RESUMO
Most eukaryotic pre-mRNAs must undergo 3'-end cleavage and polyadenylation prior to their export from the nucleus. A large number of proteins in several complexes participate in this 3'-end processing, including cleavage and polyadenylation specificity factor (CPSF) in mammals. The CPSF30 subunit contains five CCCH zinc fingers (ZFs), with ZF2-ZF3 being required for the recognition of the AAUAAA poly(A) signal. ZF4-ZF5 recruits the hFip1 subunit of CPSF, although the details of this interaction have not been characterized. Here we report the crystal structure of human CPSF30 ZF4-ZF5 in complex with residues 161-200 of hFip1 at 1.9 Å resolution, illuminating the molecular basis for their interaction. Unexpectedly, the structure reveals one hFip1 molecule binding to each ZF4 and ZF5, with a conserved mode of interaction. Our mutagenesis studies confirm that the CPSF30-hFip1 complex has 1:2 stoichiometry in vitro. Mutation of each binding site in CPSF30 still allows one copy of hFip1 to bind, while mutation of both sites abrogates binding. Our fluorescence polarization binding assays show that ZF4 has higher affinity for hFip1, with a Kd of 1.8 nM. We also demonstrate that two copies of the catalytic module of poly(A) polymerase (PAP) are recruited by the CPSF30-hFip1 complex in vitro, and both hFip1 binding sites in CPSF30 can support polyadenylation.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/química , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Modelos Moleculares , Fatores de Poliadenilação e Clivagem de mRNA/química , Sítios de Ligação , Fator de Especificidade de Clivagem e Poliadenilação/genética , Escherichia coli/genética , Humanos , Mutação , Ligação Proteica , Estrutura Quaternária de Proteína , Dedos de Zinco/fisiologia , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3'-processing of the pre-mRNA and transcription termination. Here, we present a genome-wide analysis of the 3'-transition in budding yeast. We find that the 3'-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the polyadenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and requires the "torpedo" exonuclease Rat1 (XRN2 in human). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3'-transition can result in increased transcription at downstream genes.
Assuntos
DNA Fúngico/metabolismo , Processamento de Terminações 3' de RNA , RNA Polimerase II/metabolismo , Precursores de RNA/biossíntese , RNA Fúngico/biossíntese , RNA Mensageiro/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA Fúngico/genética , Exorribonucleases/genética , Exorribonucleases/metabolismo , Modelos Genéticos , Ligação Proteica , RNA Polimerase II/genética , Precursores de RNA/genética , RNA Fúngico/genética , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/genética , Fatores de Poliadenilação e Clivagem de mRNA/metabolismoRESUMO
Seed dormancy is an important adaptive trait to prevent germination occurring at an inappropriate time. The mechanisms governing seed dormancy and germination are complex. Here, we report that FACTOR INTERACTING WITH POLY(A) POLYMERASE 1 (FIP1), a component of the pre-mRNA 3' end processing machinery, is involved in seed dormancy and germination processes in Arabidopsis thaliana. FIP1 is mainly expressed in seeds and the knockout of FIP1 causes reduced seed dormancy, indicating that FIP1 positively influences seed dormancy. Meanwhile, fip1 mutants are insensitive to exogenous ABA during seed germination and early seedling establishment. The terms 'seed maturation' and 'response to ABA stimulus' are significantly enriched in a gene ontology analysis based on genes differentially expressed between fip1-1 and the wild type. Several of these genes, including ABI5, DOG1 and PYL12, show significantly decreased transcript levels in fip1. Genetic analysis showed that either cyp707a2 or dog1-5 partially, but in combination completely, represses the reduced seed dormancy of fip1, indicating that the double mutant cyp707a2 dog1-5 is epistatic to fip1. Moreover, FIP1 is required for CFIM59, another component of pre-mRNA 3' end processing machinery, to govern seed dormancy and germination. Overall, we identified FIP1 as a regulator of seed dormancy and germination that plays a crucial role in governing these processes through the DOG1 and ABA pathways.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Ácido Abscísico/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Germinação/genética , Mutação , Dormência de Plantas/genética , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sementes/metabolismoRESUMO
CPSF73 is the endonuclease that catalyzes the cleavage reaction for 3'-end processing of mRNA precursors (pre-mRNAs) in two distinct machineries, a canonical machinery for the majority of pre-mRNAs and a U7 snRNP (U7 machinery) for replication-dependent histone pre-mRNAs in animal cells. CPSF73 also possesses 5'-3' exonuclease activity in the U7 machinery, degrading the downstream cleavage product after the endonucleolytic cleavage. Recent studies show that CPSF73 is a potential target for developing anticancer, antimalarial, and antiprotozoal drugs, spurring interest in identifying new small-molecule inhibitors against this enzyme. CPSF73 nuclease activity has so far been demonstrated using a gel-based end-point assay, using radiolabeled or fluorescently labeled RNA substrates. By taking advantage of unique properties of the U7 machinery, we have developed a novel, real-time fluorescence assay for the nuclease activity of CPSF73. This assay is facile and high-throughput, and should also be helpful for the discovery of new CPSF73 inhibitors.
