Research article

Screening for sequence-specific RNA-BPs by comprehensive UV crosslinking

Rebecca Hartley^1,3 , Valerie Le Meuth-Metzinger^2,3 and H Beverley Osborne³

¹  Department of Anatomy and Cell Biology, College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA

²  Laboratoire de Pharmologie, UPRES 2360, Faculté de Médecine, Université de Paris 13, 74 rue Marcel Cachin, 93071 Bobingy cedex, France

³  CNRS UMR 6061 "Génétique et Développement", Université de Rennes 1, Faculté de Médecine, 2 av. Pr. Leon Bernard, CS 34317, 35043 Rennes cedex, France

author email corresponding author email

BMC Molecular Biology 2002, 3:8doi:10.1186/1471-2199-3-8

Published:	7 June 2002

Abstract

Background

Specific cis-elements and the associated trans-acting factors have been implicated in the post-transcriptional regulation of gene expression. In the era of genome wide analyses identifying novel trans-acting factors and cis-regulatory elements is a step towards understanding coordinated gene expression. UV-crosslink analysis is a standard method used to identify RNA-binding proteins. Uridine is traditionally used to radiolabel substrate RNAs, however, proteins binding to cis-elments particularly uridine poor will be weakly or not detected. We evaluate here the possibility of using UV-crosslinking with RNA substrates radiolabeled with each of the four ribonucleotides as an approach for screening for novel sequence specific RNA-binding proteins.

Results

The radiolabeled RNA substrates were derived from the 3'UTRs of the cloned Eg and c-mos Xenopus laevis maternal mRNAs. Specific, but not identical, uv-crosslinking signals were obtained, some of which corresponded to already identified proteins. A signal for a novel 90 kDa protein was observed with the c-mos 3'UTR radiolabeled with both CTP and GTP but not with UTP. The binding site of the 90 kDa RNA-binding protein was localised to a 59-nucleotide portion of the c-mos 3'UTR.

Conclusion

That the 90 kDa signal was detected with RNAs radiolabeled with CTP or GTP but not UTP illustrates the advantage of radiolabeling all four nucleotides in a UV-crosslink based screen. This method can be used for both long and short RNAs and does not require knowledge of the cis-acting sequence. It should be amenable to high throughput screening for RNA binding proteins.