ABSTRACT: Ribosome profiling performed on interspecific hybrids of Sacharromyces cerevisiae and S. paradoxus in order to identify allele-specific expression indicative of cis-regulatory divergence at the level of mRNA abundance and protein translation. Two biological replicate libraries sequenced from ribosome protected fragments as well as poly-A-selected mRNA of hybrids in addition to one biological replicate library of poly-A selected mRNA from each parental strain.
Project description:Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila melanogaster, but the importance of regulated readthrough in eukaryotes remains largely unexplored. Here, we present a ribosome profiling assay (deep sequencing of ribosome-protected mRNA fragments) for Drosophila melanogaster, and provide the first genome-wide experimental analysis of readthrough. Readthrough is far more pervasive than expected: the vast majority of readthrough events evolved within D. melanogaster and were not predicted phylogenetically. The resulting C-terminal protein extensions show evidence of selection, contain functional subcellular localization signals, and their readthrough is regulated, arguing for their importance. We further demonstrate that readthrough occurs in yeast and humans. Readthrough thus provides general mechanisms both to regulate gene expression and function, and to add plasticity to the proteome during evolution. 12 samples of Drosophila ribosome profiling and poly(A)+ mRNA-seq, including technical replicates in S2 cells, and biological replicates of 0-2 hour embryos
Project description:Genetic variants that impact gene regulation are important contributors to human phenotypic variation. For this reason, considerable efforts have been made to identify genetic associations with differences in mRNA levels of nearby genes, namely, cis expression quantitative trait loci (eQTLs). The phenotypic consequences of eQTLs are presumably due, in most cases, to their ultimate effects on protein expression levels. Yet, only few studies have quantified the impact of genetic variation on proteins levels directly. It remains unclear how faithfully eQTLs are reflected at the protein level, and whether there is a significant layer of cis regulatory variation acting primarily on translation or steady state protein levels. To address these questions, we measured ribosome occupancy by high-throughput sequencing, and relative protein levels by high-resolution quantitative mass spectrometry, in a panel of lymphoblastoid cell lines (LCLs) in which we had previously measured transcript expression using RNA sequencing. We then mapped genetic variants that are associated with changes in transcript expression (eQTLs), ribosome occupancy (rQTLs), or protein abundance (pQTLs). Most of the QTLs we detected are associated with transcript expression levels, with consequent effects on ribosome and protein levels. However, we found that eQTLs tend to have significantly reduced effect sizes on protein levels, suggesting that their potential impact on downstream phenotypes is often attenuated or buffered. Additionally, we confirmed the presence of a class of cis QTLs that specifically affect protein abundance with little or no effect on mRNA levels; most of these QTLs have little effect on ribosome occupancy, and hence may arise from differences in post-translational regulation. We measured level of translation transcriptome-wide in lymphoblastoid cell lines derived from 72 HapMap Yoruba individuals using ribosome profiling assay, for which we have transcript level, protein level (62 out of 72) and genotype information collected.
Project description:Fully assembled ribosomes exist in two populations: polysomes and monosomes. While the former has been studied extensively, to what extent translation occurs on monosomes and its importance for overall translational output remains controversial. Here, we used ribosome profiling to examine the translational status of 80S monosomes in Saccharomyces cerevisiae. We found that the vast majority of 80S monosomes are elongating, not initiating. Further, most mRNAs exhibit some degree of monosome occupancy, with monosomes predominating on nonsense-mediated decay (NMD) targets, upstream open reading frames (uORFs), canonical ORFs shorter than ~590 nucleotides and ORFs for which the total time required to complete elongation is substantially shorter than that required for initiation. Importantly, mRNAs encoding low-abundance regulatory proteins tend to be enriched in the monosome fraction. Our data highlight the importance of monosomes for the translation of highly regulated mRNAs. We examined the translational status of single 80S ribosomes using ribosome profiling, and compared these monosome footprints to both polysome ribosome footprints and general ribosome profiling. RNASeq libraries were also prepared from the overall sample input.
Project description:Protein translation is at the heart of cellular metabolism and its in-depth characterization is key for many lines of research. Recently, ribosome profiling became the state-of-the-art method to quantitatively characterize translation dynamics at a transcriptome-wide level. However, the strategy of library generation affects its outcomes. Here, we present a modified ribosomeprofiling protocol starting from yeast, human cells and vertebrate brain tissue. We use a DNA linker carrying four randomized positions at its 5’ and a reverse-transcription (RT) primer with three randomized positions to reduce artifacts during library preparation. The use of seven randomized nucleotides allows to efficiently detect library-generation artifacts. We find that the effect of polymerase chain reaction (PCR) artifacts is relatively small for global analyses when sufficient input material is used. However, when input material is limiting, our strategy improves the sensitivity of gene-specific analyses. Furthermore, randomized nucleotides alleviate the skewed frequency of specific sequences at the 3’ end of ribosome-protected fragments (RPFs) likely resulting from ligase specificity. Finally, strategies that rely on dual ligation show a high degree of gene-coverage variation. Taken together, our approach helps to remedy two of the main problems associated with ribosome-profiling data. This will facilitate the analysis of translational dynamics and increase our understanding of the influence of RNA modifications on translation. Ribosome profiling and mRNA-seq libraries from wt yeast comparing different library preparation approaches using different combinations of randomized and non-randomized linkers and RT primers.
