Abstract
BackgroundDownstream targets for a large number of RNA-binding proteins remain to be identified. The Drosophila master sex-switch protein Sex-lethal (SXL) is an RNA-binding protein that controls splicing, polyadenylation, or translation of certain mRNAs to mediate female-specific sexual differentiation. Whereas some targets of SXL are known, previous studies indicate that additional targets of SXL have escaped genetic screens.Methodology/Principal findingsHere, we have used an alternative molecular approach of GEnomic Selective Enrichment of Ligands by Exponential enrichment (GESELEX) using both the genomic DNA and cDNA pools from several Drosophila developmental stages to identify new potential targets of SXL. Our systematic analysis provides a comprehensive view of the Drosophila transcriptome for potential SXL-binding sites.Conclusion/SignificanceWe have successfully identified new SXL-binding sites in the Drosophila transcriptome. We discuss the significance of our analysis and that the newly identified binding sites and sequences could serve as a useful resource for the research community. This approach should also be applicable to other RNA-binding proteins for which downstream targets are unknown.
Highlights
For several decades, genetic analyses and biochemical characterizations have offered complementary approaches to investigate developmental processes
We have used GESELEX to search for potential RNA targets on a transcriptome-wide basis
In GESELEX, overlapping fragments corresponding to the entire Drosophila genome or to the cDNA pools from different stages of development were attached to a T7 RNA polymerase promoter using a two-step random priming process with appropriate primers
Summary
Genetic analyses and biochemical characterizations have offered complementary approaches to investigate developmental processes. Such studies have led to the identification of regulatory proteins in numerous cellular pathways. Identification of SXL binding sites in the transcriptome revealed downstream regulated genes. While these approaches have hinted existence of additional downstream targets of numerous regulatory proteins, inherent limitations of these techniques have left additional putative targets to be identified. The approach of genomic SELEX is an attempt here to uncover potential new downstream targets which could serve as reagents for future detailed investigations into cellular and developmental processes. Whereas some targets of SXL are known, previous studies indicate that additional targets of SXL have escaped genetic screens
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