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1.
Front Immunol ; 14: 1091403, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36761770

RESUMO

Regulation of mRNA polyadenylation is important for response to external signals and differentiation in several cell types, and results in mRNA isoforms that vary in the amount of coding sequence or 3' UTR regulatory elements. However, its role in differentiation of monocytes to macrophages has not been investigated. Macrophages are key effectors of the innate immune system that help control infection and promote tissue-repair. However, overactivity of macrophages contributes to pathogenesis of many diseases. In this study, we show that macrophage differentiation is characterized by shortening and lengthening of mRNAs in relevant cellular pathways. The cleavage/polyadenylation (C/P) proteins increase during differentiation, suggesting a possible mechanism for the observed changes in poly(A) site usage. This was surprising since higher C/P protein levels correlate with higher proliferation rates in other systems, but monocytes stop dividing after induction of differentiation. Depletion of CstF64, a C/P protein and known regulator of polyadenylation efficiency, delayed macrophage marker expression, cell cycle exit, attachment, and acquisition of structural complexity, and impeded shortening of mRNAs with functions relevant to macrophage biology. Conversely, CstF64 overexpression increased use of promoter-proximal poly(A) sites and caused the appearance of differentiated phenotypes in the absence of induction. Our findings indicate that regulation of polyadenylation plays an important role in macrophage differentiation.


Assuntos
Poli A , Poliadenilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Poli A/metabolismo , Diferenciação Celular , Macrófagos/metabolismo
2.
Mol Cell Biol ; 42(12): e0017422, 2022 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-36519931

RESUMO

We have previously described the role of an essential Saccharomyces cerevisiae gene, important for cleavage and polyadenylation 1 (IPA1), in the regulation of gene expression through its interaction with Ysh1, the endonuclease subunit of the mRNA 3'-end processing complex. Through a similar mechanism, the mammalian homolog ubiquitin protein ligase E3D (UBE3D) promotes the migratory and invasive potential of breast cancer cells, but its role in the regulation of gene expression during normal cellular differentiation has not previously been described. In this study, we show that CRISPR/Cas9-mediated knockout of Ube3d in 3T3-L1 cells blocks their ability to differentiate into mature adipocytes. Consistent with previous studies in other cell types, Ube3d knockout leads to decreased levels of CPSF73 and global changes in cellular mRNAs indicative of a loss of 3'-end processing capacity. Ube3d knockout cells also display decreased expression of known preadipogenic markers. Overexpression of either UBE3D or CPSF73 rescues the differentiation defect and partially restores protein levels of these markers. These results support a model in which UBE3D is necessary for the maintenance of the adipocyte-committed state via its regulation of the mRNA 3'-end processing machinery.


Assuntos
Adipócitos , Adipogenia , Ubiquitina-Proteína Ligases , Animais , Camundongos , Células 3T3-L1 , Adipócitos/metabolismo , Adipogenia/genética , Adipogenia/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Mamíferos/genética , Mamíferos/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
3.
iScience ; 25(8): 104804, 2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-35992060

RESUMO

Cleavage by the endonuclease CPSF73 and polyadenylation of nascent RNA is an essential step in co-transcriptional mRNA maturation. Recent work has surprisingly identified CPSF73 as a promising drug target for inhibiting the growth of specific cancers, triggering further studies on understanding CPSF73 regulation and functions in cells. Here, we report that a HECT-like E3 ligase, UBE3D, participates in stabilizing CPFS73 protein by preventing its ubiquitin-mediated degradation by the proteasome. Depletion of UBE3D leads to CPSF73 downregulation, a pre-mRNA cleavage defect, and dysregulated gene expression in cells. UBE3D dysfunction or chemical inactivation of CPSF73 inhibited migration and invasion as well as stem cell renewal phenotypes in vitro in triple-negative breast cancer cells. In addition, genetic overexpression of CPSF73 promoted breast cancer stemness and knocking down CPSF73 inhibited stem cell renewal properties. Together, our findings indicate that targeting the pre-mRNA processing nuclease CPSF73 has potential for breast cancer therapy.

