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Genetic analysis of the Drosophila Bloom's syndrome helicase homolog (mus309/DmBLM) indicates that DmBLM is required for the synthesis-dependent strand annealing (SDSA) pathway of homologous recombination. Here we report the first biochemical study of DmBLM. Recombinant, epitope-tagged DmBLM was expressed in Drosophila cell culture and highly purified protein was prepared from nuclear extracts. Purified DmBLM exists exclusively as a high molecular weight ( approximately 1.17 MDa) species, is a DNA-dependent ATPase, has 3'-->5' DNA helicase activity, prefers forked substrate DNAs and anneals complementary DNAs. High-affinity DNA binding is ATP-dependent and low-affinity ATP-independent interactions contribute to forked substrate DNA binding and drive strand annealing. DmBLM combines DNA strand displacement with DNA strand annealing to catalyze the displacement of one DNA strand while annealing a second complementary DNA strand.

Transcription and pre-mRNA alternative splicing are highly regulated processes that play major roles in modulating eukaryotic gene expression. It is increasingly apparent that other pathways of RNA metabolism, including small RNA biogenesis, can regulate these processes. However, a direct link between alternative pre-mRNA splicing and small RNA pathways has remained elusive. Here we show that the small RNA pathway protein Argonaute-2 (Ago-2) regulates alternative pre-mRNA splicing patterns of specific transcripts in the Drosophila nucleus using genome-wide methods in conjunction with RNAi in cell culture and Ago-2 deletion or catalytic site mutations in Drosophila adults. Moreover, we show that nuclear Argonaute-2 binds to specific chromatin sites near gene promoters and negatively regulates the transcription of the Ago-2-associated target genes. These transcriptional target genes are also bound by Polycomb group (PcG) transcriptional repressor proteins and change during development, implying that Ago-2 may regulate Drosophila development. Importantly, both of these activities were independent of the catalytic activity of Ago-2, suggesting new roles for Ago-2 in the nucleus. Finally, we determined the nuclear RNA-binding profile of Ago-2, found it bound to several splicing target transcripts, and identified a G-rich RNA-binding site for Ago-2 that was enriched in these transcripts. These results suggest two new nuclear roles for Ago-2: one in pre-mRNA splicing and one in transcriptional repression.

The spliceosome, a dynamic assembly of proteins and RNAs, catalyzes the excision of intron sequences from nascent mRNAs. Recent work has suggested that the activity and composition of the spliceosome are regulated by ubiquitination, but the underlying mechanisms have not been elucidated. Here, we report that the spliceosomal Prp19 complex modifies Prp3, a component of the U4 snRNP, with nonproteolytic K63-linked ubiquitin chains. The K63-linked chains increase the affinity of Prp3 for the U5 snRNP component Prp8, thereby allowing for the stabilization of the U4/U6.U5 snRNP. Prp3 is deubiquitinated by Usp4 and its substrate targeting factor, the U4/U6 recycling protein Sart3, which likely facilitates ejection of U4 proteins from the spliceosome during maturation of its active site. Loss of Usp4 in cells interferes with the accumulation of correctly spliced mRNAs, including those for α-tubulin and Bub1, and impairs cell cycle progression. We propose that the reversible ubiquitination of spliceosomal proteins, such as Prp3, guides rearrangements in the composition of the spliceosome at distinct steps of the splicing reaction.

Drosophila P-element somatic inhibitor protein (PSI) regulates splicing of the P-element transposase pre-mRNA by binding a pseudo-splice site upstream of the authentic splice site using four tandem KH-type RNA binding motifs. While the binding domains and specificity of PSI have been established, little is known about the contributions of each PSI KH domain to overall protein stability and RNA binding affinity. Using a construct containing only the RNA binding domain of PSI (PSI-KH03), we introduced a physiologically relevant point mutation into each KH domain of PSI individually and measured stability and RNA binding affinity of the resulting mutant proteins. Although secondary structure, as measured by circular dichroism spectroscopy, is only subtly changed for each mutant protein relative to wild type, RNA binding affinity is reduced in each case. Mutations in the second or third KH domains of the protein are significantly more deleterious to substrate recognition than mutation of the outer (first and fourth) domains. These results show that despite the ability of a single KH domain to bind RNA in some systems, PSI requires multiple tandem KH domains for specific and high-affinity recognition of substrate RNA.

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.

P transposable elements in Drosophila are mobilized via a cut-and-paste mechanism. The broken DNA ends generated during transposition can be repaired via the homology-directed synthesis-dependent strand annealing or by nonhomologous end joining (NHEJ). Genetic studies have demonstrated an interaction between the gene ( mus309 , for mutagen-sensitive) encoding the Drosophila Bloom's syndrome helicase homolog (DmBLM) and the Ku70 gene, which is involved in NHEJ. We have used RNA interference (RNAi) to knock down expression of DmBLM and one or both of the Drosophila Ku subunits, DmKu70 or DmKu80. Our results show that upon reduction of DmKu, an increase in small deletions (1–49 bp) and large deletions (≥50 bp) flanking the site of P element-induced breaks is observed, and a reduction in large deletions at these sites is found upon reduction of DmBLM. Moreover, double RNAi of DmKu and DmBLM results in an increase in small deletions characteristic of the DmKu RNAi and also partially suppresses the reduction in repair efficiency observed with DmKu RNAi. These results suggest that there are DNA double-strand break recognition and/or processing events involving DmKu and DmBLM that, when eliminated by RNAi, lead to deletions. Finally, these results raise the possibility that, unlike the situation in mammals, where BLM appears to function exclusively in the homologous repair pathway, in Drosophila , DmBLM may be directly involved in, or at least influence the double-strand break recognition that leads to the NHEJ repair pathway.

