ABSTRACT
The spliceosomal gene SF3B1 is frequently mutated in cancer. While it is known that SF3B1 hotspot mutations lead to loss of splicing factor SUGP1 from spliceosomes, the cancer-relevant SF3B1-SUGP1 interaction has not been characterized. To address this issue, we show by structural modeling that two regions flanking the SUGP1 G-patch make numerous contacts with the region of SF3B1 harboring hotspot mutations. Experiments confirmed that all the cancer-associated mutations in these regions, as well as mutations affecting other residues in the SF3B1-SUGP1 interface, not only weaken or disrupt the interaction but also alter splicing similarly to SF3B1 cancer mutations. Finally, structural modeling of a trimeric protein complex reveals that the SF3B1-SUGP1 interaction "loops out" the G-patch for interaction with the helicase DHX15. Our study thus provides an unprecedented molecular view of a protein complex essential for accurate splicing and also reveals that numerous cancer-associated mutations disrupt the critical SF3B1-SUGP1 interaction.
Subject(s)
Neoplasms , Spliceosomes , Humans , RNA, Messenger/metabolism , Spliceosomes/genetics , Spliceosomes/metabolism , RNA Splicing Factors/chemistry , RNA Splicing/genetics , Neoplasms/genetics , Neoplasms/metabolism , Mutation , Phosphoproteins/metabolismABSTRACT
Nucleoli are the major cellular compartments for the synthesis of rRNA and assembly of ribosomes, the macromolecular complexes responsible for protein synthesis. Given the abundance of ribosomes, there is a huge demand for rRNA, which indeed constitutes â¼80% of the mass of RNA in the cell. Thus, nucleoli are characterized by extensive transcription of multiple rDNA loci by the dedicated polymerase, RNA polymerase (Pol) I. However, in addition to producing rRNAs, there is considerable additional transcription in nucleoli by RNA Pol II as well as Pol I, producing multiple noncoding (nc) and, in one instance, coding RNAs. In this review, we discuss important features of these transcripts, which often appear species-specific and reflect transcription antisense to pre-rRNA by Pol II and within the intergenic spacer regions on both strands by both Pol I and Pol II. We discuss how expression of these RNAs is regulated, their propensity to form cotranscriptional R loops, and how they modulate rRNA transcription, nucleolar structure, and cellular homeostasis more generally.
Subject(s)
RNA Polymerase II , RNA Precursors , Cell Nucleolus/genetics , Cell Nucleolus/metabolism , DNA, Intergenic , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Homeostasis/genetics , Macromolecular Substances/metabolism , RNA Polymerase I/genetics , RNA Polymerase I/metabolism , RNA Polymerase II/metabolism , RNA Precursors/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Transcription, GeneticABSTRACT
Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3' termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation.
Subject(s)
3' Untranslated Regions , Polyadenylation , RNA Precursors/metabolism , RNA, Messenger/metabolism , Active Transport, Cell Nucleus , Exons , Gene Expression Regulation , Humans , RNA Precursors/genetics , RNA Stability , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U1 Small Nuclear/metabolismABSTRACT
The nucleolus is an important cellular compartment in which ribosomal RNAs (rRNAs) are transcribed and where certain stress pathways that are crucial for cell growth are coordinated. Here we report novel functions of the DNA replication and repair factor replication protein A (RPA) in control of nucleolar homeostasis. We show that loss of the DNA:RNA helicase senataxin (SETX) promotes RPA nucleolar localization, and that this relocalization is dependent on the presence of R loops. Notably, this nucleolar RPA phenotype was also observed in the presence of camptothecin (CPT)-induced genotoxic stress, as well as in SETX-deficient AOA2 patient fibroblasts. Extending these results, we found that RPA is recruited to rDNA following CPT treatment, where RPA prevents R-loop-induced DNA double-strand breaks. Furthermore, we show that loss of RPA significantly decreased 47S pre-rRNA levels, which was accompanied by increased expression of both RNAP II-mediated "promoter and pre-rRNA antisense" RNA as well as RNAP I-transcribed intragenic spacer RNAs. Finally, and likely reflecting the above, we found that loss of RPA promoted nucleolar structural disorganization, characterized by the appearance of reduced size nucleoli. Our findings both indicate new roles for RPA in nucleoli through pre-rRNA transcriptional control and also emphasize that RPA function in nucleolar homeostasis is linked to R-loop resolution under both physiological and pathological conditions.
