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RNA binding by Sxl proteins in vitro and in vivo
M E Samuels1, D Bopp, R A Colvin
1Department of Molecular Biology, Princeton University, New Jersey 08544.
Abstract:
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.
Insights
The Sex lethal (Sxl) protein binds to specific RNA sequences containing poly(U) tracts, influencing pre-mRNA splicing. Sxl protein localizes to specific chromosomal sites, including its own locus and transgene insertions, indicating its role in gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- RNA Biology
Background:
- The Sex lethal (Sxl) protein is hypothesized to regulate alternative splicing of target genes through direct interaction with pre-mRNA.
- Understanding Sxl's RNA-binding properties is crucial for elucidating its regulatory mechanisms in gene expression.
Purpose of the Study:
- To investigate the in vitro and in vivo RNA-binding characteristics of the Sxl protein.
- To determine the specific RNA sequences recognized by Sxl and its localization within the cell.
Main Methods:
- Gel shift and UV cross-linking assays using recombinant MBP-Sxl fusion protein.
- Analysis of Sxl binding to various RNA sequences, including those with poly(U) tracts and specific splicing regulatory elements.
- Immunostaining of polytene chromosomes and analysis of Sxl protein localization in nuclear extracts and under heat shock conditions.
Main Results:
- MBP-Sxl preferentially binds to RNAs containing poly(U) tracts, with an adenosine at the 5' end enhancing binding.
- Sxl protein does not appear to recognize branch point or AG dinucleotide sequences.
- Sxl forms large ribonucleoprotein (RNP) complexes and localizes to specific chromosomal sites, including the Sxl locus and heat-induced puffs.
Conclusions:
- Sxl protein directly binds to specific RNA sequences, primarily poly(U) tracts, supporting its role in pre-mRNA splicing regulation.
- Sxl's localization to specific chromosomal loci and its presence in large RNP complexes highlight its dynamic role in gene regulation and RNA processing.