4.
Trends Biochem Sci ; 46(9): 772-784, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33941430

RESUMO

Cleavage of nascent transcripts is a fundamental process for eukaryotic mRNA maturation and for the production of different mRNA isoforms. In eukaryotes, cleavage of mRNA precursors by the highly conserved endonuclease CPSF73 is critical for mRNA stability, export from the nucleus, and translation. As an essential enzyme in the cell, CPSF73 surprisingly shows promise as a drug target for specific cancers and for protozoan parasites. In this review, we cover our current understanding of CPSF73 in cleavage and polyadenylation, histone pre-mRNA processing, and transcription termination. We discuss the potential of CPSF73 as a target for novel therapeutics and highlight further research into the regulation of CPSF73 that will be critical to understanding its role in cancer and other diseases.


Assuntos
Fator de Especificidade de Clivagem e Poliadenilação , Precursores de RNA , Fator de Especificidade de Clivagem e Poliadenilação/genética , Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Endonucleases/genética , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
6.
Nucleic Acids Res ; 48(10): 5407-5425, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32356874

RESUMO

Adjusting DNA structure via epigenetic modifications, and altering polyadenylation (pA) sites at which precursor mRNA is cleaved and polyadenylated, allows cells to quickly respond to environmental stress. Since polyadenylation occurs co-transcriptionally, and specific patterns of nucleosome positioning and chromatin modifications correlate with pA site usage, epigenetic factors potentially affect alternative polyadenylation (APA). We report that the histone H3K4 methyltransferase Set1, and the histone H3K36 methyltransferase Set2, control choice of pA site in Saccharomyces cerevisiae, a powerful model for studying evolutionarily conserved eukaryotic processes. Deletion of SET1 or SET2 causes an increase in serine-2 phosphorylation within the C-terminal domain of RNA polymerase II (RNAP II) and in the recruitment of the cleavage/polyadenylation complex, both of which could cause the observed switch in pA site usage. Chemical inhibition of TOR signaling, which causes nutritional stress, results in Set1- and Set2-dependent APA. In addition, Set1 and Set2 decrease efficiency of using single pA sites, and control nucleosome occupancy around pA sites. Overall, our study suggests that the methyltransferases Set1 and Set2 regulate APA induced by nutritional stress, affect the RNAP II C-terminal domain phosphorylation at Ser2, and control recruitment of the 3' end processing machinery to the vicinity of pA sites.


Assuntos
Histona-Lisina N-Metiltransferase/fisiologia , Metiltransferases/fisiologia , Poliadenilação , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Cromatina/química , Cromatina/efeitos dos fármacos , Deleção de Genes , Regulação Fúngica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histonas , Metiltransferases/genética , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sirolimo/farmacologia , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
7.
RNA Biol ; 17(5): 689-702, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32009536

RESUMO

Mutation of the essential yeast protein Ipa1 has previously been demonstrated to cause defects in pre-mRNA 3' end processing and growth, but the mechanism underlying these defects was not clear. In this study, we show that the ipa1-1 mutation causes a striking depletion of Ysh1, the evolutionarily conserved endonuclease subunit of the 19-subunit mRNA Cleavage/Polyadenylation (C/P) complex, but does not decrease other C/P subunits. YSH1 overexpression rescues both the growth and 3' end processing defects of the ipa1-1 mutant. YSH1 mRNA level is unchanged in ipa1-1 cells, and proteasome inactivation prevents Ysh1 loss and causes accumulation of ubiquitinated Ysh1. Ysh1 ubiquitination is mediated by the Ubc4 ubiquitin-conjugating enzyme and Mpe1, which in addition to its function in C/P, is also a RING ubiquitin ligase. In summary, Ipa1 affects mRNA processing by controlling the availability of the C/P endonuclease and may represent a regulatory mechanism that could be rapidly deployed to facilitate reprogramming of cellular responses.


Assuntos
Endonucleases/metabolismo , Regulação da Expressão Gênica , RNA Mensageiro/genética , Ubiquitina/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Complexos Multiproteicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo
8.
Cell Rep ; 26(7): 1919-1933.e5, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30759400

RESUMO

The yeast protein Ipa1 was recently discovered to interact with the Ysh1 endonuclease of the pre-mRNA cleavage and polyadenylation (C/P) machinery, and Ipa1 mutation impairs 3'end processing. We report that Ipa1 globally promotes proper transcription termination and poly(A) site selection, but with variable effects on genes depending upon the specific configurations of polyadenylation signals. Our findings suggest that the role of Ipa1 in termination is mediated through interaction with Ysh1, since Ipa1 mutation leads to decrease in Ysh1 and poor recruitment of the C/P complex to a transcribed gene. The Ipa1 association with transcriptionally active chromatin resembles that of elongation factors, and the mutant shows defective Pol II elongation kinetics in vivo. Ysh1 overexpression in the Ipa1 mutant rescues the termination defect, but not the mutant's sensitivity to 6-azauracil, an indicator of defective elongation. Our findings support a model in which an Ipa1/Ysh1 complex helps coordinate transcription elongation and 3' end processing.


