Global analysis of positive and negative pre-mRNA splicing regulators in Drosophila

Abstract

To gain insight into splicing regulation, we developed a microarray to assay all annotated alternative splicing events in Drosophila melanogaster and identified the alternative splice events controlled by four splicing regulators: dASF/SF2, B52/SRp55, hrp48, and PSI. The number of events controlled by each of these factors was found to be highly variable: dASF/SF2 strongly affects >300 splicing events, whereas PSI strongly affects only 43 events. Pairwise analysis also revealed many instances of splice site usage affected by multiple factors and provides the framework to understand the network controlling the alternatively spliced mRNA isoforms that compose the Drosophila transcriptome. Higher eukaryotes exploit alternative pre-mRNA splicing to diversify their proteome and to regulate gene expression with developmental stage-and tissue-specificity (Maniatis and Tasic 2002). Therefore, a comprehensive understanding of an organism's gene expression program must include an understanding of alternative splicing (Black 2003). Toward this end, a well-conserved core of pre-mRNA splicing regulatory factors has been identified in all metazoan organisms (Moore et al. 1993;Jurica and Moore 2003). However, the majority of the interactions between these regulators and their target premRNAs remain unknown.Historically, it has been challenging to identify genes specifically regulated by individual splicing factors. Despite tremendous effort, several years separated the initial identification of the hnRNP proteins PSI and hrp48 as regulators of the P-transposase pre-mRNA (Siebel et al. 1994) and the identification of a single additional targeted cellular gene (Burnette et al. 1999;Labourier et al. 2002). Recent advances in microarray technology now permit monitoring of various aspect of RNA processing and maturation (Lee and Roy 2004). In particular, highdensity microarrays have been successfully used to monitor pre-mRNA processing events in yeast (Clark et al. 2002;Burckin et al. 2005), to identify new instances of alternative splicing in human and Drosophila (Hu et al. 2001;Johnson et al. 2003;Wang et al. 2003;Stolc et al. 2004) and to monitor alternative splicing levels of cassette exons in different mouse and human tissues (Pan et al. 2004;Relogio et al. 2005). Here we describe the development of a new Drosophila microarray platform and its use to monitor all the annotated pre-mRNA splicing junctions specifically controlled by four canonical splicing regulators, the hnRNPs PSI and hrp48 as well as the argine/serine-rich (SR) proteins dASF/SF2 and dSRp55/ B52. This study identified tens to hundreds of distinct splice events modulated by each of these splicing factors and reveals the amount of coregulation and antagonism between each. Results A microarray for monitoring alternative splicing in Drosophila melanogasterIn order to rapidly and efficiently identify target genes and specific splicing events regulated by specific splicing factors, we have developed a microarray for monitoring changes of all the known alternatively s...

Genomic mRNA Profiling Reveals Compensatory Mechanisms for the Requirement of the Essential Splicing Factor U2AF

“…This shift is necessary to accommodate the complex landscape of cis-and trans-acting factors from a genomic perspective, which is not captured by model introns or genes. Recent RNA interference and microarray analyses in Drosophila and S. cerevisiae pointed to the possibility of a rich diversity of requirements for splicing factors/regulators (6,7,22,38,40,41). The present study underscores the importance of how weakening (or strengthening) one or more factors in a system can influence the need for others.…”

The gene encoding the splicing factor SF2/ASF is a proto-oncogene

“…1 and Supplementary Table 1). These observations markedly expand the number of known endogenous alternatively spliced gene targets of SR proteins 20,[35][36][37][38] .…”

Sex-Specific Splicing in Drosophila: Widespread Occurrence, Tissue Specificity and Evolutionary Conservation

“…We therefore examined the splicing of six known regulators of alternative splicing: mub, Rm62, Psi, B52, sqd, and ps (Park et al 2004;Blanchette et al 2005;Robida et al 2007). All six genes had probes on the array and showed significant sex bias for the constitutive exons (supplemental Table 6).…”