Visualization of RNA binding proteins by sequential gel shift and ultraviolet cross-linking.
RNA binding proteins partially constitute the ribonucleoprotein 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 T1; 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.
Iyer
R K
RK
School of Life and Health Sciences, University of Delaware, Newark 19716.
Levinger
L F
LF
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D004331:Drosophila melanogaster / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D008970:Molecular Weight; D012313:RNA / Q000235:genetics; D016601:RNA-Binding Proteins; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014466:Ultraviolet Rays
D002352:Carrier Proteins; D016601:RNA-Binding Proteins; D012313:RNA