Assuntos
Bioensaio , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Histonas/metabolismo , Processamento de Terminações 3' de RNA , Precursores de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U7/metabolismo , Sistema Livre de Células , Fator de Especificidade de Clivagem e Poliadenilação/química , Fator de Especificidade de Clivagem e Poliadenilação/genética , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fluorescência , Corantes Fluorescentes/química , Histonas/química , Histonas/genética , Humanos , Modelos Moleculares , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Proteólise , Precursores de RNA/química , Precursores de RNA/genética , Rodaminas/química , Ribonucleoproteína Nuclear Pequena U7/química , Ribonucleoproteína Nuclear Pequena U7/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Alternative polyadenylation (APA) is widespread in neuronal development and activity-mediated neural plasticity. However, the underlying molecular mechanisms are largely unknown. We used systematic genetic studies and genome-wide surveys of the transcriptional landscape to identify a context-dependent regulatory pathway controlling APA in the Caenorhabditis elegans nervous system. Loss of function in ssup-72, a Ser5 phosphatase for the RNA polymerase II (Pol II) C-terminal domain (CTD), dampens transcription termination at a strong intronic polyadenylation site (PAS) in unc-44/ankyrin yet promotes termination at the weak intronic PAS of the MAP kinase dlk-1. A nuclear protein, SYDN-1, which regulates neuronal development, antagonizes the function of SSUP-72 and several nuclear polyadenylation factors. This regulatory pathway allows the production of a neuron-specific isoform of unc-44 and an inhibitory isoform of dlk-1. Dysregulation of the unc-44 and dlk-1 mRNA isoforms in sydn-1 mutants impairs neuronal development. Deleting the intronic PAS of unc-44 results in increased pre-mRNA processing of neuronal ankyrin and suppresses sydn-1 mutants. These results reveal a mechanism by which regulation of CTD phosphorylation controls coding region APA in the nervous system.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/crescimento & desenvolvimento , Fosfoproteínas Fosfatases/metabolismo , Animais , Anquirinas/genética , Anquirinas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Mutação , Neurônios/enzimologia , Fosfoproteínas Fosfatases/genética , Poliadenilação , Ligação ProteicaRESUMO
Alternative polyadenylation (APA) determines stability, localization and translation potential of the majority of mRNA in eukaryotic cells. The heterodimeric mammalian cleavage factor II (CF IIm) is required for pre-mRNA 3' end cleavage and is composed of the RNA kinase hClp1 and the termination factor hPcf11; the latter protein binds to RNA and the RNA polymerase II carboxy-terminal domain. Here, we used siRNA mediated knockdown and poly(A) targeted RNA sequencing to analyze the role of CF IIm in gene expression and APA in estrogen receptor positive MCF7 breast cancer cells. Identified gene ontology terms link CF IIm function to regulation of growth factor activity, protein heterodimerization and the cell cycle. An overlapping requirement for hClp1 and hPcf11 suggested that CF IIm protein complex was involved in the selection of proximal poly(A) sites. In addition to APA shifts within 3' untranslated regions (3'-UTRs), we observed shifts from promoter proximal regions to the 3'-UTR facilitating synthesis of full-length mRNAs. Moreover, we show that several truncated mRNAs that resulted from APA within introns in MCF7 cells cosedimented with ribosomal components in an EDTA sensitive manner suggesting that those are translated into protein. We propose that CF IIm contributes to the regulation of mRNA function in breast cancer.
Assuntos
Neoplasias da Mama/genética , Poliadenilação/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Regiões 3' não Traduzidas/genética , Linhagem Celular Tumoral , Humanos , Células MCF-7 , Poli A/genética , Ligação Proteica/genética , RNA Polimerase II/genética , Precursores de RNA/genética , RNA Mensageiro/genéticaRESUMO
Circular RNAs with covalently linked ends are generated from many eukaryotic protein-coding genes when the pre-mRNA splicing machinery backsplices. These mature transcripts are resistant to digestion by exonucleases and typically have much longer half-lives than their associated linear mRNAs. Circular RNAs thus have great promise as sensitive biomarkers, including for detection of transcriptional activity. Here, we show that circular RNAs can serve as markers of readthrough transcription events in Drosophila and human cells, thereby revealing mechanistic insights into RNA polymerase II transcription termination as well as pre-mRNA 3' end processing. We describe methods that take advantage of plasmids that generate a circular RNA when an upstream polyadenylation signal fails to be used and/or RNA polymerase II fails to terminate. As a proof-of-principle, we show that RNAi-mediated depletion of well-established transcription termination factors, including the RNA endonuclease Cpsf73, results in increased circular RNA output from these plasmids in Drosophila and human cells. This method is generalizable as a circular RNA can be easily encoded downstream of any genomic region of interest. Circular RNA biomarkers, therefore, have great promise for identifying novel cellular factors and conditions that impact transcription termination processes.
