ABSTRACT
Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. Here, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by different conformational states connected to the RNA-binding status of UPF1. Our findings rationalize a key event in metazoan NMD and advance our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.
Subject(s)
Endonucleases , Nonsense Mediated mRNA Decay , RNA Helicases , RNA-Binding Proteins , Trans-Activators , Humans , Cryoelectron Microscopy , Endonucleases/metabolism , Endonucleases/genetics , Endoribonucleases , Models, Molecular , Protein Binding , RNA Helicases/metabolism , RNA Helicases/genetics , RNA Helicases/chemistry , RNA, Messenger/metabolism , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/chemistry , Trans-Activators/metabolism , Trans-Activators/genetics , Trans-Activators/chemistry , Transcription Factors/metabolism , Transcription Factors/genetics , RNA-Binding MotifsABSTRACT
Unnatural base pairs (UBPs) strikingly augment the natural genetic alphabet. The development of particular hydrophobic UBPs even allows insertion and stable propagation in bacteria. Those UBPs expand the chemical scope of DNA and RNA, and thus, could enable the evolution of novel aptamers or ribozymes by in vitro selection (systematic evolution of ligands by exponential enrichment, SELEX). However, the application of such UBPs in reverse transcription (rtc), which is a key step for RNA-based SELEX, has not been reported so far. The implication of Romesberg's NaM:TPT3 base pair in rtc reactions is presented by testing five commercially available reverse transcriptases (RTs). The employed RTs predominantly pause at the site of the unnatural nucleotide rTPT3 not being able to accept the dNaM building block as a substrate. This allows verification of the unnatural base position in RNA and an estimation of their abundance. In contrast, primer extension from an rNaM-containing template results in considerably more full-length cDNA. Furthermore, RTs that could potentially be able to handle an expanded genetic alphabet based on NaM:TPT3 are presented.
