Project description:Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). Here, we exploit a systematic CRISPR-based screening strategy to identify hundreds of non-canonical CDSs that are essential for cellular growth and whose disruption elicit specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds that are presented by the HLA system. Interestingly, we find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same mRNA, thus revealing the diverse use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.
Project description:Accurate annotations of protein coding regions are essential for understanding how genetic information is translated into biological functions. The recent development of ribosome footprint profiling provides an important new tool for measuring translation. Here we describe riboHMM, a new method that uses ribosome footprint data along with gene expression and sequence information to accurately infer translated sequences. We applied our method to human lymphoblastoid cell lines and identified 7,863 previously unannotated coding sequences, including 445 translated sequences in pseudogenes and 2,442 translated upstream open reading frames. We observed an enrichment of harringtonine-treated ribosome footprints at the inferred initiation sites, validating many of the novel coding sequences. In aggregate, the novel sequences exhibit significant signatures of purifying selection indicative of protein-coding function, suggesting that many of the novel sequences are functional. We observed that nearly 40% of bicistronic transcripts showed significant negative correlation in the levels of translation of their two coding sequences, suggesting a key regulatory role for these novel translated sequences. Despite evidence for their functional importance, the novel peptide sequences were detected by mass spectrometry at a lower rate than predicted based on data from annotated proteins, thus suggesting that many of the novel peptide products may be relatively short-lived. Our work illustrates the value of ribosome profiling for improving coding annotations, and significantly expands the set of known coding regions.
Project description:We present a genome-wide assessment of small open reading frames (smORF) translation by ribosomal profiling of polysomal fractions in Drosophila S2 cell. In this way, mRNAs bound by multiple ribosomes and hence actively translated can be isolated and distinguished from mRNAs bound by sporadic, putatively non-productive single ribosomes or ribosomal subunits. Ribosomal profiling of large and small polysomal fractions in Drosophila S2 cells to assess translation of smORFs
Project description:The Epstein-Barr virus (EBV) genome encodes several hundred transcripts. Using total RNA sequencing and ribosome profiling, we have characterized the transcriptional and translational scope of B cells infected with EBV. We could show that viral transcripts are translated at variable efficiency and that several viral genes show ribosome recruitment to the 5’ leader region of mRNAs. We used two different virus strains with differing in vitro characteristics to study EBV translation and could show that in cells infected with the weakly replicating EBV strain some lytic genes showed evidence of monosomal ribosome recruitment mainly in the 5’ leader region and on start codons in the absence of protein production. Finally, we could identify 25 novel upstream open reading frames that potentially regulate the translation efficiency of some viral genes.
Project description:We present a genome-wide assessment of small open reading frames (smORF) translation by ribosomal profiling of polysomal fractions in Drosophila S2 cell. In this way, mRNAs bound by multiple ribosomes and hence actively translated can be isolated and distinguished from mRNAs bound by sporadic, putatively non-productive single ribosomes or ribosomal subunits.