing regulation.
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Processamento Alternativo , Núcleo Celular/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Núcleo Celular/química , Sequência Conservada , Citoplasma/química , Citoplasma/enzimologia , Expressão Gênica , Células HeLa , Humanos , Cinética , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Serine/Arginine splicing factor 1 (SRSF1) is an RNA binding protein abundantly expressed in most tissues. The pleiotropic functions of SRSF1 exert multiple roles in gene expression by regulating major steps in transcription, processing, export through the nuclear pores and translation of nascent RNA transcripts. The aim of this review is to highlight recent findings in the functions of this protein and to describe its role in immune system development, functions and regulation.
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Sistema Imunitário , Expressão Gênica , Humanos , Proteínas de Ligação a RNA/genética , Fatores de Processamento de Serina-Arginina/genéticaRESUMO
SR/RS domains are found in almost all eukaryotic genomes from C. elegans to human. These domains are thought to mediate interactions between proteins but also between proteins and RNA in complex networks associated with mRNA splicing, chromatin structure, transcription, cell cycle and cell structure. A precise and tight regulation of their function is achieved through phosphorylation of a number of serine residues within the SR/RS motifs by the Serine-Arginine protein kinases (SRPKs) that lead to delicate structural alterations. Given that coronavirus N proteins also contain SR/RS domains, we formulate the hypothesis that the viruses exploit the properties of these motifs to promote unpacking of viral RNA and virion assembly.
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Segment 8 mRNAs of influenza virus A/Brevig Misson/1918/1 (H1N1) are poorly spliced compared to segment 8 mRNAs of influenza virus A/Netherlands/178/95 (H3N2). Using oligonucleotide-mediated protein pull down with oligos spanning the entire length of segment 8 of either influenza virus H1N1 or influenza virus H3N2 we identified cellular RNA binding proteins that interacted with oligonucleotides derived from either H1N1 or H3N2 sequences. When the identified hot spots for RNA binding proteins in H1N1 segment 8 mRNAs were replaced by H3N2 sequences, splicing efficiency increased significantly. Replacing as few as three nucleotides of the H1N1 mRNA with sequences from H3N2 mRNA, enhanced splicing of the H1N1 mRNAs. Cellular proteins U2AF65 and HuR interacted preferentially with the 3'-splice site of H3N2 and overexpression of HuR reduced the levels of unspliced H1N1 mRNAs, suggesting that U2AF65 and HuR contribute to control of influenza virus mRNA splicing.