Subject(s)
R-Loop Structures , Replication Protein A , Cell Nucleolus/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , DNA, Ribosomal/genetics , DNA, Ribosomal/metabolism , Humans , Multifunctional Enzymes , RNA Helicases/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Replication Protein A/genetics , Replication Protein A/metabolism , Transcription, GeneticABSTRACT
Dysregulation of the DNA/RNA-binding protein FUS causes certain subtypes of ALS/FTD by largely unknown mechanisms. Recent evidence has shown that FUS toxic gain of function due either to mutations or to increased expression can disrupt critical cellular processes, including mitochondrial functions. Here, we demonstrate that in human cells overexpressing wild-type FUS or expressing mutant derivatives, the protein associates with multiple mRNAs, and these are enriched in mRNAs encoding mitochondrial respiratory chain components. Notably, this sequestration leads to reduced levels of the encoded proteins, which is sufficient to bring about disorganized mitochondrial networks, reduced aerobic respiration and increased reactive oxygen species. We further show that mutant FUS associates with mitochondria and with mRNAs encoded by the mitochondrial genome. Importantly, similar results were also observed in fibroblasts derived from ALS patients with FUS mutations. Finally, we demonstrate that FUS loss of function does not underlie the observed mitochondrial dysfunction, and also provides a mechanism for the preferential sequestration of the respiratory chain complex mRNAs by FUS that does not involve sequence-specific binding. Together, our data reveal that respiratory chain complex mRNA sequestration underlies the mitochondrial defects characteristic of ALS/FTD and contributes to the FUS toxic gain of function linked to this disease spectrum.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/physiopathology , Gene Expression Regulation/genetics , Mitochondria/pathology , RNA, Messenger/metabolism , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolism , Cell Line , Cell Respiration/genetics , Cells, Cultured , Electron Transport/genetics , Genome, Mitochondrial , Humans , Mitochondria/genetics , Mutation , Protein Aggregation, Pathological/genetics , Protein Binding/geneticsABSTRACT
SF3B1, which encodes an essential spliceosomal protein, is frequently mutated in myelodysplastic syndromes (MDS) and many cancers. However, the defect of mutant SF3B1 is unknown. Here, we analyzed RNA sequencing data from MDS patients and confirmed that SF3B1 mutants use aberrant 3' splice sites. To elucidate the underlying mechanism, we purified complexes containing either wild-type or the hotspot K700E mutant SF3B1 and found that levels of a poorly studied spliceosomal protein, SUGP1, were reduced in mutant spliceosomes. Strikingly, SUGP1 knockdown completely recapitulated the splicing errors, whereas SUGP1 overexpression drove the protein, which our data suggest plays an important role in branchsite recognition, into the mutant spliceosome and partially rescued splicing. Other hotspot SF3B1 mutants showed similar altered splicing and diminished interaction with SUGP1. Our study demonstrates that SUGP1 loss is a common defect of spliceosomes with disease-causing SF3B1 mutations and, because this defect can be rescued, suggests possibilities for therapeutic intervention.