Assuntos
Endonucleases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica
9.
Science ; 353(6306)2016 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-27708008

RESUMO

We generated a global genetic interaction network for Saccharomyces cerevisiae, constructing more than 23 million double mutants, identifying about 550,000 negative and about 350,000 positive genetic interactions. This comprehensive network maps genetic interactions for essential gene pairs, highlighting essential genes as densely connected hubs. Genetic interaction profiles enabled assembly of a hierarchical model of cell function, including modules corresponding to protein complexes and pathways, biological processes, and cellular compartments. Negative interactions connected functionally related genes, mapped core bioprocesses, and identified pleiotropic genes, whereas positive interactions often mapped general regulatory connections among gene pairs, rather than shared functionality. The global network illustrates how coherent sets of genetic interactions connect protein complex and pathway modules to map a functional wiring diagram of the cell.


Assuntos
Redes Reguladoras de Genes , Genes Fúngicos/fisiologia , Pleiotropia Genética/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Epistasia Genética , Genes Essenciais
10.
Sci Adv ; 2(11): e1501662, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28138513

RESUMO

The complexity by which cells regulate gene and protein expression is multifaceted and intricate. Regulation of 3' untranslated region (UTR) processing of mRNA has been shown to play a critical role in development and disease. However, the process by which cells select alternative mRNA forms is not well understood. We discovered that the Saccharomyces cerevisiae lysine demethylase, Jhd2 (also known as KDM5), recruits 3'UTR processing machinery and promotes alteration of 3'UTR length for some genes in a demethylase-dependent manner. Interaction of Jhd2 with both chromatin and RNA suggests that Jhd2 affects selection of polyadenylation sites through a transcription-coupled mechanism. Furthermore, its mammalian homolog KDM5B (also known as JARID1B or PLU1), but not KDM5A (also known as JARID1A or RBP2), promotes shortening of CCND1 transcript in breast cancer cells. Consistent with these results, KDM5B expression correlates with shortened CCND1 in human breast tumor tissues. In contrast, both KDM5A and KDM5B are involved in the lengthening of DICER1. Our findings suggest both a novel role for this family of demethylases and a novel targetable mechanism for 3'UTR processing.


Assuntos
Regiões 3' não Traduzidas , Neoplasias da Mama/enzimologia , Histona Desmetilases com o Domínio Jumonji/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Neoplasias da Mama/genética , Ciclina D1/genética , Ciclina D1/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Células MCF-7 , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Proteína 2 de Ligação ao Retinoblastoma/genética , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Ribonuclease III/genética , Ribonuclease III/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Mol Cell Biol ; 34(21): 3955-67, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25135474

RESUMO

Almost all eukaryotic mRNAs must be polyadenylated at their 3' ends to function in protein synthesis. This modification occurs via a large nuclear complex that recognizes signal sequences surrounding a poly(A) site on mRNA precursor, cleaves at that site, and adds a poly(A) tail. While the composition of this complex is known, the functions of some subunits remain unclear. One of these is a multidomain protein called Mpe1 in the yeast Saccharomyces cerevisiae and RBBP6 in metazoans. The three conserved domains of Mpe1 are a ubiquitin-like (UBL) domain, a zinc knuckle, and a RING finger domain characteristic of some ubiquitin ligases. We show that mRNA 3'-end processing requires all three domains of Mpe1 and that more than one region of Mpe1 is involved in contact with the cleavage/polyadenylation factor in which Mpe1 resides. Surprisingly, both the zinc knuckle and the RING finger are needed for RNA-binding activity. Consistent with a role for Mpe1 in ubiquitination, mutation of Mpe1 decreases the association of ubiquitin with Pap1, the poly(A) polymerase, and suppressors of mpe1 mutants are linked to ubiquitin ligases. Furthermore, an inhibitor of ubiquitin-mediated interactions blocks cleavage, demonstrating for the first time a direct role for ubiquitination in mRNA 3'-end processing.


