Coordination of -1 programmed ribosomal frameshifting by transcript and nascent chain features revealed by deep mutational scanning

Abstract

Programmed ribosomal frameshifting (PRF) is a translational recoding mechanism that enables the synthesis of multiple polypeptides from a single transcript. During translation of the alphavirus structural polyprotein, the efficiency of −1PRF is coordinated by a ‘slippery’ sequence in the transcript, an adjacent RNA stem–loop, and a conformational transition in the nascent polypeptide chain. To characterize each of these effectors, we measured the effects of 4530 mutations on −1PRF by deep mutational scanning. While most mutations within the slip-site and stem–loop reduce the efficiency of −1PRF, the effects of mutations upstream of the slip-site are far more variable. We identify several regions where modifications of the amino acid sequence of the nascent polypeptide impact the efficiency of −1PRF. Molecular dynamics simulations of polyprotein biogenesis suggest the effects of these mutations primarily arise from their impacts on the mechanical forces that are generated by the translocon-mediated cotranslational folding of the nascent polypeptide chain. Finally, we provide evidence suggesting that the coupling between cotranslational folding and −1PRF depends on the translation kinetics upstream of the slip-site. These findings demonstrate how −1PRF is coordinated by features within both the transcript and nascent chain.

Abracadabra, One Becomes Two: The Importance of Context in Viral −1 Programmed Ribosomal Frameshifting

“…Further refining this idea with a focus on HIV-1, Léger et al provided a possible mechanistic explanation showing that the 3 nucleotide preceding the slippery sequence, those occupied by the ribosomal E-Site upon slipping (A,B, and C in the A-BCX-XXY-YYZ) can increase or decrease the relative rate of frameshifting ( 52 ). These findings are largely supported by the recent work of Carmody et al whose deep mutational scan of the ~300 nucleotide upstream of the slippery sequence in Sindbis virus (SINV) shows that altering the identity of these nucleotides can dramatically impact the observed rate of −1 PRF ( 53 ). These findings raise questions then about how MFE design choices could directly impact observed frameshifting efficiencies and mechanisms.…”

Ribosomal Frameshifting Selectively Modulates the Biosynthesis, Assembly, and Function of a Misfolded CFTR Variant

“…5 However, it remains unclear how the conformational effects of ΔF508 result in direct modifications of translation and vice versa. In light of the recent discovery that conformational transitions in the nascent chain can stimulate -1PRF, 14,15,30 we searched the CFTR transcript for structural features that might promote ribosomal frameshifting. Our findings identified a slippery sequence (SSC) and stem-loop (SL2) that stimulate ribosomal frameshifting and the premature translational termination (Fig.…”

“…Our recent findings show that the mechanical forces generated by cotranslational folding can stimulate -1PRF, 14,15 which minimally requires a slippery sequence within the mRNA and an adjacent stem-loop. 17 To identify potential RF sites in the CFTR transcript we first searched for consensus or near-consensus X1 XXY4 YYZ7 slippery sequences, where XXX and YYY are nucleobase triplets and Z can be any nucleobase.…”

“…We recently found that cotranslational folding mechanically modulates a translational recoding mechanism known as -1 programmed ribosomal frameshifting (-1PRF) during viral polyprotein biosynthesis. 14,15 These findings reveal that, under certain circumstances, the ribosome exhibits an enhanced propensity to slip into alternative reading frames in response to conformational transitions in the nascent polypeptide chain. 16 In the following, we evaluate whether similar feedback occurs during CFTR biosynthesis.…”

mutscan - a flexible R package for efficient end-to-end analysis of multiplexed assays of variant effect data

“…They have been used to examine activities of proteins, such as protein-protein interaction (PPI) (6)(7)(8), E3 ubiquitin ligase activity (8), protein abundance (7,9), receptor binding (10), aggregation (11,12), and activity within signaling pathways (13). The functional assays used to achieve enrichment or depletion of variants are equally diverse and include for example fitness or cell growth (6,7,11,12), different reporter assays coupled with fluorescence-activated cell sorting (FACS) (9,10,13,14), and protein display (8,15).…”