The majority of the genes in higher eukaryotic genomes are interrupted by non-coding sequences, introns, that are removed to allow the coding sequences or exons, to be spliced together producing functional mRNAs. The splicing reaction is performed by a large multi-component machinery, the spliceosome, consisting of ~200 proteins and 5 different small nuclear RNAs (snRNAs). One surprising observation that has arisen from the recent sequencing of higher eukaryotic genomes is that there is little correlation between the number of genes in a given genome and organismal complexity. It has been proposed that the paucity of genes in higher eukaryotes could be compensated by the production of different mRNA isoforms, where portions of the initial pre-mRNA are differentially included in the final mRNA, through a mechanism called alternative splicing of pre-mRNA. Alternative splicing has been found to be almost ubiquitous among multi-exon genes in mammals (Wang et al., 2008
). These mRNA isoforms frequently encode different proteins, and can differ in their 5′ or 3′ untranslated regions (UTR) to include cis-acting elements required for the correct expression of the corresponding protein (for review see Hughes, 2006
Most of our understanding of the mechanisms controlling pre-mRNA splicing comes from molecular approaches focused on single-gene studies. These studies have led to the molecular characterization of only a relatively small number of alternatively spliced genes but provide a general understanding of the basic mechanisms of splicing and its regulation.
The sequences recognized by the splicing machinery are highly degenerate and frequently embed in introns that are significantly larger than the flanking exons, and necessitate auxiliary proteins to promote their use. Conversely, several protein factors have been found to modify the interaction of the splicing machinery with the pre-mRNA. Several of these regulatory protein factors have been characterized and found to recognize cis-acting elements within pre-mRNAs that generally fall into two major categories, splicing enhancers and splicing silencers, found in either exons or introns (reviewed in Black, 2003
; Cartegni et al., 2002
; House and Lynch, 2008
; Pagani and Baralle, 2004
; Smith and Valcarcel, 2000
Two group of proteins with apparently antagonistic effects on splicing regulation have been well characterized to date: the SR family and the hnRNP proteins. Several SR proteins recognize splicing enhancers and stimulate the use of nearby splice sites through interactions with components of the basal splicing machinery, like the U2AF factor and the U1 snRNP 70K protein (for review see Matlin et al., 2005
). HnRNP proteins, which have more than 20 different members in most mammals and ten to fifteen members in Drosophila (Dreyfuss et al., 2002
) tend to interact with splicing silencers (for review see Matlin et al., 2005
). Some hnRNP proteins prevent binding of other splicing factors to the pre-mRNA by forming complexes with RNA and/or other proteins and prevent the association with RNA splicing control elements (Zhu et al., 2001
). HnRNP proteins also mediate long range interactions between distant RNA regions flanking alternative exons, thus looping out the intervening region of the pre-mRNA and preventing splicing of the excluded RNA region (Blanchette and Chabot, 1999
; Martinez-Contreras et al., 2006
). In several cases hnRNP proteins have been found to antagonize, both in vitro and in vivo, the activity of SR proteins (Mayeda et al., 1993
; Zahler et al., 2004
; Zhu et al., 2001
High-density oligonucleotide microarray technologies provide the opportunity to profile RNA splicing patterns and the interaction of RNA-binding proteins with RNA transcripts at the whole-transcriptome level, which offers an insight into the post-transcriptional control of gene expression at the level of mRNA maturation and alternative splicing (for review see Blencowe, 2006
). We have developed a splicing-sensitive microarray platform to monitor changes in alternative splicing in Drosophila (Blanchette et al., 2005
). Our first analysis identified the alternatively spliced genes controlled by four different splicing regulators, two SR proteins SC35 and B52/SRp55 and two hnRNP members from different protein families, PSI and hrp48. This initial analysis suggested that very few genes are co-regulated by these SR proteins and hnRNP proteins while identifying a significant number of genes co-regulated by members of the same protein family. These experiments raised several important questions, namely the extent of co-regulated genes within a family of splicing regulators, the specificity, organization and distribution of the cis-acting elements recognized by those proteins, and the molecular mechanisms used to regulate these alternative splicing events.
The hnRNP A/B family of proteins is well conserved from C. elegans to humans and is believed to carry out similar functions across animal phyla. In this work, we have studied four closely related members of the five known Drosophila hnRNP A/B family, hrp36, hrp38, hrp40, and hrp48. Using splicing-sensitive microarrays, between 200 and 300 genes were detected as specifically regulated by each individual hrp protein. The specific RNA sequence motifs recognized by individual hrp proteins were identified using purified proteins and in vitro selection (SELEX) and found to be different for all four proteins tested. The identified binding sites were significantly over-represented in the genes specifically, as well as co-regulated, by the four hnRNP proteins indicative of a complex regulatory network. The genome-wide RNA binding distributions of the four hrp proteins were characterized using a newly developed nuclear RNA-protein (RNP) complex immunopurification approach in conjunction with Drosophila whole-genome tiling arrays (RIP-Chip). Discrete binding locations on expressed RNA transcripts were characterized for all four proteins and found to be predominantly intronic for hrp36, hrp38 and hrp40, while largely exonic binding regions were found for hrp48. Finally, similarly to what was found in our previous analysis, there was no significant overlap between genes regulated by the four hrp proteins and two of the eight different Drosophila SR proteins. In summary, our data suggest that hnRNP proteins bind and control alternative splicing of specific subsets of pre-mRNAs through sequence-specific binding to their target RNAs. Together these studies provide mechanistic insights into how these subsets of specific pre-mRNAs are regulated in vivo.