Informational Suppression to Probe RNA:RNA Interactions in the Context of Ribonucleoproteins: U1 and 5' Splice-Site...

Informational suppression is a method to map specific RNA:RNA interactions by taking advantage of the rules of base complementarity. First, a predicted Watson-Crick base pair is broken by single-nucleotide substitution which disrupts the RNA's structure and/or function. Second, the base pair is restored by mutating the opposing nucleotide, thereby rescuing structure and/or function. This method applies to RNP:RNA interactions such as 5' splice-site (5'ss) base-pairing to the 5' end of U1 small nuclear RNA as part of a small nuclear RNP. Our protocol aims to determine the 5'ss:U1 base-pairing register for natural 5'ss, because for distinct 5'ss sequences the nucleotides on each strand can be aligned differently. This methodology includes cloning of a wild-type splicing minigene and introduction of 5'ss variants by PCR mutagenesis. A U1-expression plasmid is mutated to construct "suppressor U1" snRNAs with restored base-pairing to mutant 5'ss in different registers. Cells are transfected with combinations of minigenes and suppressor U1s, and the splicing patterns are analyzed by reverse transcription and semiquantitative PCR, followed by gel electrophoresis. The identity of suppressor U1s that rescue splicing for specific mutations indicates the register used in that 5'ss. We also provide tips to adapt this protocol to other minigenes or registers.