Assuntos
Domínios RING Finger/fisiologia , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Sequência de Aminoácidos , Sequência Conservada , Proteínas Associadas a Pancreatite , Poliadenilação , Polinucleotídeo Adenililtransferase/metabolismo , Estabilidade de RNA , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinação , Fatores de Poliadenilação e Clivagem de mRNA/genética
12.
Genome Res ; 23(10): 1690-703, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23788651

RESUMO

Systemic response to DNA damage and other stresses is a complex process that includes changes in the regulation and activity of nearly all stages of gene expression. One gene regulatory mechanism used by eukaryotes is selection among alternative transcript isoforms that differ in polyadenylation [poly(A)] sites, resulting in changes either to the coding sequence or to portions of the 3' UTR that govern translation, stability, and localization. To determine the extent to which this means of regulation is used in response to DNA damage, we conducted a global analysis of poly(A) site usage in Saccharomyces cerevisiae after exposure to the UV mimetic, 4-nitroquinoline 1-oxide (4NQO). Two thousand thirty-one genes were found to have significant variation in poly(A) site distributions following 4NQO treatment, with a strong bias toward loss of short transcripts, including many with poly(A) sites located within the protein coding sequence (CDS). We further explored one possible mechanism that could contribute to the widespread differences in mRNA isoforms. The change in poly(A) site profile was associated with an inhibition of cleavage and polyadenylation in cell extract and a decrease in the levels of several key subunits in the mRNA 3'-end processing complex. Sequence analysis identified differences in the cis-acting elements that flank putatively suppressed and enhanced poly(A) sites, suggesting a mechanism that could discriminate between variable and constitutive poly(A) sites. Our analysis indicates that variation in mRNA length is an important part of the regulatory response to DNA damage.


Assuntos
Dano ao DNA , Genoma Fúngico , Isoformas de RNA/metabolismo , Saccharomyces cerevisiae/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Óxidos N-Cíclicos/farmacologia , Etiquetas de Sequências Expressas , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Fases de Leitura Aberta , Poliadenilação , Isoformas de RNA/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Análise de Sequência de RNA
13.
J Biol Chem ; 288(27): 19750-9, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23689372

RESUMO

Proper RNA polymerase II (Pol II) transcription termination is essential to generate stable transcripts, to prevent interference at downstream loci, and to recycle Pol II back to the promoter (1-3). As such, termination is an intricately controlled process that is tightly regulated by a variety of different cis- and trans-acting factors (4, 5). Although many eukaryotic termination factors have been identified to date, the details of the precise molecular mechanisms governing termination remain to be elucidated. We devised an in vitro transcription system to study specific Pol II termination. We show for the first time that the exonucleolytic Rat1·Rai1 complex can elicit the release of stalled Pol II in vitro and can do so in the absence of other factors. We also find that Rtt103, which interacts with the Pol II C-terminal domain (CTD) and with Rat1, can rescue termination activity of an exonucleolytically deficient Rat1 mutant. In light of our findings, we posit a model whereby functional nucleolytic activity is not the feature of Rat1 that ultimately promotes termination. Degradation of the nascent transcript allows Rat1 to pursue Pol II in a guided fashion and arrive at the site of RNA exit from Pol II. Upon this arrival, however, it is perhaps the specific and direct contact between Rat1 and Pol II that transmits the signal to terminate transcription.


Assuntos
Exorribonucleases/metabolismo , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas/fisiologia , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Terminação da Transcrição Genética/fisiologia , Exorribonucleases/genética , Modelos Biológicos , Complexos Multiproteicos/genética , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Polimerase II/genética , Estabilidade de RNA/fisiologia , Proteínas de Ligação a RNA , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
14.
RNA ; 17(4): 652-64, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21282348