Assuntos
Poliadenilação , RNA Circular , Biomarcadores , Poliadenilação/genética , RNA/genética , RNA/metabolismo , Splicing de RNA/genética , RNA Circular/genéticaRESUMO
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.
Assuntos
Regiões 3' não Traduzidas/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , RNA Mensageiro/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Poliadenilação/genética , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Nearly all eukaryotic messenger RNA precursors must undergo cleavage and polyadenylation at their 3'-end for maturation. A crucial step in this process is the recognition of the AAUAAA polyadenylation signal (PAS), and the molecular mechanism of this recognition has been a long-standing problem. Here, we report the cryo-electron microscopy structure of a quaternary complex of human CPSF-160, WDR33, CPSF-30, and an AAUAAA RNA at 3.4-Å resolution. Strikingly, the AAUAAA PAS assumes an unusual conformation that allows this short motif to be bound directly by both CPSF-30 and WDR33. The A1 and A2 bases are recognized specifically by zinc finger 2 (ZF2) of CPSF-30 and the A4 and A5 bases by ZF3. Interestingly, the U3 and A6 bases form an intramolecular Hoogsteen base pair and directly contact WDR33. CPSF-160 functions as an essential scaffold and preorganizes CPSF-30 and WDR33 for high-affinity binding to AAUAAA. Our findings provide an elegant molecular explanation for how PAS sequences are recognized for mRNA 3'-end formation.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Proteínas Nucleares/metabolismo , Poli A/metabolismo , Poliadenilação , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Fator de Especificidade de Clivagem e Poliadenilação/química , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Proteínas Nucleares/química , Poli A/química , Conformação Proteica , Precursores de RNA/química , RNA Mensageiro/químicaRESUMO
The nuclear poly(A) binding protein (PABPN1) has been suggested, on the basis of biochemical evidence, to play a role in mRNA polyadenylation by strongly increasing the processivity of poly(A) polymerase. While experiments in metazoans have tended to support such a role, the results were not unequivocal, and genetic data show that the S. pombe ortholog of PABPN1, Pab2, is not involved in mRNA polyadenylation. The specific model in which PABPN1 increases the rate of poly(A) tail elongation has never been examined in vivo. Here, we have used 4-thiouridine pulse-labeling to examine the lengths of newly synthesized poly(A) tails in human cells. Knockdown of PABPN1 strongly reduced the synthesis of full-length tails of â¼250 nucleotides, as predicted from biochemical data. We have also purified S. pombe Pab2 and the S. pombe poly(A) polymerase, Pla1, and examined their in vitro activities. Whereas PABPN1 strongly increases the activity of its cognate poly(A) polymerase in vitro, Pab2 was unable to stimulate Pla1 to any significant extent. Thus, in vitro and in vivo data are consistent in supporting a role of PABPN1 but not S. pombe Pab2 in the polyadenylation of mRNA precursors.
Assuntos
Poli A/genética , Proteína I de Ligação a Poli(A)/genética , Proteínas de Ligação a Poli(A)/genética , Polinucleotídeo Adenililtransferase/genética , Precursores de RNA/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Poli A/biossíntese , Proteína I de Ligação a Poli(A)/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Poliadenilação , Polinucleotídeo Adenililtransferase/metabolismo , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidade da Espécie , Especificidade por SubstratoRESUMO
The development of RNA imaging techniques and the establishment of systems biology approaches, together with the improvement of large-scale RNA-protein crosslinking immunoprecipitation protocols have enormously expanded our knowledge of RNA networks and the function of RNA-binding proteins in metazoans and model yeasts. In pathogenic fungi, the biological role of the vast majority of RNA-binding proteins and non-coding RNAs is still largely unknown. However, many RNA-dependent mechanisms which shape fungal pathogenicity have been defined. Here, advances made in this field are reviewed and further theories of biological significance are discussed in the light of latest findings.