Subject(s)
Leukemia, Erythroblastic, Acute/metabolism , Mutation , Myelodysplastic Syndromes/metabolism , Phosphoproteins/metabolism , RNA Splicing Factors/metabolism , RNA Splicing , Spliceosomes/metabolism , Gene Expression Regulation, Neoplastic , Genetic Predisposition to Disease , HEK293 Cells , Humans , K562 Cells , Leukemia, Erythroblastic, Acute/genetics , Leukemia, Erythroblastic, Acute/pathology , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Phenotype , Phosphoproteins/genetics , Protein Binding , RNA Splicing Factors/genetics , Spliceosomes/genetics , Spliceosomes/pathologyABSTRACT
The cleavage and polyadenylation specificity factor (CPSF) complex plays a central role in the formation of mRNA 3' ends, being responsible for the recognition of the poly(A) signal sequence, the endonucleolytic cleavage step, and recruitment of poly(A) polymerase. CPSF has been extensively studied for over three decades, and its functions and those of its individual subunits are becoming increasingly well-defined, with much current research focusing on the impact of these proteins on the normal functioning or disease/stress states of cells. In this review, we provide an overview of the general functions of CPSF and its subunits, followed by a discussion of how they exert their functions in a surprisingly diverse variety of biological processes and cellular conditions. These include transcription termination, small RNA processing, and R-loop prevention/resolution, as well as more generally cancer, differentiation/development, and infection/immunity.
Subject(s)
Cleavage And Polyadenylation Specificity Factor , RNA, Messenger , Cleavage And Polyadenylation Specificity Factor/metabolism , Cleavage And Polyadenylation Specificity Factor/genetics , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism , Animals , Polyadenylation , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Transcription Termination, Genetic , RNA 3' End ProcessingABSTRACT
Certain long non-coding RNAs (lncRNAs) are known to contain small open reading frames that can be translated. Here we describe a much larger 25 kDa human protein, "Ribosomal IGS Encoded Protein" (RIEP), that remarkably is encoded by the well-characterized RNA polymerase (RNAP) II-transcribed nucleolar "promoter and pre-rRNA antisense" lncRNA (PAPAS). Strikingly, RIEP, which is conserved throughout primates but not found in other species, predominantly localizes to the nucleolus as well as mitochondria, but both exogenously expressed and endogenous RIEP increase in the nuclear and perinuclear regions upon heat shock (HS). RIEP associates specifically with the rDNA locus, increases levels of the RNA:DNA helicase Senataxin, and functions to sharply reduce DNA damage induced by heat shock. Proteomics analysis identified two mitochondrial proteins, C1QBP and CHCHD2, both known to have mitochondrial and nuclear functions, that we show interact directly, and relocalize following heat shock, with RIEP. Finally, it is especially notable that the rDNA sequences encoding RIEP are multifunctional, giving rise to an RNA that functions both as RIEP messenger RNA (mRNA) and as PAPAS lncRNA, as well as containing the promoter sequences responsible for rRNA synthesis by RNAP I. Our work has thus not only shown that a nucleolar "non-coding" RNA in fact encodes a protein, but also established a novel link between mitochondria and nucleoli that contributes to the cellular stress response.
Subject(s)
RNA, Long Noncoding , Animals , Humans , RNA, Long Noncoding/metabolism , Transcription, Genetic , DNA, Ribosomal/genetics , Cell Nucleolus/metabolism , RNA Polymerase I/metabolism , RNA Polymerase II/metabolism , Ribosomal Proteins/metabolism , RNA, Untranslated/metabolism , RNA, Ribosomal/genetics , Carrier Proteins/metabolism , Mitochondrial Proteins/metabolism , DNA-Binding Proteins/metabolism , Transcription Factors/metabolismABSTRACT
Alternative splicing (AS) plays important roles in embryonic stem cell (ESC) differentiation. In this study, we first identified transcripts that display specific AS patterns in pluripotent human ESCs (hESCs) relative to differentiated cells. One of these encodes T-cell factor 3 (TCF3), a transcription factor that plays important roles in ESC differentiation. AS creates two TCF3 isoforms, E12 and E47, and we identified two related splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNPs) H1 and F (hnRNP H/F), that regulate TCF3 splicing. We found that hnRNP H/F levels are high in hESCs, leading to high E12 expression, but decrease during differentiation, switching splicing to produce elevated E47 levels. Importantly, hnRNP H/F knockdown not only recapitulated the switch in TCF3 AS but also destabilized hESC colonies and induced differentiation. Providing an explanation for this, we show that expression of known TCF3 target E-cadherin, critical for maintaining ESC pluripotency, is repressed by E47 but not by E12.
