ABSTRACT
Pre-mRNA splicing is an essential process orchestrated by the spliceosome, a dynamic complex assembled stepwise on pre-mRNA. We have previously identified that USH1G protein SANS regulates pre-mRNA splicing by mediating the intranuclear transfer of the spliceosomal U4/U6.U5 tri-snRNP complex. During this process, SANS interacts with the U4/U6 and U5 snRNP-specific proteins PRPF31 and PRPF6 and regulates splicing, which is disturbed by variants of USH1G/SANS causative for human Usher syndrome (USH), the most common form of hereditary deaf-blindness. Here, we aim to gain further insights into the molecular interaction of the splicing molecules PRPF31 and PRPF6 to the CENTn domain of SANS using fluorescence resonance energy transfer assays in cells and in silico deep learning-based protein structure predictions. This demonstrates that SANS directly binds via two distinct conserved regions of its CENTn to the two PRPFs. In addition, we provide evidence that these interactions occur sequentially and a conformational change of an intrinsically disordered region to a short α-helix of SANS CENTn2 is triggered by the binding of PRPF6. Furthermore, we find that pathogenic variants of USH1G/SANS perturb the binding of SANS to both PRPFs, implying a significance for the USH1G pathophysiology.
Subject(s)
RNA Splicing Factors , Spliceosomes , Usher Syndromes , Humans , Eye Proteins/metabolism , Nerve Tissue Proteins/metabolism , Ribonucleoprotein, U4-U6 Small Nuclear/metabolism , RNA Precursors/genetics , RNA Splicing , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , Spliceosomes/metabolism , Transcription Factors/metabolism , HEK293 CellsABSTRACT
The spliceosome machinery is composed of multimeric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the tri-snRNP (small ribonucleoprotein) spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. High-resolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth-related gene products.
Subject(s)
Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Alternative Splicing , Cell Line , Cell Line, Tumor , Cell Proliferation , Humans , Protein Isoforms , RNA Splicing Factors , SpliceosomesABSTRACT
Cilia are critical to numerous biological functions, both in development and everyday homeostatic processes. Diseases arising from genetic mutations that cause cilia dysfunction are termed ciliopathies. Several ubiquitously expressed splicing factors have been implicated in the condition Retinitis Pigmentosa (RP), a group of diseases characterised by the progressive degeneration of the retina. In many types of RP the disease affects the modified primary cilium of the photoreceptor cells and thus, these types of RP are considered ciliopathies. Here, we discuss sequence variants found within a number of these splicing factors, the resulting phenotypes, and the mechanisms underpinning disease pathology. Additionally, we discuss recent evidence investigating why RP patients with mutations in globally expressed splicing factors present with retina-specific phenotypes.
Subject(s)
Cilia/genetics , Ciliopathies/genetics , Genetic Predisposition to Disease/genetics , Mutation , RNA Splicing Factors/genetics , Retinitis Pigmentosa/genetics , Animals , Cilia/metabolism , Cilia/pathology , Ciliopathies/metabolism , Humans , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Splicing , RNA Splicing Factors/metabolism , Retina/metabolism , Retina/pathology , Retinitis Pigmentosa/metabolism , Spliceosomes/genetics , Spliceosomes/metabolismABSTRACT
The de novo assembly and post-splicing reassembly of the U4/U6.U5 tri-snRNP remain to be investigated. We report here that ZIP, a protein containing a CCCH-type zinc finger and a G-patch domain, as characterized by us previously, regulates pre-mRNA splicing independent of RNA binding. We found that ZIP physically associates with the U4/U6.U5 tri-small nuclear ribonucleoprotein (tri-snRNP). Remarkably, the ZIP-containing tri-snRNP, which has a sedimentation coefficient of â¼35S, is a tri-snRNP that has not been described previously. We also found that the 35S tri-snRNP contains hPrp24, indicative of a state in which the U4/U6 di-snRNP is integrating with the U5 snRNP. We found that the 35S tri-snRNP is enriched in the Cajal body, indicating that it is an assembly intermediate during 25S tri-snRNP maturation. We showed that the 35S tri-snRNP also contains hPrp43, in which ATPase/RNA helicase activities are stimulated by ZIP. Our study identified, for the first time, a tri-snRNP intermediate, shedding new light on the de novo assembly and recycling of the U4/U6.U5 tri-snRNP.