Assuntos
Fungos/metabolismo , Fungos/patogenicidade , RNA Fúngico/metabolismo , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética , Estabilidade de RNA/genética , Virulência/genéticaRESUMO
BACKGROUND: HSF1 (heat shock factor 1) is a transcription factor that is found to facilitate malignant cancer development and proliferation. In cancer cells, HSF1 mediates a set of genes distinct from heat shock that contributes to malignancy. This set of genes is known as the HSF1 Cancer Signature genes or simply HSF1-CanSig genes. HSF1-CanSig genes function and operate differently than typical cancer-causing genes, yet it is involved in fundamental oncogenic processes. RESULTS: By utilizing expression data from 9241 cancer patients, we identified that human chromosome 8q21-24 is a location hotspot for the most frequently overexpressed HSF1-CanSig genes. Intriguingly, the strength of the HSF1 cancer program correlates with the number of overexpressed HSF1-CanSig genes in 8q, illuminating the essential role of HSF1 in mediating gene expression in different cancers. Chromosome 8q21-24 is found under selective pressure in preserving gene order as it exhibits strong synteny among human, mouse, rat, and bovine, although the biological significance remains unknown. Statistical modeling, hierarchical clustering, and gene ontology-based pathway analyses indicate crosstalk between HSF1-mediated responses and pre-mRNA 3' processing in cancers. CONCLUSIONS: Our results confirm the unique role of chromosome 8q mediated by the master regulator HSF1 in cancer cases. Additionally, this study highlights the connection between cellular processes triggered by HSF1 and pre-mRNA 3' processing in cancers.
Assuntos
Biomarcadores Tumorais/genética , Cromossomos Humanos Par 8 , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição de Choque Térmico/genética , Neoplasias/genética , Animais , Bovinos , Redes Reguladoras de Genes , Genoma Humano , Fatores de Transcrição de Choque Térmico/metabolismo , Humanos , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Regiões Promotoras Genéticas , Processamento Pós-Transcricional do RNA , Ratos , Células Tumorais CultivadasRESUMO
Alternative polyadenylation (APA) is a crucial mechanism for regulating gene expression during pre-mRNA 3' processing. Pre-mRNA 3' end processing factors is the main factor involved in this process. However, pre-mRNA 3' end processing factors in different cancer expression profiles and the relationship between pre-mRNA 3' end processing factors and tumor microenvironment and the prognosis of the same patient is still unclear. In this study, we conducted a comprehensive exploration of the core pre-mRNA 3' end processing factors across various cancer types by utilizing common cancer database, and revealing a robust correlation between the expression of these core factors and tumor characteristics. Leveraging advanced bioinformatics databases, we evaluated the expression levels and prognostic relevance of pre-mRNA 3' end processing factors across pan-cancer tissues. Our extensive pan-cancer analysis revealed unique expression patterns of pre-mRNA 3' end processing factors in both tumor and adjacent non-tumorous tissues. Notably, we found a significant correlation between the expression levels of pre-mRNA 3' end processing factors and patient prognosis. Furthermore, we identified strong associations between pre-mRNA 3' end processing factors expression and various factors, such as stromal, immune, RNA stemness, and DNA stemness scores across pan-cancer tissues. Our data also highlighted a link between the expression of pre-mRNA 3' end processing factors and sensitivity to specific drugs, including pyrazoloacndine, amonaflide, and chelerythrinede, among others. We found four key pre-mRNA 3' end processing factors that play a crucial role in mRNA preprocessing. Our study illuminates the potential promotion and inhibition role of pre-mRNA 3' end processing regulators in the progression of cancer, CPSF2, CPSF3, CSTF2, SYMPK offering valuable insights for future research investigations on these regulators as diagnostic markers and therapeutic targets across pan-cancer.
Assuntos
Neoplasias , Precursores de RNA , Microambiente Tumoral , Humanos , Microambiente Tumoral/genética , Neoplasias/genética , Neoplasias/patologia , Prognóstico , Precursores de RNA/genética , Precursores de RNA/metabolismo , Regulação Neoplásica da Expressão Gênica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Processamento de Terminações 3' de RNA/genética , Biologia Computacional/métodos , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , PoliadenilaçãoRESUMO
Replication-dependent (RD) core histone mRNA produced during S-phase is the only known metazoan protein-coding mRNA presenting a 3' stem-loop instead of the otherwise universal polyA tail. A metallo ß-lactamase (MBL) fold enzyme, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to be the sole endonuclease responsible for 3' end processing of both mRNA classes. We report cellular, genetic, biochemical, substrate selectivity, and crystallographic studies providing evidence that an additional endoribonuclease, MBL domain containing protein 1 (MBLAC1), is selective for 3' processing of RD histone pre-mRNA during the S-phase of the cell cycle. Depletion of MBLAC1 in cells significantly affects cell cycle progression thus identifying MBLAC1 as a new type of S-phase-specific cancer target.