RESUMO
Quality control of mRNA represents an important regulatory mechanism for gene expression in eukaryotes. One component of this quality control is the nuclear retention and decay of misprocessed RNAs. Previously, we demonstrated that mature mRNAs containing a 5' splice site (5'SS) motif, which is typically found in misprocessed RNAs such as intronic polyadenylated (IPA) transcripts, are nuclear retained and degraded. Using high-throughput sequencing of cellular fractions, we now demonstrate that IPA transcripts require the zinc finger protein ZFC3H1 for their nuclear retention and degradation. Using reporter mRNAs, we demonstrate that ZFC3H1 promotes the nuclear retention of mRNAs with intact 5'SS motifs by sequestering them into nuclear speckles. Furthermore, we find that U1-70K, a component of the spliceosomal U1 snRNP, is also required for the nuclear retention of these reporter mRNAs and likely functions in the same pathway as ZFC3H1. Finally, we show that the disassembly of nuclear speckles impairs the nuclear retention of reporter mRNAs with 5'SS motifs. Our results highlight a splicing independent role of U1 snRNP and indicate that it works in conjunction with ZFC3H1 in preventing the nuclear export of misprocessed mRNAs by sequestering them into nuclear speckles.
Assuntos
Sítios de Splice de RNA , Ribonucleoproteína Nuclear Pequena U1 , Salpicos Nucleares , Sítios de Splice de RNA/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
Mutations in RanBP2 (also known as Nup358), one of the main components of the cytoplasmic filaments of the nuclear pore complex, contribute to the overproduction of acute necrotizing encephalopathy (ANE1)-associated cytokines. Here we report that RanBP2 represses the translation of the interleukin 6 (IL6) mRNA, which encodes a cytokine that is aberrantly up-regulated in ANE1. Our data indicates that soon after its production, the IL6 messenger ribonucleoprotein (mRNP) recruits Argonautes bound to let-7 microRNA. After this mRNP is exported to the cytosol, RanBP2 sumoylates mRNP-associated Argonautes, thereby stabilizing them and enforcing mRNA silencing. Collectively, these results support a model whereby RanBP2 promotes an mRNP remodelling event that is critical for the miRNA-mediated suppression of clinically relevant mRNAs, such as IL6.
Assuntos
Proteínas Argonautas/genética , Fatores de Iniciação em Eucariotos/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Chaperonas Moleculares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Regiões 3' não Traduzidas/genética , Proteínas Argonautas/metabolismo , Linhagem Celular Tumoral , Fatores de Iniciação em Eucariotos/metabolismo , Células HEK293 , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , MicroRNAs/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pancreatite Necrosante Aguda/genética , Pancreatite Necrosante Aguda/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , SumoilaçãoRESUMO
While splicing has been shown to enhance nuclear export, it has remained unclear whether mRNAs generated from intronless genes use specific machinery to promote their export. Here, we investigate the role of the major nuclear pore basket protein, TPR, in regulating mRNA and lncRNA nuclear export in human cells. By sequencing mRNA from the nucleus and cytosol of control and TPR-depleted cells, we provide evidence that TPR is required for the efficient nuclear export of mRNAs and lncRNAs that are generated from short transcripts that tend to have few introns, and we validate this with reporter constructs. Moreover, in TPR-depleted cells reporter mRNAs generated from short transcripts accumulate in nuclear speckles and are bound to Nxf1. These observations suggest that TPR acts downstream of Nxf1 recruitment and may allow mRNAs to leave nuclear speckles and properly dock with the nuclear pore. In summary, our study provides one of the first examples of a factor that is specifically required for the nuclear export of intronless and intron-poor mRNAs and lncRNAs.