RESUMO

Synthesis of the poly(A) tail of mRNA in Saccharomyces cerevisiae requires recruitment of the polymerase Pap1 to the 3' end of cleaved pre-mRNA. This is made possible by the tethering of Pap1 to the Cleavage/Polyadenylation Factor (CPF) by Fip1. We have recently reported that Fip1 is an unstructured protein in solution, and proposed that it might maintain this conformation as part of CPF, when bound to Pap1. However, the role that this feature of Fip1 plays in 3' end processing has not been investigated. We show here that Fip1 has a flexible linker in the middle of the protein, and that removal or replacement of the linker affects the efficiency of polyadenylation. However, the point of tethering is not crucial, as a fusion protein of Pap1 and Fip1 is fully functional in cells lacking genes encoding the essential individual proteins, and directly tethering Pap1 to RNA increases the rate of poly(A) addition. We also find that the linker region of Fip1 provides a platform for critical interactions with other parts of the processing machinery. Our results indicate that the Fip1 linker, through its flexibility and protein/protein interactions, allows Pap1 to reach the 3' end of the cleaved RNA and efficiently initiate poly(A) addition.


Assuntos
Sistemas de Transporte de Aminoácidos/metabolismo , Poliadenilação , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Sequência de Bases , Proteínas Associadas a Pancreatite , Estrutura Terciária de Proteína , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/genética
15.
PLoS One ; 3(9): e3273, 2008 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-18818768

RESUMO

The production of a functional mRNA is regulated at every step of transcription. An area not well-understood is the transition of RNA polymerase II from elongation to termination. The S. cerevisiae SR-like protein Npl3 functions to negatively regulate transcription termination by antagonizing the binding of polyA/termination proteins to the mRNA. In this study, Npl3 is shown to interact with the CTD and have a direct stimulatory effect on the elongation activity of the polymerase. The interaction is inhibited by phosphorylation of Npl3. In addition, Casein Kinase 2 was found to be required for the phosphorylation of Npl3 and affect its ability to compete against Rna15 (Cleavage Factor I) for binding to polyA signals. Our results suggest that phosphorylation of Npl3 promotes its dissociation from the mRNA/RNAP II, and contributes to the association of the polyA/termination factor Rna15. This work defines a novel role for Npl3 in elongation and its regulation by phosphorylation.


Assuntos
Proteínas Nucleares/metabolismo , RNA Polimerase II/química , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ligação Competitiva , Caseína Quinase II/metabolismo , Domínio Catalítico , Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Fosforilação , Poli A/química , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Transcrição Gênica , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
16.
RNA ; 13(10): 1756-64, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17684230

RESUMO

Multiple steps in mRNA processing and transcription are coupled. Notably, the processing of mRNA 3' ends is linked to transcription termination by RNA polymerase II. Previously, we found that the yeast hnRNP protein Npl3 can negatively regulate 3' end mRNA formation and termination at the GAL1 gene. Here we show that overexpression of the Hrp1 or Rna14 subunits of the CF IA polyadenylation factor increases recognition of a weakened polyadenylation site. Genetic interactions of mutant alleles of NPL3 or HRP1 with RNA15 also indicate antagonism between these factors. Npl3 competes with Rna15 for binding to a polyadenylation precursor and inhibits cleavage and polyadenylation in vitro. These results suggest that an important function of hnRNP proteins is to ensure the fidelity of mRNA processing. Our results support a model in which balanced competition of Npl3 with mRNA processing factors may promote recognition of proper polyadenylation sites while suppressing cryptic sites.


Assuntos
Proteínas Nucleares/metabolismo , Poliadenilação , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Saccharomyces cerevisiae/genética
17.
J Biol Chem ; 278(35): 33000-10, 2003 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-12819204

RESUMO

Messenger RNA 3'-end formation is functionally coupled to transcription by RNA polymerase II. By tagging and purifying Ref2, a non-essential protein previously implicated in mRNA cleavage and termination, we isolated a multiprotein complex, holo-CPF, containing the yeast cleavage and polyadenylation factor (CPF) and six additional polypeptides. The latter can form a distinct complex, APT, in which Pti1, Swd2, a type I protein phosphatase (Glc7), Ssu72 (a TFIIB and RNA polymerase II-associated factor), Ref2, and Syc1 are associated with the Pta1 subunit of CPF. Systematic tagging and purification of holo-CPF subunits revealed that yeast extracts contain similar amounts of CPF and holo-CPF. By purifying holo-CPF from strains lacking Ref2 or containing truncated subunits, subcomplexes were isolated that revealed additional aspects of the architecture of APT and holo-CPF. Chromatin immunoprecipitation was used to localize Ref2, Ssu72, Pta1, and other APT subunits on small nucleolar RNA (snoRNA) genes and primarily near the polyadenylation signals of the constitutively expressed PYK1 and PMA1 genes. Use of mutant components of APT revealed that Ssu72 is important for preventing readthrough-dependent expression of downstream genes for both snoRNAs and polyadenylated transcripts. Ref2 and Pta1 similarly affect at least one snoRNA transcript.


