Abstract
Ribosome profiling spectra bear rich information on translation control and dynamics. Yet, due to technical biases in library generation, extracting quantitative measures of discrete translation events has remained elusive. Using maximum likelihood statistics and data set from Escherichia coli we develop a robust method for neutralizing technical biases (e.g. base specific RNase preferences in ribosome-protected mRNA fragments (RPF) generation), which allows for correct estimation of translation times at single codon resolution. Furthermore, we validated the method with available datasets from E. coli treated with antibiotic to inhibit isoleucyl-tRNA synthetase, and two datasets from Saccharomyces cerevisiae treated with two RNases with distinct cleavage signatures. We demonstrate that our approach accounts for RNase cleavage preferences and provides bias-corrected translation times estimates. Our approach provides a solution to the long-standing problem of extracting reliable information about peptide elongation times from highly noisy and technically biased ribosome profiling spectra.
Highlights
Ribosome profiling couples cell-wide profiling of the positions of translating ribosomes on messenger at single codon resolution (1) with deep sequencing (2) and has provided new insights into regulation of protein synthesis across species (reviewed in (3–5))
Interpretation of the ribosome-protected mRNA fragments (RPFs) in terms of elongation times at single codon resolution requires (i) ribosomal arrest to be faster than the single peptide elongation steps, (ii) precise estimation of the distance of the ribosomal A site from the 5 - or 3 -ends of RPFs, (iii) neutralization of sequencedependent biases in the experimental protocol (3,6)
There is a clear connection between the expected number, λi j, of experimentally detected ribosomes with a particular codon j of open reading frames from gene i (ORFi) in the A site, and the expected codon translation time τi j (Equation 1)
Summary
Ribosome profiling (or Ribo-Seq) couples cell-wide profiling of the positions of translating ribosomes on messenger (mRNA) at single codon resolution (1) with deep sequencing (2) and has provided new insights into regulation of protein synthesis across species (reviewed in (3–5)). Interpretation of the RPFs in terms of elongation times at single codon resolution requires (i) ribosomal arrest to be faster than the single peptide elongation steps, (ii) precise estimation of the distance of the ribosomal A site (that is the ribosomal site accepting aminoacyl-tRNA-elongation factor complex) from the 5 - or 3 -ends of RPFs, (iii) neutralization of sequencedependent biases in the experimental protocol (i.e. nuclease cleavage, amplification in the library preparation) (3,6). Fulfillment of these criteria enables determining translation time for any particular codon in the transcriptome. Using the more precise MNase cleavage at the 3 end to infer the A-site codon position improves the resolution of bacterial ribosome profiling sets (6,7), yet the bias in RPF generation due to the nucleotide-dependent specificity of the MNase persists
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