Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the
RNA-binding properties of Sxl
protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion
protein demonstrated preferential binding to RNAs containing
poly(U) tracts, and the
protein footprinted over the
poly(U) region. The
protein did not appear to recognize either branch point or AG dinucleotide sequences, but an
adenosine residue at the 5' end of the
poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site
RNA; the doubly shifted form may have been stabilized by
protein-
protein interactions. Consistent with its proposed role in
pre-mRNA processing, in nuclear extracts Sxl was found in large
ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl
protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl
protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl
protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive
small nuclear RNP protein was observed at several heat puffs following
shock.