Assuntos
Proteínas de Transporte/química , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , DNA Recombinante/metabolismo , Eletroforese em Gel de Poliacrilamida , Modelos Biológicos , Peptídeos/química , Fosfoproteínas Fosfatases/metabolismo , Poliadenilação , Testes de Precipitina , Ligação Proteica , Proteína Fosfatase 1 , Estrutura Terciária de Proteína , RNA/metabolismo , Proteínas de Ligação a RNA/química , Saccharomyces cerevisiae , Transcrição Gênica , beta-Galactosidase/metabolismo
18.
Genes Dev ; 17(8): 1030-42, 2003 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-12704082

RESUMO

Transcription and processing of pre-mRNA are coupled events. By using a combination of biochemical, molecular, and genetic methods, we have found that the phylogenetically conserved transcription factor Ssu72 is a component of the cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae. Our results demonstrate that Ssu72 is required for 3' end cleavage of pre-mRNA but is dispensable for poly(A) addition and RNAP II termination. The in vitro cleavage defect caused by depletion of Ssu72 from cells can be rescued by addition of recombinant Ssu72. Ssu72 interacts physically and genetically with the Pta1 subunit of CPF. Overexpression of PTA1 causes synthetic lethality in an ssu72-3 mutant. Moreover, Sub1, which has been implicated in transcription initiation and termination, also interacts with Pta1, and overexpression of SUB1 suppresses the growth and processing defect of a pta1 mutation. Physical interactions of Ssu72 and Sub1 with Pta1 are mutually exclusive. Based on the interactions of Ssu72 and Sub1 with both the Pta1 of CPF and the TFIIB component of the initiation complex, we present a model describing how these novel connections between the transcription and 3' end processing machineries might facilitate transitions in the RNAP II transcription cycle.


Assuntos
Proteínas de Transporte/genética , RNA Polimerase II/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fator de Transcrição TFIIB/genética , Fator de Transcrição TFIIB/metabolismo , Transcrição Gênica , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/metabolismo , Proteínas de Transporte/metabolismo , Cromatografia de Afinidade , Primers do DNA/química , Escherichia coli/genética , Técnicas In Vitro , Mutagênese Sítio-Dirigida , Mutação/genética , Fosfoproteínas Fosfatases , Poli A , Reação em Cadeia da Polimerase , Testes de Precipitina , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Supressão Genética , Regiões Terminadoras Genéticas , Transativadores/metabolismo , Ativação Transcricional , Fatores de Poliadenilação e Clivagem de mRNA/química , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
19.
Nucleic Acids Res ; 31(6): 1744-52, 2003 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-12626716

RESUMO

Yth1, a subunit of yeast Cleavage Polyadenylation Factor (CPF), contains five CCCH zinc fingers. Yth1 was previously shown to interact with pre-mRNA and with two CPF subunits, Brr5/Ysh1 and the polyadenylation-specific Fip1, and to act in both steps of mRNA 3' end processing. In the present study, we have identified new domains involved in each interaction and have analyzed the consequences of mutating these regions on Yth1 function in vivo and in vitro. We have found that the essential fourth zinc finger (ZF4) of Yth1 is critical for interaction with Fip1 and RNA, but not for cleavage, and a single point mutation in ZF4 impairs only polyadenylation. Deletion of the essential N-terminal region that includes the ZF1 or deletion of ZF4 weakened the interaction with Brr5 in vitro. In vitro assays showed that the N-terminus is necessary for both processing steps. Of particular importance, we find that the binding of Fip1 to Yth1 blocks the RNA-Yth1 interaction, and that this inhibition requires the Yth1-interacting domain on Fip1. Our results suggest a role for Yth1 not only in the execution of cleavage and poly(A) addition, but also in the transition from one step to the other.


Assuntos
Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Dedos de Zinco/genética , Fatores de Poliadenilação e Clivagem de mRNA/genética , Sítios de Ligação/genética , Teste de Complementação Genética/métodos , Mutação , Fenótipo , Poli A/genética , Poli A/metabolismo , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo
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