Subject(s)
Alternative Splicing , Basic Helix-Loop-Helix Transcription Factors/genetics , Cadherins/metabolism , Embryonic Stem Cells/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism , Antigens, CD , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cadherins/genetics , Cell Differentiation/genetics , Cell Line , Embryonic Stem Cells/cytology , Exons , Gene Expression Regulation , Humans , RNA Precursors/chemistry , RNA, Messenger/chemistry , Regulatory Sequences, Ribonucleic AcidABSTRACT
The RNA polymerase II largest subunit C-terminal domain consists of repeated YSPTSPS heptapeptides. The role of tyrosine-1 (Tyr1) remains incompletely understood, as, for example, mutating all Tyr1 residues to Phe (Y1F) is lethal in vertebrates but a related mutant has only a mild phenotype in S. pombe. Here we show that Y1F substitution in budding yeast resulted in a strong slow-growth phenotype. The Y1F strain was also hypersensitive to several different cellular stresses that involve MAP kinase signaling. These phenotypes were all linked to transcriptional changes, and we also identified genetic and biochemical interactions between Tyr1 and both transcription initiation and termination factors. Further studies uncovered defects related to MAP kinase I (Slt2) pathways, and we provide evidence that Slt2 phosphorylates Tyr1 in vitro and in vivo. Our study has thus identified Slt2 as a Tyr1 kinase, and in doing so provided links between stress response activation and Tyr1 phosphorylation.
Subject(s)
Gene Expression Regulation, Fungal , Mitogen-Activated Protein Kinases/metabolism , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Stress, Physiological , Cyclin-Dependent Kinase 8/genetics , Cyclin-Dependent Kinase 8/metabolism , Genotype , Mediator Complex/genetics , Mediator Complex/metabolism , Mitogen-Activated Protein Kinases/genetics , Mutation , Phenotype , Phosphorylation , Protein Domains , RNA Polymerase II/chemistry , RNA Polymerase II/genetics , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction , Time Factors , Transduction, Genetic , TyrosineABSTRACT
SF3B1 is the most frequently mutated spliceosomal gene in cancer. Several hotspot mutations are known to disrupt the interaction of SF3B1 with another splicing factor, SUGP1, resulting in the RNA missplicing that characterizes mutant SF3B1 cancers. Properties of SUGP1, especially the presence of a G-patch motif, a structure known to function by activating DEAH-box RNA helicases, suggest the requirement of such an enzyme in SUGP1 function in splicing. However, the identity of this putative helicase has remained an important unanswered question. Here, using a variety of protein-protein interaction assays, we identify DHX15 as the critical helicase. We further show that depletion of DHX15 or expression of any of several DHX15 mutants, including one implicated in acute myeloid leukemia, partially recapitulates the splicing defects of mutant SF3B1. Moreover, a DHX15-SUGP1 G-patch fusion protein is able to incorporate into the spliceosome to rescue the splicing defects of mutant SF3B1. We also present the crystal structure of the human DHX15-SUGP1 G-patch complex, which reveals the molecular basis of their direct interaction. Our data thus demonstrate that DHX15 is the RNA helicase that functions with SUGP1 and additionally provide important insight into how mutant SF3B1 disrupts splicing in cancer.