Project description:During translation elongation, the ribosome ratchets along its mRNA template, incorporating each new amino acid and translocating from one codon to the next. The elongation cycle requires dramatic structural rearrangements of the ribosome. We show here that deep sequencing of ribosome-protected mRNA fragments reveals not only the position of each ribosome but also, unexpectedly, its particular stage of the elongation cycle. Sequencing reveals two distinct populations of ribosome footprints, 28-30 nucleotides and 20-22 nucleotides long, representing translating ribosomes in distinct states, differentially stabilized by specific elongation inhibitors. We find that the balance of small and large footprints varies by codon and is correlated with translation speed. The ability to visualize conformational changes in the ribosome during elongation, at single-codon resolution, provides a new way to study the detailed kinetics of translation and a new probe with which to identify the factors that affect each step in the elongation cycle. Ribosome profiling, or sequencing of ribosome-protected mRNA fragments, in yeast. We assay ribosome footprint sizes and positions in three conditions: untreated yeast (3 replicates) and yeast treated with translation inhibitors cycloheximide (2 replicates) and anisomycin (2 biological replicates, one technical replicate). We also treat yeast with 3-aminotriazole to measure the effect of limited histidine tRNAs on ribosome footprint size and distribution (two treatment durations).
Project description:Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide‐encoding genes in vertebrates, providing an entry point to define their function in vivo. Ribosome profiling experiments at five timepoints across zebrafish development in WT embryos
Project description:Elucidating the extent and consequences of genetic differences between humans is essential for understanding phenotypic diversity and personalized medicine. Although variation in RNA levels, transcription factor binding and chromatin have been explored, little is known about global variation in translation and its genetic determinants among humans. We used ribosome profiling, RNA sequencing, and mass spectrometry to perform an integrated analysis in lymphoblastoid cell lines from a diverse group of individuals. We find significant differences in RNA levels, translation, and protein abundance suggesting diverse mechanisms of personalized gene expression control. Combined analysis of RNA expression and ribosome occupancy improves the identification of individual protein level differences. Finally, we identify genetic differences that specifically modulate ribosome occupancy - many of these differences lie close to start codons and upstream ORFs. Our results reveal a new level of gene expression variation among humans and indicate that genetic variants can cause changes in protein levels through effects on translation. Ribosome profiling and RNA sequencing experiments from human lymphoblastoid cells
Project description:Poly(A) tails are critical for mRNA stability and translation. However, recent studies have challenged this view, showing that poly(A) tail length and translation efficiency are decoupled in non-embryonic cells. Using TAIL-seq and ribosome profiling, we investigate poly(A) tail dynamics and translational control in the somatic cell cycle. We find dramatic changes in poly(A) tail lengths of cell cycle regulatory genes like CDK1, TOP2A, and FBXO5, explaining their translational repression in M phase. We also find that poly(A) tail length is coupled to translation when the poly(A) tail is <20 nucleotides. However, as most genes have >20 nucleotide poly(A) tails, their translation is regulated mainly via poly(A) tail length-independent mechanisms during the cell cycle. Specifically, we find that terminal oligopyrimidine (TOP) tract-containing transcripts escape global translational suppression in M phase and are actively translated. Our quantitative and comprehensive data provide a revised view of translational control in the somatic cell cycle. HeLa cells were synchronized at S or M phase, and subject to RNA-seq, ribosome profiling and TAIL-seq analysis.
Project description:In general, RNA-binding proteins act to modulate gene expression at transcript level through degradation or at protein level through translation. To elucidate the effect of Whi3, a yeast RNA binding protein, on gene expression, we performed ribosome profiling experiment on whi3 mutant and wildtype cells. Comparison ribosome profiling and RNA-seq data between whi3 mutant and wildtype cells
Project description: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 codons 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. Ribosome-footprint profiling and RNA-seq (total RNA, poly(A) selected, RiboMinus treated, or Ribo-Zero treated) from log-phase S. cerevisiae. The study includes a reanalysis of the two Samples from GSE53313. The reanalyzed data is linked to the Series record.