Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes

Ingolia, Nicholas T.; Brar, Gloria A.; Stern-Ginossar, Noam; Harris, Michael S.; Talhouarne, Gaëlle J.S.; Jackson, Sarah E.; Wills, Mark R.; Weissman, Jonathan S.

doi:10.1016/j.celrep.2014.07.045

Cited by 576 publications

(662 citation statements)

References 51 publications

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“…Improving the quantification by measuring ribosome density along the mRNA body and, in doing so, in a cell-type-specific manner can make TRAP combined with ribosome footprinting a very powerful tool. In a recent paper 34 , this combination was performed in human embryonic kidney 293 cells. The authors ran nuclease footprinting followed by affinity purification of an inducible biotinylated form of RpL10A using streptavidin beads.…”

Section: Discussionmentioning

confidence: 99%

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of <em>Drosophila</em> Embryos

Bertin

Renaud

Aradhya

et al. 2015

JoVE

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Measuring levels of mRNAs in the process of translation in individual cells provides information on the proteins involved in cellular functions at a given point in time. The protocol dubbed Translating Ribosome Affinity Purification (TRAP) is able to capture this mRNA translation process in a cell-type-specific manner. Based on the affinity purification of polysomes carrying a tagged ribosomal subunit, TRAP can be applied to translatome analyses in individual cells, making it possible to compare cell types during the course of developmental processes or to track disease development progress and the impact of potential therapies at molecular level. Here we report an optimized version of the TRAP protocol, called TRAP-rc (rare cells), dedicated to identifying engaged-in-translation RNAs from rare cell populations. TRAP-rc was validated using the Gal4/UAS targeting system in a restricted population of muscle cells in Drosophila embryos. This novel protocol allows the recovery of cell-type-specific RNA in sufficient quantities for global gene expression analytics such as microarrays or RNA-seq. The robustness of the protocol and the large collections of Gal4 drivers make TRAP-rc a highly versatile approach with potential applications in cell-specific genomewide studies.

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Section: Discussionmentioning

confidence: 99%

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of <em>Drosophila</em> Embryos

Bertin

Renaud

Aradhya

et al. 2015

JoVE

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“…Prior to the development of Ribo-Seq, translated uORFs with regulatory functions were often identified by mutant screening or evolutionary conservation (Hill and Morris, 1993;Delbecq et al, 1994;Wiese et al, 2004;Imai et al, 2006;Hayden and Jorgensen, 2007). Although Ribo-Seq data have been used in the identification of translated uORFs (Fritsch et al, 2012;Liu et al, 2013;Ingolia et al, 2014), the application of Ribo-Seq data to the identification of ribosome stalling uORFs has not been reported. In this study, we show that some CPuORFs overaccumulate 59-truncated RNA ends with a signature of stacked ribosomes (Figure 2A).…”

Section: Investigation Of Regulatory Uorfs Using the Rna Degradomementioning

confidence: 99%

Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants

et al. 2016

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High-throughput approaches for profiling the 59 ends of RNA degradation intermediates on a genome-wide scale are frequently applied to analyze and validate cleavage sites guided by microRNAs (miRNAs). However, the complexity of the RNA degradome other than miRNA targets is currently largely uncharacterized, and this limits the application of RNA degradome studies. We conducted a global analysis of 59-truncated mRNA ends that mapped to coding sequences (CDSs) of Arabidopsis thaliana, rice (Oryza sativa), and soybean (Glycine max). Based on this analysis, we provide multiple lines of evidence to show that the plant RNA degradome contains in vivo ribosome-protected mRNA fragments. We observed a 3-nucleotide periodicity in the position of free 59 RNA ends and a bias toward the translational frame. By examining conserved peptide upstream open reading frames (uORFs) of Arabidopsis and rice, we found a predominance of 59 termini of RNA degradation intermediates that were separated by a length equal to a ribosome-protected mRNA fragment. Through the analysis of RNA degradome data, we discovered uORFs and CDS regions potentially associated with stacked ribosomes in Arabidopsis. Furthermore, our analysis of RNA degradome data suggested that the binding of Arabidopsis ARGONAUTE7 to a noncleavable target site of miR390 might directly hinder ribosome movement. This work demonstrates an alternative use of RNA degradome data in the study of ribosome stalling.

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“…Advances in high-throughput approaches (e.g., ChIP-seq, ATAC-seq) have allowed comprehensive annotation of cis-acting DNA sequences that may influence transcription. Similarly, identifying cis-acting RNA elements that influence translation has been accelerated through the application of Ribosome profiling (Ribo-seq) (Ingolia et al 2014), which measures ribosome occupancy throughout the transcriptome at near nucleotide precision. Common to these approaches is the need for careful statistical control and validation of proposed regulatory elements.…”

Section: [Supplemental Materials Is Available For This Article]mentioning

confidence: 99%

Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data

et al. 2017

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Upstream open reading frames (uORFs), located in transcript leaders (5′ UTRs), are potent cis-acting regulators of translation and mRNA turnover. Recent genome-wide ribosome profiling studies suggest that thousands of uORFs initiate with non-AUG start codons. Although intriguing, these non-AUG uORF predictions have been made without statistical control or validation; thus, the importance of these elements remains to be demonstrated. To address this, we took a comparative genomics approach to study AUG and non-AUG uORFs. We mapped transcription leaders in multiple Saccharomyces yeast species and applied a novel machine learning algorithm (uORF-seqr) to ribosome profiling data to identify statistically significant uORFs. We found that AUG and non-AUG uORFs are both frequently found in Saccharomyces yeasts. Although most non-AUG uORFs are found in only one species, hundreds have either conserved sequence or position within Saccharomyces. uORFs initiating with UUG are particularly common and are shared between species at rates similar to that of AUG uORFs. However, non-AUG uORFs are translated less efficiently than AUG-uORFs and are less subject to removal via alternative transcription initiation under normal growth conditions. These results suggest that a subset of non-AUG uORFs may play important roles in regulating gene expression.

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Ribosome Profiling Reveals Pervasive Translation Outside of Annotated Protein-Coding Genes

Cited by 576 publications

References 51 publications

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of <em>Drosophila</em> Embryos

TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of <em>Drosophila</em> Embryos

Global Analysis of Truncated RNA Ends Reveals New Insights into Ribosome Stalling in Plants

Conserved non-AUG uORFs revealed by a novel regression analysis of ribosome profiling data

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