William Baez

RiboProP: A Probabilistic Ribosome Positioning Algorithm for Ribosome Profiling

MOTIVATION Ribosome profiling has been widely used to study translation in a genome-wide fashion. It requires deep sequencing of ribosome protected mRNA fragments followed by mapping of fragments to the reference genome. For applications such as identification of ribosome pausing sites, it is not enough to map a fragment to a given gene, but the exact position of the ribosome represented by the fragment must be identified for each mRNA fragment. The assignment of the correct ribosome position is complicated by the broad length distribution of the ribosome protected fragments caused by the known sequence bias of micrococcal nuclease (MNase), the most widely used nuclease for digesting mRNAs in bacteria. Available mapping algorithms suffer from either MNase bias or low accuracy in characterizing the ribosome pausing kinetics. RESULTS In this paper, we introduce a new computational method for mapping the ribosome protected fragments to ribosome locations. We first develop a mathematical model of the interplay between MNase digestion and ribosome protection of the mRNAs. We then use the model to reconstruct the ribosome occupancy profile on a per gene level. We demonstrate that our method has the capability of mitigating the sequence bias introduced by MNase and accurately locating ribosome pausing sites at codon resolution. We believe that our method can be broadly applied to ribosome profiling studies on bacteria where codon resolution is necessary. AVAILABILITY AND IMPLEMENTATION Source code implementing our approach can be downloaded under GPL3 license at http://bioserv.mps.ohio-state.edu/RiboProP. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Loss of fragile histidine triad (Fhit) protein expression alters the translation of cancer-associated mRNAs.

ObjectivesIn > 50% of cancers tumor development involves the early loss of Fhit (fragile histidine triad) protein expression, yet the mechanistic pathway(s) by which Fhit mediates its tumor suppressor functions are not fully understood. Earlier attempts to identify a Fhit-deficient gene expression profile relied on total cellular RNA and microarray analysis. The data here used RNA sequencing (RNA-Seq) of Fhit-negative and Fhit-positive cells as proof of principle for the impact of Fhit on specific mRNAs, and to lay the foundation for a study using ribosome profiling to identify mRNAs whose translation is affected by FHIT loss.Data descriptionRNA-Seq was performed on RNA from lines of Fhit-expressing and Fhit-deficient lung cancer cells. This identified changes in the levels of mRNAs for a number of cell survival and cell cycle progression genes. Polysome profile analysis performed on cytoplasmic extracts from Fhit-negative and Fhit-positive cells showed changes in the sedimentation of select mRNAs consistent with changes in translation efficiency. The impact of differential Fhit expression on the turnover of selected cancer-linked mRNAs was determined by RT-qPCR of cytoplasmic RNA isolated at intervals after treating cells with a transcription inhibitor.