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Alternative premessenger RNA (pre-mRNA) splicing is a post-transcriptional mechanism for controlling gene expression. Splicing patterns are determined by both RNA-binding proteins and nuclear pre-mRNA structure. Here, we analyzed pre-mRNA splicing patterns, RNA-binding sites, and RNA structures near these binding sites coordinately controlled by two splicing factors: the heterogeneous nuclear ribonucleoprotein hnRNPA1 and the RNA helicase DDX5. We identified thousands of alternative pre-mRNA splicing events controlled by these factors by RNA sequencing (RNA-seq) following RNAi. Enhanced cross-linking and immunoprecipitation (eCLIP) on nuclear extracts was used to identify protein–RNA-binding sites for both proteins in the nuclear transcriptome. We found a significant overlap between hnRNPA1 and DDX5 splicing targets and that they share many closely linked binding sites as determined by eCLIP analysis. In vivo SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical RNA structure probing data were used to model RNA structures near several exons controlled and bound by both proteins. Both sequence motifs and in vivo UV cross-linking sites for hnRNPA1 and DDX5 were used to map binding sites in their RNA targets, and often these sites flanked regions of higher chemical reactivity, suggesting an organized nature of nuclear pre-mRNPs. This work provides a first glimpse into the possible RNA structures surrounding pre-mRNA splicing factor-binding sites.

Alternative splicing of precursor mRNA (pre-mRNA) is a strategy employed by most eukaryotes to increase transcript and proteomic diversity. Many metazoan splicing factors are members of multigene families, with each member having different functions. How these highly related proteins evolve unique properties has been unclear. Here we characterize the evolution and function of a new Drosophila splicing factor, termed LS2 (Large Subunit 2), that arose from a gene duplication event of dU2AF50, the large subunit of the highly conserved heterodimeric general splicing factor U2AF (U2-associated factor). The quickly evolving LS2 gene has diverged from the splicing-promoting, ubiquitously expressed dU2AF50 such that it binds a markedly different RNA sequence, acts as a splicing repressor, and is preferentially expressed in testes. Target transcripts of LS2 are also enriched for performing testes-related functions. We therefore propose a path for the evolution of a new splicing factor in Drosophila that regulates specific pre-mRNAs and contributes to transcript diversity in a tissue-specific manner.

The pre-mRNA splicing factor U2AF (U2 snRNP auxiliary factor) has an essential role in 3' splice site selection. U2AF binds the intron pyrimidine tract between the branchpoint and the 3' splice site and recruits U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF65) and small (hU2AF35) subunits. Both subunits contain a domain enriched in arginine-serine dipeptide repeats termed an RS domain. The two U2AF RS domains have been assigned essential and independent roles in spliceosome assembly in vitro-the hU2AF65 RS domain is required to target U2 snRNP to the branch site and the hU2AF35 RS domain is necessary for protein-protein interactions with constitutive and alternative splicing factors. We have investigated the functional requirements for the RS domains on the Drosophila U2AF homolog in vivo. In sharp contrast to its essential role in U2 snRNP recruitment in vitro, the RS domain on the Drosophila large subunit homolog (dU2AF50) was completely dispensable in vivo. Prompted by this unexpected result, we analyzed the RS domain on the Drosophila small subunit homolog (dU2AF38). Despite its requirement for enhancer-dependent splicing activity in vitro, the dU2AF38 RS domain was also inessential in vivo. Finally, we have tested whether the Drosophila U2AF heterodimer requires any RS domain. Flies mutant for both the small and large subunits could not be rescued by dU2AF50deltaRS and dU2AF38deltaRS transgenes. Therefore, in contrast to the separate roles assigned to the U2AF RS domains in vitro, our genetic data suggest that they may have redundant functions in vivo.

PSI is an RNA-binding protein involved in repressing splicing of the P element third intron in Drosophila somatic cell extracts. PSI produced in bacteria restores splicing inhibition to an extract relieved of inhibitory activity, indicating that PSI plays a direct role in somatic inhibition. Sequence analysis of cDNAs encoding PSI reveals three KH RNA-binding domains, a conserved motif also found in the yeast splicing regulator MER1. Notably, PSI is expressed highly in somatic embryonic nuclei but is undetectable in germ-line cells. In contrast, hrp48, another protein implicated in somatic inhibition, is found in the nucleus and cytoplasm of both tissues. The splicing inhibitory properties and soma-specific expression of PSI may be sufficient to explain the germ-line-specific transposition of P elements.

Somatic inhibition restricts splicing of the Drosophila P-element third intron (IVS3) to the germ line. We have exploited this simple system to provide a model for a mechanism of alternative pre-mRNA splicing. Biochemical complementation experiments revealed that Drosophila somatic extracts inhibited U1 snRNP binding to the 5' splice site. Using sensitive RNase protection and modification-interference assays, we found that U1 snRNP bound to a pseudo-5' splice site in the 5' exon and that multiprotein complexes bound to an adjacent site. Binding of these factors appeared to mediate the inhibitory effect, because mutations in the pseudo-5' splice sites blocked binding and activated splicing in vitro. Likewise, wild-type, but not mutant, 5' exon RNA titrated inhibitory factors away from the pre-mRNA and activated splicing. Thus, we have defined the pseudo-5' splice sites as crucial components of the regulatory element, correlated the inhibitory activity with specific RNA binding factors from Drosophila somatic cells, and provided a mechanistic description of somatic inhibition. Because the inhibitory activity involves general splicing functions such as protein recognition of 5' splice site sequences and changes in the distribution of bound U1 snRNP, our data may also provide insights into how splice sites are selected.

The P transposable element of Drosophila melanogaster has a complex array of cis-acting DNA sequences necessary for efficient transposition. At the 3' end these sequences extend over more than 150 bp and include 11- and 31-bp sequences found repeated in inverted orientation at the 5' end. The P element's 5' end, however, cannot function as its 3' end. When two 3' P-element ends are present, the more proximal end is used preferentially. We found also that the duplication of the target site does not appear to play a role in forward transposition.