Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation

Abstract

SUMMARY Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and condos matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a 10-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5’-untranslated regions. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast.

Understanding biases in ribosome profiling experiments reveals signatures of translation dynamics in yeast

“…Experiments without CHX, on the other hand, report positive correlations of varying magnitude (Fig 1D, 0x Gerashchenko NAR points and purple labels). Experiments by Pop [36], Lareau [26], Nedialkova [29], Guydosh [35] and Gardin [33] produce weak to moderate correlations, but experiments by Gerashchenko [34], Jan [41], Williams [42], Weinberg [38], and Young [37] produce fairly strong and highly statistically significant correlations.…”

“…Conversely, a mean relative enrichment value lower than 1 indicates that translation of a codon is faster than its surroundings. We compared the mean relative A-site enrichments for all 61 non-stop codons between the original CHX-pretreatment data of Ingolia et al [15], an experiment we performed using the same CHX-pretreatment protocol, and data from experiments without CHX pretreatment by Gerashchenko [34] and by Weinberg et al [38] (Fig 1B). A-site occupancies are strongly positively correlated between experiments that use CHX pretreatment (upper left panel) and between experiments that do not (lower right panel).…”

“…An alternative experimental protocol uses an optimized harvesting and flash-freezing process that allows CHX pretreatment to be omitted [15,17,26,29,[33][34][35][36][37][38]. Experiments performed with this protocol have revealed that treatment with CHX affects several high-level characteristics of footprinting data, including the distribution of lengths of nuclease-protected fragments in mammalian cells [39] and the amount of enrichment in ribosome density at the 5' end of coding sequences in yeast [34].…”

“…Experiments performed with this protocol have revealed that treatment with CHX affects several high-level characteristics of footprinting data, including the distribution of lengths of nuclease-protected fragments in mammalian cells [39] and the amount of enrichment in ribosome density at the 5' end of coding sequences in yeast [34]. In contrast to data produced using CHX pretreatment, several studies using this alternative protocol have reported that non-optimal codons are in fact translated more slowly [33,38]. The source of this discrepancy between the statistical properties of measured ribosome positions with and without CHX pretreatment has been unclear, leading to uncertainty as to which measurements correspond to actual properties of in vivo translation dynamics.…”

Translation initiation factor eIF4G1 preferentially binds yeast transcript leaders containing conserved oligo-uridine motifs

“…Several well-characterized TL features can repress translation to varying degrees. Inhibitory features include upstream open reading frames, which divert ribosomes from the main open reading frame of an mRNA (Hinnebusch 2005;Arribere and Gilbert 2013;Pelechano et al 2013); stable RNA secondary structures, which block or impede ribosome recruitment or scanning (Kozak 1990;Ding et al 2014;Weinberg et al 2016); and target sites for certain RNA-binding proteins, which lead to translational repression and mRNA decay of specific sets of messages (Hentze et al 2004;Beckmann et al 2005). In contrast, relatively little is known about TL features that can act as general translational enhancers, although such elements could contribute substantially to the wide range of TEs observed in eukaryotic cells (Gilbert 2010;Shatsky et al 2014;Lee et al 2015).…”