RESUMO
In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90-R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA(+) adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs.
Assuntos
Proteínas Nucleares/química , Ribonucleoproteínas Nucleares Pequenas/química , Ribonucleoproteínas Nucleolares Pequenas/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Cristalografia por Raios X , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteômica/métodos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Fatores de TranscriçãoRESUMO
The nuclear cap-binding complex (CBC) stimulates multiple steps in several RNA maturation pathways, but how it functions in humans is incompletely understood. For small, capped RNAs such as pre-snRNAs, the CBC recruits PHAX. Here, we identify the CBCAP complex, composed of CBC, ARS2 and PHAX, and show that both CBCAP and CBC-ARS2 complexes can be reconstituted from recombinant proteins. ARS2 stimulates PHAX binding to the CBC and snRNA 3'-end processing, thereby coupling maturation with export. In vivo, CBC and ARS2 bind similar capped noncoding and coding RNAs and stimulate their 3'-end processing. The strongest effects are for cap-proximal polyadenylation sites, and this favors premature transcription termination. ARS2 functions partly through the mRNA 3'-end cleavage factor CLP1, which binds RNA Polymerase II through PCF11. ARS2 is thus a major CBC effector that stimulates functional and cryptic 3'-end processing sites.
Assuntos
Modelos Genéticos , Complexo Proteico Nuclear de Ligação ao Cap/fisiologia , Proteínas Nucleares/fisiologia , Proteínas de Transporte Nucleocitoplasmático/fisiologia , Fosfoproteínas/fisiologia , Processamento de Terminações 3' de RNA , Células HeLa , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/química , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Poli A/química , Poli A/metabolismoRESUMO
Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome and the cap-binding complex (CBC). The CBC associates with the ARS2 protein to form CBC-ARS2 (CBCA) and then further connects, together with the ZC3H18 protein, to the nuclear exosome targeting (NEXT) complex, thus forming CBC-NEXT (CBCN). RNA immunoprecipitation using CBCN factors as well as the analysis of combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5' cap links transcription termination to exosomal RNA degradation through CBCN.