Christina Akirtava, Gemma E May, C Joel McManus
Protein synthesis is a vital process that is highly regulated at the initiation step of translation. Eukaryotic 5′ transcript leaders (TLs) contain a variety of cis-acting features that influence translation and messenger RNA stability. However, the relative influences of these features in natural TLs are poorly characterized. To address this, we used massively parallel reporter assays (MPRAs) to quantify RNA levels, ribosome loading, and protein levels from 11,027 natural yeast TLs in vivo and systematically compared the relative impacts of their sequence features on gene expression. We found that yeast TLs influence gene expression over two orders of magnitude. While a leaky scanning model using Kozak contexts (−4 to +1 around the AUG start) and upstream AUGs (uAUGs) explained half of the variance in expression across TLs, the addition of other features explained ∼80% of gene expression variation. Our analyses detected key cis-acting sequence features, quantified their effects in vivo, and compared their roles to motifs reported from an in vitro study of ribosome recruitment. In addition, our work quantitated the effects of alternative transcription start site usage on gene expression in yeast. Thus, our study provides new quantitative insights into the roles of TL cis-acting sequences in regulating gene expression.
Christina Akirtava, Gemma E May, C Joel McManus. Deciphering the landscape of cis-acting sequences in natural yeast transcript leaders. 2025-03-12. https://doi.org/10.1093/nar/gkaf165
A two-replicate polysome-library sequencing assay measured relative ribosome loading of reporter transcripts carrying natural 5′ transcript leaders from S. cerevisiae and S. paradoxus. Polysome-gradient reads were pooled into translating and nontranslating classes and reported as the percentage of translated reads for each construct.
Three biological replicates of an episomal dual-fluorescence reporter library tested natural 5′ transcript leaders from Saccharomyces cerevisiae and Saccharomyces paradoxus upstream of YFP, with mCherry as an internal control. Cells were FACS-binned by the YFP/mCherry ratio and construct counts were used to estimate normalized protein output for each transcript leader.
Targeted RNA-seq measured reporter RNA relative to plasmid DNA for the same episomal dual-fluorescence library of natural S. cerevisiae and S. paradoxus 5′ transcript leaders. Three biological replicates are reported as RNA/DNA activity values, with the matched FACS-seq library QC applied to keep the element set consistent.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 7 files (ZIP)Akirtava_et_al_2024_Rev1_Supp_Mat.pdfAkirtava_et_al_2024_Supp_Data_Rev1.xlsxgkaf165_supplemental_files.zipPRJNA721222_bioproject.xmlPRJNA721222_runinfo.csvSAMN18740588_89_biosample.xmlsource_manifest.txt