Subject(s)
Alternative Splicing , Antigens, Neoplasm/metabolism , Organelle Biogenesis , RNA Helicases/metabolism , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolism , Spliceosomes/metabolism , Ubiquitin-Specific Proteases/metabolism , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/genetics , Coiled Bodies/chemistry , Coiled Bodies/enzymology , Coiled Bodies/metabolism , HeLa Cells , Humans , Immunoprecipitation , MCF-7 Cells , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Molecular Weight , Mutation , Negative Staining , Oligopeptides/genetics , Oligopeptides/metabolism , Protein Multimerization , Protein Stability , RNA Helicases/chemistry , RNA Helicases/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/genetics , Ribonucleoprotein, U5 Small Nuclear/chemistry , Ribonucleoprotein, U5 Small Nuclear/metabolism , Spliceosomes/chemistry , Spliceosomes/enzymology , Ubiquitin-Specific Proteases/chemistry , Ubiquitin-Specific Proteases/geneticsABSTRACT
Prp31 is one of the key tri-snRNP components essential for pre-mRNA splicing although its exact molecular function is not well studied. In a previous study, suppressor mutations were identified in the PRP31 ortholog in two spontaneous suppressors of Fgprp4 mutant deleted of the only kinase of the spliceosome in Fusarium graminearum. To further characterize the function of FgPrp31 and its relationship with FgPrp4 kinase, in this study we identified additional suppressor mutations in FgPrp31 and determined the suppressive effects of selected mutations. In total, 28 of the 35 suppressors had missense or nonsense mutations in the C terminus 465-594 aa (CT130) region of FgPrp31. The other 7 had missense or deletion mutations in the 7-64 aa region. The nonsense mutation at R464 in FgPRP31 resulted in the truncation of CT130 that contains all the putative Prp4 kinase-phosphorylation sites reported in humans, and partially rescued intron splicing defects of Fgprp4. The CT130 of FgPrp31 displayed self-inhibitory interaction with the N-terminal 1-463 (N463) region, which was reduced or abolished by the L532P, D534G, or G529D mutation in yeast two-hybrid assays. The N463 region, but not full-length FgPrp31, interacted with the N-terminal region of FgBrr2, one main U5 snRNP protein. The L532P mutation in FgPrp31 increased its interaction with FgBrr2. In contrast, suppressor mutations in FgPrp31 reduced its interaction with FgPrp6, another key component of tri-snRNP. Furthermore, we showed that FgPrp31 was phosphorylated by FgPrp4 in vivo. Site-directed mutagenesis analysis showed that phosphorylation at multiple sites in FgPrp31 is necessary to suppress Fgprp4, and S520 and S521 are important FgPrp4-phosphorylation sites. Overall, these results indicated that phosphorylation by FgPrp4 at multiple sites may release the self-inhibitory binding of FgPrp31 and affect its interaction with other components of tri-snRNP during spliceosome activation.
