A guard protein mediated quality control mechanism monitors 5’-capping of pre-mRNAs

Abstract

Efficient gene expression requires properly matured mRNAs for functional transcript translation. Several factors including the guard proteins monitor maturation and act as nuclear retention factors for unprocessed pre-mRNAs. Here we show that the guard protein Npl3 monitors 5’-capping. In its absence, uncapped transcripts resist degradation, because the Rat1–Rai1 5’-end degradation factors are not efficiently recruited to these faulty transcripts. Importantly, in npl3Δ, these improperly capped transcripts escape this quality control checkpoint and leak into the cytoplasm. Our data suggest a model in which Npl3 associates with the Rai1 bound pre-mRNAs. In case the transcript was properly capped and is thus CBC (cap binding complex) bound, Rai1 dissociates from Npl3 allowing the export factor Mex67 to interact with this guard protein and support nuclear export. In case Npl3 does not detect proper capping through CBC attachment, Rai1 binding persists and Rat1 can join this 5’-complex to degrade the faulty transcript.

Single-molecule quantitation of RNA-binding protein occupancy and stoichiometry defines a role for Yra1 (Aly/REF) in nuclear mRNP organization

“…Upon RNase A treatment, most spots were lost for almost all RBPs tested, while ~50% of Npl3 spots were not RNase A sensitive ( Figures 1B and S4 ). Npl3 was reported to directly bind the CBC, 45 which likely accounts for the presence of a large fraction of RNase-insensitive Npl3 spots in a Cbp80-PrA pull-down. Still, even considering the RNase-insensitive fraction, Npl3 showed a high level of enrichment compared to other RBPs.…”

“…This may reflect the existence of Npl3 in cytoplasmic mRNPs and/or non-coding RNPs (e.g., with small nuclear [sn]RNAs, small nucleolar [sno]RNAs, or long non-coding [lnc]RNAs), as Npl3 has functions in translation and binds non-coding RNAs. 46 , 47 Based on the interactions of Npl3 with the CBC complex and its role in pre-mRNA capping quality control, 45 it is also possible that Npl3 would be detected with Cbp80 in nascent and abortive transcripts that lack other RBPs found in mature mRNPs. It is expected that these multi-functional features of Npl3 account for the frequent association of Npl3 with Cbp80 within RNase-sensitive complexes.…”

Proteostasis regulation through ribosome quality control and no‐go‐decay

“…Cellular mRNAs are subjected to multiple checkpoints, co‐ and post‐transcriptionally, before entering the cytoplasm and engaging in protein synthesis (as previously reviewed by Hilleren & Parker, 1999; Karamyshev & Karamysheva, 2018; Keene, 2007; Peck et al, 2019; Rougemaille et al, 2008; Tutucci & Stutz, 2011; Wolin & Maquat, 2019). As a result of these primary quality control checkpoints, mRNAs with serious aberrations, such as the lack of 5′ cap or the poly‐A tail, are retained and degraded within the nucleus through nuclear mRNA surveillance mechanisms (Klama et al, 2022; Singh et al, 2018; Stutz, 2003). Additionally, cells have co‐translational quality control mechanisms to detect mRNAs bearing subtle defective sequences, such as the insertion of or mutation of a stop codon (Bengtson & Joazeiro, 2010; Choe et al, 2016; Dimitrova et al, 2009; Powers et al, 2020; Shoemaker & Green, 2012).…”

A potential function for the helicase Dbp5 in cytoplasmic quality control

“…Eukaryotic mRNAs have to complete several different quality control checkpoints to ensure the production of correct polypeptides. In nuclear mRNA quality control, the transcript is monitored for correct splicing, 5’ capping, and 3’ polyadenylation, mediated by a small number of guard proteins that retain the transcripts until processing is completed and then allow the export receptor Mex67 to bind for the export into the cytoplasm (1-6). On defective transcripts, Mex67 binding is prevented and the guard proteins recruit the degradation machinery instead.…”