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Quantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs

ABSTRACT: We report an efficient, high-throughput method to optimize translation initiation from modified mRNAs. Using direct analysis of ribosome targeting, DART, we identify 5′ untranslated sequences that mediate 250-fold effects on ribosome recruitment, show that incorporation of m1Ψ significantly affects activity, and identify more active 5′ UTRs that surpass current mRNA vaccines. DART is broadly applicable to dissect translation initiation mechanisms of human genes and optimize mRNA regulatory sequences for desired protein outputs

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE256185 | GEO | 2024/12/19

REPOSITORIES: GEO

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Direct analysis of ribosome targeting illuminates thousand-fold regulation of translation initiation

Project description:Translational control shapes the proteome in normal and pathophysiological conditions. Current high-throughput approaches reveal large differences in mRNA-specific translation activity but cannot identify the causative mRNA features. We developed direct analysis of ribosome targeting (DART) and used it to dissect regulatory elements within 5′ untranslated regions that confer thousand-fold differences in ribosome recruitment in biochemically accessible cell lysates. Using DART, we identified novel translational enhancers and silencers, determined a functional role for most alternative 5′ UTR isoforms expressed in yeast, and revealed a general mode of increased translation via direct binding to a core translation factor. DART enables systematic assessment of the translational regulatory potential of 5′ UTR variants, whether native or disease-associated, and will facilitate engineering of mRNAs for optimized protein production in various systems.

2021-08-20 | GSE182290 | GEO

Genome-Wide Analysis In Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling

Project description:Techniques for systematically monitoring protein translation have lagged far behind methods for measuring mRNA levels. Here we present a ribosome profiling strategy, based on deep sequencing of ribosome protected mRNA fragments, that enables genome-wide investigation of translation with sub-codon resolution. We used this technique to monitor translation in budding yeast under both rich and starvation conditions. These studies defined the protein sequences being translated and found extensive translational control both for determining absolute protein abundance and for responding to environmental stress. We also observed distinct phases during translation involving a large decrease in ribosome density going from early to late peptide elongation as well as wide-spread, regulated initiation at non-AUG codons. Ribosome profiling is readily adaptable to other organisms, making high-precision investigation of protein translation experimentally accessible. Examine replicates of ribosome footprints and mRNA abundance in biological replicates of log-phase growth and acute amino acid starvation

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Project description:Translational control is a key determinant of protein abundance, which in turns defines the physiology and pathology of human cells. Initiation of translation is highly regulated in eukaryotes and is considered as the rate-limiting step of protein synthesis. mRNA secondary structures in 5’ untranslated region (UTR) and associated helicases have been characterised as key determinants of translation initiation. Nevertheless the transcriptome-wide contribution of non-canonical secondary structures, such as RNA G-quadruplexes (rG4s), to the translation of human mRNAs remains largely unappreciated. Here we use a ribosome profiling strategy to investigate the translational landscape associated to rG4s-containing mRNAs and the contribution of two rG4s-specialised DExH-box helicases, DHX9 and DHX36, to translation initiation in human cells. We show that rG4-forming sequences in 5’-UTR is associated with decreased translation efficiency which correlate with an increased ribosome density within the 5’-UTRs. We found that rG4s contribute to the translation of upstream open reading frames, and as a consequence, thwart the translation of the associated protein coding sequences (CDS). Depletion of the DHX36 and DHX9 helicases demonstrated that the formation of the rG4 structural motif rather than its nucleotide sequence mediate translation initiation. Our findings unveil a role for non-canonical structures in defining alternative 5’ starts for human mRNAs translation initiation.

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Quantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs

Project description:Quantitative profiling of human translation initiation reveals elements that potently regulate endogenous and therapeutically modified mRNAs

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Genome-Wide Analysis In Vivo of Translation with Nucleotide Resolution Using Ribosome Profiling

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