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

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.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.It is now well documented that gene expression can be regulated post-transcriptionally and a number of RNA sequence elements have been identified that play a role in targeting a mRNA for a particular mode of regulation. For instance, the stability, the translation and the localisation of a mRNA are regulated by cis-elements, many of which are situated in the 3' untranslated region (3'UTR), but such sequence elements are not restricted to the 3'UTR (for review see [1]).In most cases, regulatory cis-elements are associated with specific RNA-binding proteins (RNA-BP). Several functional pairs of cis-element and trans-acting factor have been identified, for instance the Iron responsive element and the Iron Responsive Protein (reviewed in [2]), the bruno responsive element and bruno [3,4], the Cytoplasmic polyadenylation element (CPE) and the CPE-binding protein [5-7], the Embryo Deadenylation ElemeNt (EDEN) and the EDEN-binding protein [8,9] and lastly, the nanos responsive element and pumilio [10,11]. In at least one instance, the identified cis-element can associate with several factors that confer opposing behaviours: AU-rich elemen