Subject(s)
Amino Acid Sequence , Fungal Proteins/metabolism , Fusarium/metabolism , Mutation, Missense , Protein Serine-Threonine Kinases/metabolism , Sequence Deletion , Amino Acid Substitution , Fungal Proteins/genetics , Fusarium/genetics , Phosphorylation/genetics , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/geneticsABSTRACT
BACKGROUND: Pre-mRNA splicing involves the stepwise assembly of a pre-catalytic spliceosome, followed by its catalytic activation, splicing catalysis and disassembly. Formation of the pre-catalytic spliceosomal B complex involves the incorporation of the U4/U6.U5 tri-snRNP and of a group of non-snRNP B-specific proteins. While in Saccharomyces cerevisiae the Prp38 and Snu23 proteins are recruited as components of the tri-snRNP, metazoan orthologs of Prp38 and Snu23 associate independently of the tri-snRNP as members of the B-specific proteins. The human spliceosome contains about 80 proteins that lack obvious orthologs in yeast, including most of the B-specific proteins apart from Prp38 and Snu23. Conversely, the tri-snRNP protein Spp381 is one of only five S. cerevisiae splicing factors without a known human ortholog. RESULTS: Using InParanoid, a state-of-the-art method for ortholog inference between pairs of species, and systematic BLAST searches we identified the human B-specific protein MFAP1 as a putative ortholog of the S. cerevisiae tri-snRNP protein Spp381. Bioinformatics revealed that MFAP1 and Spp381 share characteristic structural features, including intrinsic disorder, an elongated shape, solvent exposure of most residues and a trend to adopt α-helical structures. In vitro binding studies showed that human MFAP1 and yeast Spp381 bind their respective Prp38 proteins via equivalent interfaces and that they cross-interact with the Prp38 proteins of the respective other species. Furthermore, MFAP1 and Spp381 both form higher-order complexes that additionally include Snu23, suggesting that they are parts of equivalent spliceosomal sub-complexes. Finally, similar to yeast Spp381, human MFAP1 partially rescued a growth defect of the temperature-sensitive mutant yeast strain prp38-1. CONCLUSIONS: Human B-specific protein MFAP1 structurally and functionally resembles the yeast tri-snRNP-specific protein Spp381 and thus qualifies as its so far missing ortholog. Our study indicates that the yeast Snu23-Prp38-Spp381 triple complex was evolutionarily reprogrammed from a tri-snRNP-specific module in yeast to the B-specific Snu23-Prp38-MFAP1 module in metazoa, affording higher flexibility in spliceosome assembly and thus, presumably, in splicing regulation.
Subject(s)
Contractile Proteins/genetics , Extracellular Matrix Proteins/genetics , RNA Splicing Factors/metabolism , RNA Splicing , Humans , Nuclear Proteins/genetics , RNA Precursors/metabolism , Ribonucleoprotein, U4-U6 Small Nuclear , Ribonucleoproteins, Small Nuclear/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , SpliceosomesABSTRACT
Exon definition is the predominant initial spliceosome assembly pathway in higher eukaryotes, but it remains much less well-characterized compared to the intron-defined assembly pathway. Addition in trans of an excess of 5'ss containing RNA to a splicing reaction converts a 37S exon-defined complex, formed on a single exon RNA substrate, into a 45S B-like spliceosomal complex with stably integrated U4/U6.U5 tri-snRNP. This 45S complex is compositonally and structurally highly similar to an intron-defined spliceosomal B complex. Stable tri-snRNP integration during B-like complex formation is accompanied by a major structural change as visualized by electron microscopy. The changes in structure and stability during transition from a 37S to 45S complex can be induced in affinity-purified cross-exon complexes by adding solely the 5'ss RNA oligonucleotide. This conformational change does not require the B-specific proteins, which are recruited during this stabilization process, or site-specific phosphorylation of hPrp31. Instead it is triggered by the interaction of U4/U6.U5 tri-snRNP components with the 5'ss sequence, most importantly between Prp8 and nucleotides at the exon-intron junction. These studies provide novel insights into the conversion of a cross-exon to cross-intron organized spliceosome and also shed light on the requirements for stable tri-snRNP integration during B complex formation.
Subject(s)
RNA Splice Sites/genetics , RNA-Binding Proteins/genetics , Ribonucleoprotein, U4-U6 Small Nuclear/genetics , Spliceosomes/genetics , Cell Line, Tumor , Exons/genetics , Eye Proteins/genetics , HeLa Cells , Humans , Introns/genetics , Phosphorylation/genetics , RNA Splicing/geneticsABSTRACT
INTRODUCTION: Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. METHODS: We used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31-adRP, unaffected individuals and a CRISPR/Cas9 isogenic control. RESULTS: To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle and other RP-linked tri-snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild-type levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival. CONCLUSIONS: Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients.
