RESUMO
As ribosomes translate the genetic code, they can encounter a variety of obstacles that hinder their progress. If ribosomes stall for prolonged times, cells suffer due to the loss of translating ribosomes and the accumulation of aberrant protein products. Thus to protect cells, stalled ribosomes experience a series of reactions to relieve the stall and degrade the offending mRNA, a process known as No-Go mRNA Decay (NGD). While much of the machinery for NGD is known, the precise ordering of events and factors along this pathway has not been tested. Here, we deploy C. elegans to unravel the coordinated events comprising NGD. Utilizing a novel reporter and forward and reverse genetics, we identify the machinery required for NGD. Our subsequent molecular analyses define a functional requirement for ubiquitination on at least two ribosomal proteins (eS10 and uS10), and we show that ribosomes lacking ubiquitination sites on eS10 and uS10 fail to perform NGD in vivo. We show that the nuclease NONU-1 acts after the ubiquitin ligase ZNF-598, and discover a novel requirement for the ribosome rescue factors HBS-1/PELO-1 in mRNA decay via NONU-1. Taken together, our work demonstrates mechanisms by which ribosomes signal to effectors of mRNA repression, and we delineate links between repressive factors working toward a well-defined NGD pathway.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Animais , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Caenorhabditis elegans/genética , Ribossomos/genética , Ubiquitinação , Estabilidade de RNA/genética , RNA Mensageiro/genética , Biossíntese de ProteínasRESUMO
Nonsense-mediated mRNA decay (NMD) protects cells from the toxic and potentially dominant effects of truncated proteins. Targeting of mRNAs with early stop codons is mediated by the ribosome and spatiotemporally aligned with translation termination. Previously we identified a novel NMD intermediate: ribosomes stalled on cleaved stop codons, raising the possibility that NMD begins even prior to ribosome removal from the stop codon. Here we show that this intermediate is the result of mRNA cleavage by the endonuclease SMG-6. Our work supports a model in which ribosomes stall secondary to SMG-6 mRNA cleavage in Caenorhabditis elegans and humans, i.e. that the novel NMD intermediate occurs after a prior ribosome elicits NMD. Our genetic analysis of C. elegans' SMG-6 supports a central role for SMG-6 in metazoan NMD, and provides a context for evaluating its function in other metazoans.