Subject(s)
Neoplasms , RNA Helicases , RNA Splicing Factors , RNA Splicing , Humans , DNA Helicases , Genes, Regulator , Phosphoproteins , RNA Helicases/genetics , RNA Splicing/genetics , RNA Splicing Factors/genetics , Spliceosomes/geneticsABSTRACT
SF3B1 is the most frequently mutated RNA splicing factor in cancer, including in â¼25% of myelodysplastic syndromes (MDS) patients. SF3B1-mutated MDS, which is strongly associated with ringed sideroblast morphology, is characterized by ineffective erythropoiesis, leading to severe, often fatal anemia. However, functional evidence linking SF3B1 mutations to the anemia described in MDS patients harboring this genetic aberration is weak, and the underlying mechanism is completely unknown. Using isogenic SF3B1 WT and mutant cell lines, normal human CD34 cells, and MDS patient cells, we define a previously unrecognized role of the kinase MAP3K7, encoded by a known mutant SF3B1-targeted transcript, in controlling proper terminal erythroid differentiation, and show how MAP3K7 missplicing leads to the anemia characteristic of SF3B1-mutated MDS, although not to ringed sideroblast formation. We found that p38 MAPK is deactivated in SF3B1 mutant isogenic and patient cells and that MAP3K7 is an upstream positive effector of p38 MAPK. We demonstrate that disruption of this MAP3K7-p38 MAPK pathway leads to premature down-regulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation, erythroid hyperplasia, and ultimately apoptosis. Our findings thus define the mechanism leading to the severe anemia found in MDS patients harboring SF3B1 mutations.
Subject(s)
Anemia/metabolism , Erythropoiesis , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Signaling System , Mutation , Myelodysplastic Syndromes/metabolism , Phosphoproteins/metabolism , RNA Splicing Factors/metabolism , Anemia/genetics , Anemia/pathology , Cell Differentiation/genetics , Erythroid Cells/metabolism , Erythroid Cells/pathology , Humans , K562 Cells , MAP Kinase Kinase Kinases/genetics , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Phosphoproteins/genetics , RNA Splicing Factors/genetics , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Neurodegeneration is a leading cause of death in the developed world and a natural, albeit unfortunate, consequence of longer-lived populations. Despite great demand for therapeutic intervention, it is often the case that these diseases are insufficiently understood at the basic molecular level. What little is known has prompted much hopeful speculation about a generalized mechanistic thread that ties these disparate conditions together at the subcellular level and can be exploited for broad curative benefit. In this review, we discuss a prominent theory supported by genetic and pathological changes in an array of neurodegenerative diseases: that neurons are particularly vulnerable to disruption of RNA-binding protein dosage and dynamics. Here we synthesize the progress made at the clinical, genetic, and biophysical levels and conclude that this perspective offers the most parsimonious explanation for these mysterious diseases. Where appropriate, we highlight the reciprocal benefits of cross-disciplinary collaboration between disease specialists and RNA biologists as we envision a future in which neurodegeneration declines and our understanding of the broad importance of RNA processing deepens.
Subject(s)
Neurodegenerative Diseases/metabolism , RNA-Binding Proteins/metabolism , Aging , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Microsatellite Repeats/genetics , Neurodegenerative Diseases/genetics , Neurons/metabolism , Organelles/metabolism , RNA/metabolism , RNA Processing, Post-Transcriptional , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolism , RNA-Binding Proteins/geneticsABSTRACT
Many long noncoding RNAs (lncRNAs) are unstable and rapidly degraded in the nucleus by the nuclear exosome. An exosome adaptor complex called NEXT (nuclear exosome targeting) functions to facilitate turnover of some of these lncRNAs. Here we show that knockdown of one NEXT subunit, Mtr4, but neither of the other two subunits, resulted in accumulation of two types of lncRNAs: prematurely terminated RNAs (ptRNAs) and upstream antisense RNAs (uaRNAs). This suggested a NEXT-independent Mtr4 function, and, consistent with this, we isolated a distinct complex containing Mtr4 and the zinc finger protein ZFC3H1. Strikingly, knockdown of either protein not only increased pt/uaRNA levels but also led to their accumulation in the cytoplasm. Furthermore, all pt/uaRNAs examined associated with active ribosomes, but, paradoxically, this correlated with a global reduction in heavy polysomes and overall repression of translation. Our findings highlight a critical role for Mtr4/ZFC3H1 in nuclear surveillance of naturally unstable lncRNAs to prevent their accumulation, transport to the cytoplasm, and resultant disruption of protein synthesis.
