RESUMO
Stress response mechanisms that modulate the dynamics of tRNA degradation and accumulation from the cytoplasm to the nucleus have been studied in yeast, the rat hepatoma and human cells. In the current study, we investigated tRNA degradation and accumulation in HeLa cells under various forms of stress. We found that initiator tRNA(Met) (tRNA(iMet)) was specifically degraded under heat stress. Two exonucleases, Xrn1 and Xrn2, are involved in the degradation of tRNA(iMet) in the cytoplasm and the nucleus, respectively. In addition to degradation, we observed accumulation of tRNA(iMet) in the nucleus. We also found that the mammalian target of rapamycin (mTOR), which regulates tRNA trafficking in yeast, is partially phosphorylated at Ser2448 in the presence of rapamycin and/or during heat stress. Our results suggest phosphorylation of mTOR may correlate with accumulation of tRNA(iMet) in heat-treated HeLa cells.
Assuntos
Núcleo Celular/metabolismo , Exorribonucleases/metabolismo , Temperatura Alta , Proteínas Associadas aos Microtúbulos/metabolismo , RNA de Transferência de Metionina/metabolismo , Sequência de Bases , Células HeLa , Humanos , Dados de Sequência Molecular , RNA de Transferência de Metionina/química , Serina-Treonina Quinases TOR/metabolismoRESUMO
UPF1 eliminates aberrant mRNAs harboring premature termination codons, and regulates the steady-state levels of normal physiological mRNAs. Although genome-wide studies of UPF1 targets performed, previous studies did not distinguish indirect UPF1 targets because they could not determine UPF1-dependent altered RNA stabilities. Here, we measured the decay rates of the whole transcriptome in UPF1-depleted HeLa cells using BRIC-seq, an inhibitor-free method for directly measuring RNA stability. We determined the half-lives and expression levels of 9,229 transcripts. An amount of 785 transcripts were stabilized in UPF1-depleted cells. Among these, the expression levels of 76 transcripts were increased, but those of the other 709 transcripts were not altered. RNA immunoprecipitation showed UPF1 bound to the stabilized transcripts, suggesting that UPF1 directly degrades the 709 transcripts. Many UPF1 targets in this study were newly identified. This study clearly demonstrates that direct determination of RNA stability is a powerful approach for identifying targets of RNA degradation factors.
Assuntos
Códon sem Sentido , Estabilidade de RNA , RNA Mensageiro/genética , Transativadores/genética , Transativadores/metabolismo , Transcriptoma , Linhagem Celular Tumoral , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Helicases , Interferência de RNA , RNA Interferente Pequeno , Análise de Sequência de RNARESUMO
MALAT-1, a long non-coding RNA, is associated with metastasis, but its role in the metastatic process remains unknown. Here, we show that short-interfering RNA-mediated MALAT-1 silencing impaired in vitro cell motility of lung cancer cells and influenced the expression of numerous genes. In these genes, knockdown of any one of CTHRC1, CCT4, HMMR, or ROD1 clearly inhibited cell migration. In MALAT-1 knockdown cells, pre-mRNA levels were decreased in some but not all genes. Thus, our findings suggest that MALAT-1 is a novel class of non-coding RNA that promotes cell motility through transcriptional and post-transcriptional regulation of motility related gene expression.