Project description:To test how inosine in a codon is translated, we synthesized short reporter-transcripts coding for an N-terminal Flag-tag and a test-peptide containing inosine in one defined codon. All codons containing inosines in at least one position were tested. Codons that would lead to ambiguous translation products were omitted. The transcripts were generated using in vitro transcription with ITP instead of GTP and subsequently in vitro translated using rabbit reticulocyte lysate and resulting peptides were analyzed by LC-MS/MS. The templates for in vitro transcription were generated using linearized DNA plasmids.
Project description:Cisplatin induces both acute and chronic nephrotoxicity during chemotherapy in cancer patients. Here, we report the study of single-nucleus sequencing (snRNA-seq) of cisplatin-induced nephrotoxicity.
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:Active protein translation can be assessed and measured using ribosome profiling sequencing strategies. Existing approaches make use of sequence fragment length or frame occupancy to differentiate between active translation and background noise, however they do not consider additional characteristics inherent to the technology which limits their overall accuracy. Here, we present an analytical tool that models the overall tri-nucleotide periodicity of ribosomal occupancy using a classifier based on spectral coherence. Our software, SPECtre, examines the relationship of normalized ribosome profiling read coverage over a rolling series of windows along a transcript against an idealized reference signal. A comparison of SPECtre against current methods on existing and new data shows a marked improvement in accuracy for detecting active translation and exhibits overall high sensitivity at a low false discovery rate. Classification of actively translated transcripts in ribosome profiling data derived from human neuroblastoma SH-SY5Y cells, and data previously published derived from mouse embryonic stem cells and zebrafish embryos.