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Cell Rep. 2018 Oct 23;25(4):1097-1108.e5. doi: 10.1016/j.celrep.2018.09.084.

Active Ribosome Profiling with RiboLace.

Author information

1
Centre for Integrative Biology, University of Trento, Via Sommarive, 9 Povo, Italy; IMMAGINA Biotechnology s.r.l., Via alla cascata 56/c, Povo, Italy. Electronic address: mclamer@immaginabiotech.com.
2
Centre for Integrative Biology, University of Trento, Via Sommarive, 9 Povo, Italy.
3
Institute of Biophysics, CNR Unit at Trento, Via Sommarive, 18 Povo, Italy.
4
Fondazione Bruno Kessler-LaBSSAH, Via Sommarive, 18 Povo, Trento, Italy.
5
IMMAGINA Biotechnology s.r.l., Via alla cascata 56/c, Povo, Italy.
6
Institute of Biophysics, CNR Unit at Trento, Via Sommarive, 18 Povo, Italy; Department of Information Engineering and Computer Science, University of Trento, Povo, Italy.
7
Department of Physics, University of Trento, Povo, Italy.
8
Edinburgh Medical School: Biomedical Sciences, University of Edinburgh, Edinburgh, UK.
9
Institute of Biophysics, CNR Unit at Trento, Via Sommarive, 18 Povo, Italy. Electronic address: gabriella.viero@cnr.it.

Abstract

Ribosome 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.

KEYWORDS:

polysomal profiling; protein synthesis; proteome; puromycin; ribosome; ribosome profiling; translation; translational control; translatome

PMID:
30355487
DOI:
10.1016/j.celrep.2018.09.084
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