Strategies and Challenges in Identifying Function for Thousands of sORF-Encoded Peptides in Meiosis

Abstract

Recent genomic analyses have revealed pervasive translation from formerly unrecognized short open reading frames (sORFs) during yeast meiosis. Despite their short length, which has caused these regions to be systematically overlooked by traditional gene annotation approaches, meiotic sORFs share many features with classical genes, implying the potential for similar types of cellular functions. We found that sORF expression accounts for approximately 10-20% of the cellular translation capacity in yeast during meiotic differentiation and occurs within well-defined time windows, suggesting the production of relatively abundant peptides with stage-specific meiotic roles from these regions. Here, we provide arguments supporting this hypothesis and discuss sORF similarities and differences, as a group, to traditional protein coding regions, as well as challenges in defining their specific functions.

Keywords: meiosis; sORFs; small peptides; yeast.

Fig.1:. Ribosome profiling through budding yeast meiosis revealed regulated translation of 2555 meiotic sORFs encoded by freestanding transcripts.

Ribosome footprints summed over each newly identified independent yeast sORF (columns) in exponentially growing cells (top rows) and cells at different stages of meiosis (below, represented by cartoons to the left of the dendogram plot). Blue and yellow represent low and high translation, respectively. A Western blot confirming the predicted meiotic expression of an individual C-terminally GFP-tagged sORF is shown on the right.

Fig. 2.. The overall amino acid composition of sORFs and known ORFs is similar, with interesting exceptions.

A) G/C and A/T content of meiotic sORFs (independent sORFs and AUG-initiated uORFs) and known ORFs. χ2 test was applied to calculate statistical significance. Start and stop codons were computationally removed from each ORF prior to analysis. B) Amino acid composition of peptides produced from sORFs (independent sORFs and AUG-initiated uORFs) relative to those of proteins translated from known ORFs. Fold enrichment of individual amino acids in sORF-encoded peptides compared to proteins translated from known ORFs. C) Enriched or disenriched amino acids for all sORFs (independent and AUG-initiated uORFs) are shown in green and red, respectively, with lines indicating the fold difference relative to known ORFs (solid line = no enrichment, dotted line = 1.33-fold change, bold dotted line = 2-fold change). For the analyses in B) and C), the content of all ORFs in each category was pooled, after computational removal of the start codon-encoded methionine.