Subject(s)
Active Transport, Cell Nucleus/genetics , Cytoplasm/metabolism , Gene Expression Regulation/genetics , RNA Helicases/metabolism , RNA, Nuclear/metabolism , Transcription Factors/metabolism , Gene Knockdown Techniques , HEK293 Cells , HeLa Cells , Humans , RNA Helicases/genetics , RNA Stability , Transcription Factors/geneticsABSTRACT
The GGGGCC hexanucleotide expansion in C9orf72 (C9) is the most frequent known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), yet a clear understanding of how C9 fits into the broader context of ALS/FTD pathology has remained lacking. The repetitive RNA derived from the C9 repeat is known to sequester hnRNPH, a splicing regulator, into insoluble aggregates, resulting in aberrant alternative splicing. Furthermore, hnRNPH insolubility and altered splicing of a robust set of targets have been observed to correlate in C9 and sporadic ALS/FTD patients alike, suggesting that changes along this axis are a core feature of disease pathogenesis. Here, we characterize previously uncategorized RNA splicing defects involving widespread intron retention affecting almost 2000 transcripts in C9ALS/FTD brains exhibiting a high amount of sequestered, insoluble hnRNPH. These intron retention events appear not to alter overall expression levels of the affected transcripts but rather the protein-coding regions. These retained introns affect transcripts in multiple cellular pathways predicted to be involved in C9 as well as sporadic ALS/FTD etiology, including the proteasomal and autophagy systems. The retained intron pre-mRNAs display a number of characteristics, including enrichment of hnRNPH-bound splicing enhancer motifs and a propensity for G-quadruplex (G-Q) formation, linking the defective splicing directly to high amounts of sequestered hnRNPH. Together, our results reveal previously undetected splicing defects in high insoluble hnRNPH-associated C9ALS brains, suggesting a feedback between effective RNA-binding protein dosage and protein quality control in C9, and perhaps all, ALS/FTD.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , C9orf72 Protein/genetics , Frontotemporal Dementia/genetics , Gene Regulatory Networks , Sequence Analysis, RNA/methods , Aged , Aged, 80 and over , Amyotrophic Lateral Sclerosis/metabolism , Brain/metabolism , C9orf72 Protein/metabolism , Case-Control Studies , Female , Frontotemporal Dementia/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Introns , Male , Middle Aged , Proteostasis , RNA SplicingABSTRACT
The gene encoding the core spliceosomal protein SF3B1 is the most frequently mutated gene encoding a splicing factor in a variety of hematologic malignancies and solid tumors. SF3B1 mutations induce use of cryptic 3' splice sites (3'ss), and these splicing errors contribute to tumorigenesis. However, it is unclear how widespread this type of cryptic 3'ss usage is in cancers and what is the full spectrum of genetic mutations that cause such missplicing. To address this issue, we performed an unbiased pan-cancer analysis to identify genetic alterations that lead to the same aberrant splicing as observed with SF3B1 mutations. This analysis identified multiple mutations in another spliceosomal gene, SUGP1, that correlated with significant usage of cryptic 3'ss known to be utilized in mutant SF3B1 expressing cells. Remarkably, this is consistent with recent biochemical studies that identified a defective interaction between mutant SF3B1 and SUGP1 as the molecular defect responsible for cryptic 3'ss usage. Experimental validation revealed that five different SUGP1 mutations completely or partially recapitulated the 3'ss defects. Our analysis suggests that SUGP1 mutations in cancers can induce missplicing identical or similar to that observed in mutant SF3B1 cancers.
