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Functional analysis of SNF, the Drosophila U1A/U2B″ homolog: Identification of dispensable and indispensable motifs for both snRNP assembly and function in vivo

  • SHANE M. STITZINGER (a1), TERESA R. CONRAD (a1), ALICE M. ZACHLIN (a1) and HELEN K. SALZ (a1)

Abstract

In Drosophila, the spliceosomal protein SNF fulfills the functions of two vertebrate proteins, U1 snRNP-U1A and U2 snRNP-U2B″. The structure and sequence of SNF, U1A, and U2B″ are nearly identical with two RNA recognition motifs (RRM) separated by a short linker region, yet they have different RNA-binding properties: U1A binds U1 snRNA, U2B″ binds U2 snRNA, and SNF binds both snRNAs. Structure/function studies on the human proteins have identified motifs in the N-terminal RRM that are critical for RNA-binding specificity but have failed to identify a function for the C-terminal RRM. Interestingly, SNF is chimeric in these motifs, suggesting a basis for its dual specificity. Here, we test the importance of these motifs by introducing site-directed mutations in the snf coding region and examining the effects of these mutations on assembly into the snRNP and on snf function in vivo. We found that an N-terminal RRM mutant protein predicted to eliminate RNA binding still assembles into snRNPs and is capable of rescuing snf 's lethal phenotype only if the normally dispensable C-terminal RRM is present. We also found that the mixed motif in the “RNA-specificity” domain is necessary for SNF's dual function whereas the mixed motif in the U2A′-protein-binding region is not. Finally, we demonstrate that animals carrying a snf mutation that converts SNF from a bifunctional protein to a U1 snRNP-specific protein are viable. This unexpected result suggests that SNF's presence within the U2 snRNP is not essential for splicing.

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Corresponding author

Reprint requests to: Helen K. Salz, Department of Genetics, Case Western Reserve University, Cleveland, Ohio 44106-4955, USA; e-mail: hks@po.cwru.edu.

Keywords

Functional analysis of SNF, the Drosophila U1A/U2B″ homolog: Identification of dispensable and indispensable motifs for both snRNP assembly and function in vivo

  • SHANE M. STITZINGER (a1), TERESA R. CONRAD (a1), ALICE M. ZACHLIN (a1) and HELEN K. SALZ (a1)

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