Accounting for biases in riboprofiling data indicates a major role for proline in stalling translation

Abstract

The recent advent of ribosome profiling-sequencing of short ribosome-bound fragments of mRNA-has offered an unprecedented opportunity to interrogate the sequence features responsible for modulating translational rates. Nevertheless, numerous analyses of the first riboprofiling data set have produced equivocal and often incompatible results. Here we analyze three independent yeast riboprofiling data sets, including two with much higher coverage than previously available, and find that all three show substantial technical sequence biases that confound interpretations of ribosomal occupancy. After accounting for these biases, we find no effect of previously implicated factors on ribosomal pausing. Rather, we find that incorporation of proline, whose unique side-chain stalls peptide synthesis in vitro, also slows the ribosome in vivo. We also reanalyze a method that implicated positively charged amino acids as the major determinant of ribosomal stalling and demonstrate that it produces false signals of stalling in low-coverage data. Our results suggest that any analysis of riboprofiling data should account for sequencing biases and sparse coverage. To this end, we establish a robust methodology that enables analysis of ribosome profiling data without prior assumptions regarding which positions spanned by the ribosome cause stalling.

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

“…This calculation quantifies the expectation that tRNAs expressed at lower abundances or that involve non-standard base pairing in their codon-anticodon interaction should require longer to translate. Consistent with previous reports [13,23,31,32], all CHX experiments report weak experiments from many different studies in yeast, grouped by hierarchical clustering. Red indicates positive correlation and blue indicates negative correlation.…”

“…The exact mechanism of this inhibition, however, is not completely understood, with recent studies suggesting that CHX binds to a ribosome's E-site along with a deacylated tRNA to block further translocation [19,20]. The majority of the rapidly growing body of ribosome profiling experiments in yeast have followed this original CHX-pretreatment protocol [13,[21][22][23][24][25][26][27][28][29][30]. Several groups have applied a variety of conceptually similar computational methods to the data produced by these experiments to infer the average speed with which each codon identity is translated.…”

“…These so-called wobble pairings are thought to modulate the speed of decoding [9][10][11][12]. Once a tRNA has arrived and base-paired, the speed of peptide bond formation between the C-terminal amino acid in the nascent chain and an incoming amino acid may be influenced by chemical properties of these amino acids [13]. The relative contributions of these effects to overall rates of translation remain poorly understood.…”

Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing

“…complexes, biased conversion of the mRNA fragments into complementary DNAs, and procedures for aligning the ribosomeprotected base sequences (9,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31). These considerations underscore the importance of profiling the translation progression directly.…”

Mapping the non-standardized biases of ribosome profiling

“…Prior to this, each fragment is polyadenylated at its 3′ ends with poly(A)-polymerase (Ingolia et al, 2009), which serves as a priming site for the reverse transcription. Polyadenylation was also introduced to produce uniform 3′ ends of all fragments and to reduce the bias in the ligation (Ingolia, 2010), however the sequenced fragments are enriched in adenines at their 3′ termini (Artieri and Fraser, 2014). Furthermore, in the circularization procedure, an additional preference for adenine at the first 5′-position is observed (Lamm et al, 2011;Artieri and Fraser, 2014): it does not depend on the polyA-tails of the fragments and the origin of this bias is unknown.…”