Visualization of RNA binding proteins by sequential gel shift and ultraviolet cross-linking

Abstract

RNA binding proteins partially constitute theribonucleoprotein or protein machinery for RNA processing (splicing, polyadenylation and 3′ end formation), transport, and storage. We have devised a novel method for the detection of RNA binding proteins in vitro. The template for transcription is a cloned Drosophila melanogaster 5S rRNA gene. The method is a two-dimensional gel analysis involving: in vitro transcription of ³²P-labeled 5S rRNA using a cellular S-100; resolution of labeled RNA protein complexes from unbound RNA on a first-dimension mobility shift gel; cross-linking of RNA to protein in gel by ultraviolet irradiation; degradation of the RNA by RNase A and Tl; and analysis of ³²P-protein patterns on a second-dimension discontinuous SDS gel by autoradiography. The pattern of proteins associated with ³²P-5S rRNA is obtained by covalent transfer of ³²P-nucleotides from RNA to the proteins with which the RNA was bound. This method could be useful in the analysis of RNA maturation and processing pathways.