High-throughput interrogation of programmed ribosomal frameshifting in human cells

Abstract

Programmed ribosomal frameshifting (PRF) is the controlled slippage of the translating ribosome to an alternative frame. This process is widely employed by human viruses such as HIV and SARS coronavirus and is critical for their replication. Here, we developed a highthroughput approach to assess the frameshifting potential of a sequence. We designed and tested >12,000 sequences based on 15 viral and human PRF events, allowing us to systematically dissect the rules governing ribosomal frameshifting and discover novel regulatory inputs based on amino acid properties and tRNA availability. We assessed the natural variation in HIV gag-pol frameshifting rates by testing >500 clinical isolates and identified subtype-specific differences and associations between viral load in patients and the optimality of PRF rates. We devised computational models that accurately predict frameshifting potential and frameshifting rates, including subtle differences between HIV isolates. This approach can contribute to the development of antiviral agents targeting PRF.

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

“…PRF enables a single locus to generate protein isoforms with different C-terminal sequences by encoding in multiple frames, but without bulky sequence elements such as introns or alternative exons. PRF is thus common in viruses, where genome space is at a premium, but also plays a role in both normal and disease-associated gene expression in humans [102]. In addition to promoting expression of alternative protein isoforms, -1 PRF can also mediate suppression of gene expression by shifting ribosomes into a frame with a premature stop codon [103].…”

Programmed −1 ribosomal frameshifting from the perspective of the conformational dynamics of mRNA and ribosomes

“…In contrast with its prevalence and critical role in viruses, −1 PRF is rare in the human genome [101] . This fact suggests that −1 PRF is a potential target for drug- or host-factor-based antiviral therapeutic strategies [102] , [103] , [104] , [105] , [106] , [107] .…”

Dynamic changes in tRNA modifications and abundance during T cell activation

“…S4-3A). The plasmids were generous gift from Martin Mikl, Weizmann institute, Rehovot, Israel (49). The cells were analyzed using ATTUNE FACS (ThermoFisher) 48 h after transfection.…”

“…S4-3). We have recently created a library of such dual fluorescent proteins connected with diverse candidate linkers (49). To select from this library one biologically meaningful linker sequence, we returned to the biology of activated T cells.…”