RESUMO
The nuclear Cap-Binding Complex (CBC), consisting of Nuclear Cap-Binding Protein 1 (NCBP1) and 2 (NCBP2), associates with the nascent 5'cap of RNA polymerase II transcripts and impacts RNA fate decisions. Recently, the C17orf85 protein, also called NCBP3, was suggested to form an alternative CBC by replacing NCBP2. However, applying protein-protein interaction screening of NCBP1, 2 and 3, we find that the interaction profile of NCBP3 is distinct. Whereas NCBP1 and 2 identify known CBC interactors, NCBP3 primarily interacts with components of the Exon Junction Complex (EJC) and the TRanscription and EXport (TREX) complex. NCBP3-EJC association in vitro and in vivo requires EJC core integrity and the in vivo RNA binding profiles of EJC and NCBP3 overlap. We further show that NCBP3 competes with the RNA degradation factor ZC3H18 for binding CBC-bound transcripts, and that NCBP3 positively impacts the nuclear export of polyadenylated RNAs and the expression of large multi-exonic transcripts. Collectively, our results place NCBP3 with the EJC and TREX complexes in supporting mRNA expression.
Assuntos
RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , RNA/genética , Transcrição Gênica , Transporte Ativo do Núcleo Celular/genética , Núcleo Celular/genética , Éxons , Regulação da Expressão Gênica/genética , Humanos , Complexo Proteico Nuclear de Ligação ao Cap/genética , Proteínas de Ligação ao Cap de RNA/genética , RNA Polimerase II/genética , Estabilidade de RNA/genética , Transporte de RNA/genética , Fatores de Transcrição/genéticaRESUMO
Nuclear RNA metabolism is influenced by protein complexes connecting to both RNA-productive and -destructive pathways. The ZC3H18 protein binds the cap-binding complex (CBC), universally present on capped RNAs, while also associating with the nuclear exosome targeting (NEXT) complex, linking to RNA decay. To dissect ZC3H18 function, we conducted interaction screening and mutagenesis of the protein, which revealed a phosphorylation-dependent isoform. Surprisingly, the modified region of ZC3H18 associates with core histone proteins. Further examination of ZC3H18 function, by genome-wide analyses, demonstrated its impact on transcription of a subset of protein-coding genes. This activity requires the CBC-interacting domain of the protein, with some genes being also dependent on the NEXT- and/or histone-interacting domains. Our data shed light on the domain requirements of a protein positioned centrally in nuclear RNA metabolism, and they suggest that post-translational modification may modulate its function.
Assuntos
Núcleo Celular/metabolismo , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , Estabilidade de RNA/fisiologia , Proteínas de Ligação a RNA/metabolismo , RNA/biossíntese , Núcleo Celular/química , Núcleo Celular/genética , Estudo de Associação Genômica Ampla , Células HEK293 , Células HeLa , Humanos , Mutagênese , Complexo Proteico Nuclear de Ligação ao Cap/química , Complexo Proteico Nuclear de Ligação ao Cap/genética , Domínios Proteicos , RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genéticaRESUMO
Termination of transcription is important for establishing gene punctuation marks. It is also critical for suppressing many of the pervasive transcription events occurring throughout eukaryotic genomes and coupling their RNA products to efficient decay. In human cells, the ARS2 protein has been implicated in such function as its depletion causes transcriptional read-through of selected gene terminators and because it physically interacts with the ribonucleolytic nuclear RNA exosome. Here, we study the role of ARS2 on transcription and RNA metabolism genome wide. We show that ARS2 depletion negatively impacts levels of promoter-proximal RNA polymerase II at protein-coding (pc) genes. Moreover, our results reveal a general role of ARS2 in transcription termination-coupled RNA turnover at short transcription units like snRNA-, replication-dependent histone-, promoter upstream transcript- and enhancer RNA-loci. Depletion of the ARS2 interaction partner ZC3H18 mimics the ARS2 depletion, although to a milder extent, whereas depletion of the exosome core subunit RRP40 only impacts RNA abundance post-transcriptionally. Interestingly, ARS2 is also involved in transcription termination events within first introns of pc genes. Our work therefore establishes ARS2 as a general suppressor of pervasive transcription with the potential to regulate pc gene expression.