RESUMO
Pumilio paralogs, PUM1 and PUM2, are sequence-specific RNA-binding proteins that are essential for vertebrate development and neurological functions. PUM1&2 negatively regulate gene expression by accelerating degradation of specific mRNAs. Here, we determined the repression mechanism and impact of human PUM1&2 on the transcriptome. We identified subunits of the CCR4-NOT (CNOT) deadenylase complex required for stable interaction with PUM1&2 and to elicit CNOT-dependent repression. Isoform-level RNA sequencing revealed broad coregulation of target mRNAs through the PUM-CNOT repression mechanism. Functional dissection of the domains of PUM1&2 identified a conserved amino-terminal region that confers the predominant repressive activity via direct interaction with CNOT. In addition, we show that the mRNA decapping enzyme, DCP2, has an important role in repression by PUM1&2 amino-terminal regions. Our results support a molecular model of repression by human PUM1&2 via direct recruitment of CNOT deadenylation machinery in a decapping-dependent mRNA decay pathway.
Assuntos
RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Receptores CCR4/genética , Fatores de Transcrição/genética , Transcriptoma , Monofosfato de Adenosina , Sequência de Bases , Sítios de Ligação , Endorribonucleases/genética , Endorribonucleases/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Células HCT116 , Humanos , Luciferases/genética , Luciferases/metabolismo , Ligação Proteica , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores CCR4/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Many cellular functions, such as translation, require ribonucleoproteins (RNPs). The biogenesis of RNPs is a multi-step process that, depending on the RNP, can take place in many cellular compartments. Here we examine 2 different RNPs: telomerase and small Cajal body-specific RNPs (scaRNPs). Both of these RNPs are enriched in the Cajal body (CB), which is a subnuclear domain that also has high concentrations of another RNP, small nuclear RNPs (snRNPs). SnRNPs are essential components of the spliceosome, and scaRNPs modify the snRNA component of the snRNP. The CB contains many proteins, including WRAP53, SMN and coilin, the CB marker protein. We show here that coilin, SMN and coilp1, a newly identified protein encoded by a pseudogene in human, associate with telomerase RNA and a subset of scaRNAs. We also have identified a processing element within box C/D scaRNA. Our findings thus further strengthen the connection between the CB proteins coilin and SMN in the biogenesis of telomeras e and box C/D scaRNPs, and reveal a new player, coilp1, that likely participates in this process.
Assuntos
Corpos Enovelados/genética , Proteínas Nucleares/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Telomerase/genética , Animais , Corpos Enovelados/metabolismo , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/genética , Ligação Proteica , Pseudogenes , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Telomerase/metabolismoRESUMO
Small nuclear ribonucleoproteins (snRNPs), which are required for pre-mRNA splicing, contain extensively modified snRNA. Small Cajal body-specific ribonucleoproteins (scaRNPs) mediate these modifications. It is unknown how the box C/D class of scaRNPs localizes to Cajal Bodies (CBs). The processing of box C/D scaRNA is also unclear. Here, we explore the processing of box C/D scaRNA 2 and 9 by coilin. We also broaden our investigation to include WRAP53 and SMN, which accumulate in CBs, play a role in RNP biogenesis and associate with coilin. These studies demonstrate that the processing of an ectopically expressed scaRNA2 is altered upon the reduction of coilin, WRAP53 or SMN, but the extent and direction of this change varies depending on the protein reduced. We also show that box C/D scaRNP activity is reduced in a cell line derived from coilin knockout mice. Collectively, the findings presented here further implicate coilin as being a direct participant in the formation of box C/D scaRNPs, and demonstrate that WRAP53 and SMN may also play a role, but the activity of these proteins is divergent to coilin.
Assuntos
Corpos Enovelados/metabolismo , Splicing de RNA/genética , RNA Nuclear Pequeno/biossíntese , Ribonucleoproteínas Nucleares Pequenas/biossíntese , Animais , Corpos Enovelados/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Chaperonas Moleculares , Precursores de RNA/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Telomerase/genéticaRESUMO
Spliceosomal small nuclear ribonucleoproteins (snRNPs) are enriched in the Cajal body (CB). Guide RNAs, known as small Cajal body-specific RNAs (scaRNAs), direct modification of the small nuclear RNA (snRNA) component of the snRNP. The protein WRAP53 binds a sequence motif (the CAB box) found in many scaRNAs and the RNA component of telomerase (hTR) and targets these RNAs to the CB. We have previously reported that coilin, the CB marker protein, associates with certain non-coding RNAs. For a more comprehensive examination of the RNAs associated with coilin, we have sequenced the RNA isolated from coilin immunocomplexes. A striking preferential association of coilin with the box C/D scaRNAs 2 and 9, which lack a CAB box, was observed. This association varied by treatment condition and WRAP53 knockdown. In contrast, reduction of WRAP53 did not alter the level of coilin association with hTR. Additional studies showed that coilin degrades/processes scaRNA 2 and 9, associates with active telomerase and can influence telomerase activity. These findings suggest that coilin plays a novel role in the biogenesis of box C/D scaRNPs and telomerase.