Subject(s)
Computational Biology/methods , Mutation , Neoplasms/genetics , Phosphoproteins/genetics , RNA Splice Sites , RNA Splicing Factors/genetics , RNA Splicing , DNA Mutational Analysis , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Neoplasms/pathology , SpliceosomesABSTRACT
Amyotrophic lateral sclerosis (ALS) is caused by mutations in a number of genes, including the gene encoding the RNA/DNA-binding protein translocated in liposarcoma or fused in sarcoma (TLS/FUS or FUS). Previously, we identified a number of FUS target genes, among them MECP2. To investigate how ALS mutations in FUS might impact target gene expression, we examined the effects of several FUS derivatives harboring ALS mutations, such as R521C (FUS(C)), on MECP2 expression in transfected human U87 cells. Strikingly, FUS(C) and other mutants not only altered MECP2 alternative splicing but also markedly increased mRNA abundance, which we show resulted from sharply elevated stability. Paradoxically, however, MeCP2 protein levels were significantly reduced in cells expressing ALS mutant derivatives. Providing a parsimonious explanation for these results, biochemical fractionation and in vivo localization studies revealed that MECP2 mRNA colocalized with cytoplasmic FUS(C) in insoluble aggregates, which are characteristic of ALS mutant proteins. Together, our results establish that ALS mutations in FUS can strongly impact target gene expression, reflecting a dominant effect of FUS-containing aggregates.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Gene Expression Regulation/genetics , RNA-Binding Protein FUS/genetics , RNA-Binding Protein FUS/metabolism , Amyotrophic Lateral Sclerosis/physiopathology , Cell Line, Tumor , Cytoplasm/metabolism , Humans , Methyl-CpG-Binding Protein 2/genetics , Methyl-CpG-Binding Protein 2/metabolism , Mutation , Protein Transport , RNA Splicing/genetics , RNA Stability/genetics , RNA, Messenger/metabolismABSTRACT
The key RNA sequence elements and protein factors necessary for 3' processing of polyadenylated mRNA precursors are well known. Recent studies, however, have significantly reshaped current models for the protein-RNA interactions involved in poly(A) site recognition, painting a picture more complex than previously envisioned and also providing new insights into regulation of this important step in gene expression. Here we review the recent advances in this area and provide a perspective for future studies.
Subject(s)
Gene Expression Regulation , RNA Precursors/metabolism , RNA, Messenger/metabolism , Amino Acid Motifs , Cleavage And Polyadenylation Specificity Factor/metabolism , Conserved Sequence , Polyadenylation , Protein Binding , RNA Precursors/geneticsABSTRACT
Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.
Subject(s)
Breast Neoplasms/physiopathology , Chromatin Assembly and Disassembly/genetics , Gene Expression Regulation, Neoplastic , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Vascular Endothelial Growth Factor Receptor-2/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , Cell Line, Tumor , Chromosomal Proteins, Non-Histone/metabolism , HT29 Cells , Humans , MCF-7 Cells , Mutation/genetics , Nucleosomes/metabolism , Promoter Regions, Genetic/genetics , Protein Binding , Protein Conformation , Transcription Factors/metabolismABSTRACT
Polyadenylation of mRNA precursors is mediated by a large multisubunit protein complex. Here we show that RBBP6 (retinoblastoma-binding protein 6), identified initially as an Rb- and p53-binding protein, is a component of this complex and functions in 3' processing in vitro and in vivo. RBBP6 associates with other core factors, and this interaction is mediated by an unusual ubiquitin-like domain, DWNN ("domain with no name"), that is required for 3' processing activity. The DWNN is also expressed, via alternative RNA processing, as a small single-domain protein (isoform 3 [iso3]). Importantly, we show that iso3, known to be down-regulated in several cancers, competes with RBBP6 for binding to the core machinery, thereby inhibiting 3' processing. Genome-wide analyses following RBBP6 knockdown revealed decreased transcript levels, especially of mRNAs with AU-rich 3' untranslated regions (UTRs) such as c-Fos and c-Jun, and increased usage of distal poly(A) sites. Our results implicate RBBP6 and iso3 as novel regulators of 3' processing, especially of RNAs with AU-rich 3' UTRs.