Biology subjects

Gillingwater, T. H.

Publications and source records attributed to Gillingwater, T. H..

3 recordsLinked to original sources

bioRxiv2017

Active ribosome profiling with RiboLace

Ribosome profiling, or Ribo-Seq, is based around large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information concerning ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-Seq approaches lack the ability to distinguish between fragments protected by ribosomes in active translation or by inactive ribosomes. To overcome these significant limitation, we developed RiboLace: a novel method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.

molecular biology↗

bioRxiv2017

riboWaltz: optimization of ribosome P-site positioning in ribosome profiling data

Ribosome profiling is a powerful technique used to study translation at the genome-wide level, generating unique information concerning ribosome positions along RNAs. Optimal localization of ribosomes requires the proper identification of the ribosome P-site in each ribosome protected fragment, a crucial step to determine trinucleotide periodicity of translating ribosomes, and draw correct conclusions concerning where ribosomes are located. To determine the P-site within ribosome footprints at nucleotide resolution, the precise estimation of its offset with respect to the protected fragment is necessary. Here we present riboWaltz, an R package for calculation of optimal P-site offsets, diagnostic analysis and visual inspection of data. Compared to existing tools, riboWaltz shows improved accuracies for P-site estimation and neat ribosome positioning in multiple case studies.\n\nAvailability and ImplementationriboWaltz was implemented in R and is available at https://github.com/LabTranslationalArchitectomics/RiboWaltz\n\nContactgabriella.viero@cnr.it or fabio.lauria@unitn.it

bioinformatics↗

bioRxiv2017

In vivo translatome profiling reveals early defects in ribosome biology underlying SMA pathogenesis

BackgroundGenetic alterations impacting on ubiquitously expressed proteins involved in mRNA metabolism often result in neurodegenerative conditions, with increasing evidence suggesting that translational defects can contribute to disease. Spinal Muscular Atrophy (SMA) is a neuromuscular disease caused by low levels of SMN protein, whose role in disease pathogenesis remains unclear.\n\nResultsBy determining in parallel the in vivo transcriptome and translatome in SMA mice we identified a robust decrease in translational efficiency, arising during early stages of disease. Translational defects affected translation-related transcripts, were cell autonomous, and were fully rescued after treatment with antisense oligonucleotides to restore SMN levels. Defects in translation were accompanied by a decrease in the number of ribosomes in motor neurons in vivo.\n\nConclusionOur findings suggest that neuronal tissues and cells are particularly sensitive to perturbations in translation during SMA, and identify ribosome biology as an important, yet largely neglected, factor in motor neuron degeneration.

neuroscience↗