Subject(s)
Autophagy , Eye Proteins , Induced Pluripotent Stem Cells , Protein Aggregates , Retinitis Pigmentosa , Eye Proteins/genetics , Eye Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Retinal Pigment Epithelium/cytology , Retinal Pigment Epithelium/metabolism , Retinitis Pigmentosa/genetics , Retinitis Pigmentosa/metabolism , Ribonucleoproteins, Small NuclearABSTRACT
Circadian clocks regulate multiple physiological processes in the eye, but their requirement for retinal health remains unclear. We previously showed that Drosophila homologs of spliceosome proteins implicated in human retinitis pigmentosa (RP), the most common genetically inherited cause of blindness, have a role in the brain circadian clock. In this study, we report circadian phenotypes in murine models of RP. We found that mice carrying a homozygous H2309P mutation in Pre-mRNA splicing factor 8 (Prpf8) display a lengthened period of the circadian wheel-running activity rhythm. We show also that the daily cycling of circadian gene expression is dampened in the retina of Prpf8-H2309P mice. Surprisingly, molecular rhythms are intact in the eye cup, which includes the retinal pigment epithelium (RPE), even though the RPE is thought to be the primary tissue affected in this form of RP. Downregulation of Prp31, another RNA splicing factor implicated in RP, leads to period lengthening in a human cell culture model. The period of circadian bioluminescence in primary fibroblasts of human RP patients is not significantly altered. Together, these studies link a prominent retinal disorder to circadian deficits, which could contribute to disease pathology.
Subject(s)
Chronobiology Disorders/genetics , Mutation , RNA Splicing Factors/genetics , Retinitis Pigmentosa/complications , Retinitis Pigmentosa/genetics , Adult , Animals , Cells, Cultured , Chronobiology Disorders/etiology , Circadian Rhythm/genetics , Disease Models, Animal , Eye Proteins/genetics , Eye Proteins/metabolism , Fibroblasts/physiology , Humans , Luminescence , Male , Mice , Middle Aged , Retina/pathology , Retinal Pigment Epithelium/physiology , Retinitis Pigmentosa/physiopathology , Skin/cytologyABSTRACT
Proteins and RNA are often found in ribonucleoprotein particles (RNPs), where they function in cellular processes to synthesize proteins (the ribosome), chemically modify RNAs (small nucleolar RNPs), splice pre-mRNAs (the spliceosome), and, on a larger scale, sequester RNAs, degrade them, or process them (P bodies, Cajal bodies, and nucleoli). Each RNA-protein interaction is a story in itself, as both molecules can change conformation, compete for binding sites, and regulate cellular functions. Recent studies of Xist long non-coding RNP, the U4/5/6 tri-small nuclear RNP complex, and an activated state of a spliceosome reveal new features of RNA interactions with proteins, and, although their stories are incomplete, they are already fascinating.
ABSTRACT
Pat1 RNA-binding proteins, enriched in processing bodies (P bodies), are key players in cytoplasmic 5' to 3' mRNA decay, activating decapping of mRNA in complex with the Lsm1-7 heptamer. Using co-immunoprecipitation and immunofluorescence approaches coupled with RNAi, we provide evidence for a nuclear complex of Pat1b with the Lsm2-8 heptamer, which binds to the spliceosomal U6 small nuclear RNA (snRNA). Furthermore, we establish the set of interactions connecting Pat1b/Lsm2-8/U6 snRNA/SART3 and additional U4/U6.U5 tri-small nuclear ribonucleoprotein particle (tri-snRNP) components in Cajal bodies, the site of snRNP biogenesis. RNA sequencing following Pat1b depletion revealed the preferential upregulation of mRNAs normally found in P bodies and enriched in 3' UTR AU-rich elements. Changes in >180 alternative splicing events were also observed, characterized by skipping of regulated exons with weak donor sites. Our data demonstrate the dual role of a decapping enhancer in pre-mRNA processing as well as in mRNA decay via distinct nuclear and cytoplasmic Lsm complexes.