Active Ribosome Profiling with RiboLace
2018; Cell Press; Volume: 25; Issue: 4 Linguagem: Inglês
10.1016/j.celrep.2018.09.084
ISSN2639-1856
AutoresMassimiliano Clamer, Toma Tebaldi, Fabio Lauria, Paola Bernabò, Rodolfo F. Gómez‐Biagi, Marta Marchioretto, Divya T. Kandala, L. Minati, Elena Perenthaler, Daniele Gubert, Laura Pasquardini, Graziano Guella, Ewout J. N. Groen, Thomas H. Gillingwater, Alessandro Quattrone, Gabriella Viero,
Tópico(s)Machine Learning in Bioinformatics
ResumoHighlights•RiboLace isolates ribosomes in active translation by antibody-free and tag-free pull-down•RiboLace works reliably with low amounts of input material in vitro and in vivo•RiboLace provides positional data of active ribosomes with nucleotide resolution•RiboLace estimates translation levels and predicts protein levels with accuracySummaryRibosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about 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 either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a 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.Graphical abstract
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