A Specific RNA Hairpin Loop Structure Binds the RNA Recognition Motifs of the Drosophila SR Protein B52

Abstract

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.Most metazoan genes are transcribed to yield pre-mRNAs containing introns that are excised and exons that are joined to create a functional mRNA (for a review, see reference 39). This pre-mRNA splicing process is catalyzed by the spliceosome, a multicomponent protein-RNA complex consisting of small nuclear ribonuclear protein particles (snRNPs) and nonsnRNP proteins. The accurate selection and pairing of corresponding 5Ј and 3Ј splice sites occur when these factors are assembled onto the pre-mRNA in an orderly manner (for a review, see reference 32). A family of non-snRNP protein factors, termed SR proteins, have been identified as both essential splicing factors and modulators of alternative splicing that function in early spliceosome assembly (for reviews, see references 13 and 30).SR proteins are structurally and functionally related and evolutionarily conserved. This family comprises at least six members, designated SRp20, SRp30a, SRp30b, SRp40, SRp55, and SRp75, which are conserved from Drosophila melanogaster to humans (50). Among them, the human SRp30a (SF2/ASF) (16,17,25,26) and SRp30b (SC35) (14, 15) have been the most intensively studied. In addition, three other human SR proteins, SRp30c (38), p54 (8), and 9G8 (6), have been cloned recently. For Drosophila, only two SR proteins have been cloned. They are homologous to the human SRp55 and SRp20 and are designated B52 (7) and RBP1 (24), respectively. Different SR proteins appear to have overlapping but distinct functions in constitutive and alternative splicing reactions in vitro. Although individual SR proteins are able to complement splicing-deficient cytoplasmic S100 extracts (31, 50), their activities vary when they are presented with different premRNA substrates (12). In alternative splicing, one SR protein may promote preferential usage of one splice site, whe...

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“…The sequence of the flanking constant region was described previously. 4 aptamers, they do compete with each other for binding to dTBP, indicating that they probably recognize the same site.…”

“…The sequences of the template-primer system were described previously. 4) The starting pool for both selections was the 4th generation pool selected by yeast TBP. 22) Twelve cycles of selection and amplification were run for each selection.…”

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