Transposition in many organisms is regulated to control the frequency of DNA damage caused by the DNA breakage and joining reactions. However, genetic studies in prokaryotic systems have led to the isolation of mutant transposase proteins with higher or novel activities compared to those of the wild-type protein. In the course of our study of the effects of mutating potential ATM-family DNA damage checkpoint protein kinase sites in the Drosophila P-element transposase protein, we found one mutation, S129A, that resulted in an elevated level of transposase activity using in vivo recombination assays, including P-element-mediated germline transformation. In vitro assays for P-element transposase activity indicate that the S129A mutant exhibits elevated donor DNA cleavage activity when compared to the wild-type protein, whereas the strand-transfer activity is similar to that of wild type. This difference may reflect the nature of the in vitro assays and that normally in vivo the two reactions may proceed in concert. The P-element transposase protein contains 10 potential consensus phosphorylation sites for the ATM family of PI3-related protein kinases. Of these 10 sites, 8 affect transposase activity either positively or negatively when substituted individually with alanine and tested in vivo. A mutant transposase protein that contains all eight N-terminal serine and threonine residues substituted with alanine is inactive and can be restored to full activity by substitution of wild-type amino acids back at only 3 of the 8 positions. These data suggest that the activity of P-element transposase may be regulated by phosphorylation and demonstrate that one mutation, S129A, results in hyperactive transposition.

U1 snRNP is required at an early stage during assembly of the spliceosome, the dynamic ribonucleoprotein (RNP) complex that performs nuclear pre-mRNA splicing. Here, we report the purification of U1 snRNP particles from Drosophila nuclear extracts and the characterization of their biochemical properties, polypeptide contents, and splicing activities. On the basis of their antigenicity, apparent molecular weight, and by peptide sequencing, the Drosophila 70K, SNF, B, U1-C, D1, D2, D3, E, F, and G proteins are shown to be integral components of these particles. Sequence database searches revealed that both the U1-specific and the Sm proteins are extensively conserved between human and Drosophila snRNPs. Furthermore, both species possess a conserved intrinsic U1-associated kinase activity with identical substrate specificity in vitro. Finally, our results demonstrate that a second type of functional U1 particle, completely lacking the U1/U2-specific protein SNF and the associated protein kinase activity, can be isolated from cultured Kc cell or Canton S embryonic nuclear extracts. This work describes the first characterization of a purified Drosophila snRNP particle and reinforces the view that their activity and composition, with the exception of the atypical bifunctional U1-A/U2-B" SNF protein, are highly conserved in metazoans.

Drosophila P-element transposition is regulated by a maternally inherited state known as P cytotype. An important aspect of P cytotype is transcriptional repression of the P-element promoter. P cytotype can also repress non-P-element promoters within P-element ends, suggesting that P cytotype repression might involve chromatin-based transcriptional silencing. To learn more about the role of chromatin in P cytotype repression, we have been studying the P strain Lk-P(1A). This strain contains two full-length P elements inserted in the heterochromatic telomere-associated sequences (TAS elements) at cytological location 1A. Mutations in the Polycomb group gene (Pc-G gene), Enhancer of zeste (E(z)), whose protein product binds at 1A, resulted in a loss of Lk-P(1A) cytotype control. E(z) mutations also affected the trans-silencing of heterologous promoters between P-element termini by P-element transgenes inserted in the TAS repeats. These data suggest that pairing interactions between P elements, resulting in exchange of chromatin structures, may be a mechanism for controlling the expression and activity of P elements.

The heterodimeric pre-mRNA splicing factor, U2AF (U2 snRNP auxiliary factor), plays a critical role in 3′ splice site selection. Although the U2AF subunits associate in a tight complex, biochemical experiments designed to address the requirement for both subunits in splicing have yielded conflicting results. We have taken a genetic approach to assess the requirement for the Drosophila U2AF heterodimer in vivo. We developed a novel Escherichia coli copurification assay to map the domain on the Drosophila U2AF large subunit (dU2AF50) that interacts with the Drosophila small subunit (dU2AF38). A 28-amino-acid fragment on dU2AF50 that is both necessary and sufficient for interaction with dU2AF38 was identified. Using the copurification assay, we scanned this 28-amino-acid interaction domain for mutations that abrogate heterodimer formation. A collection of these dU2AF50 point mutants was then tested in vivo for genetic complementation of a recessive lethal dU2AF50 allele. A mutation that completely abolished interaction with dU2AF38 was incapable of complementation, whereas dU2AF50 mutations that did not effect heterodimer formation rescued the recessive lethal dU2AF50 allele. Analysis of heterodimer formation in embryo extracts derived from these interaction mutant lines revealed a perfect correlation between the efficiency of subunit association and the ability to complement the dU2AF50 recessive lethal allele. These data indicate that Drosophila U2AF heterodimer formation is essential for viability in vivo, consistent with a requirement for both subunits in splicing in vitro.

We developed in vitro assays to study the first step of the P-element transposition reaction: donor DNA cleavage. We found that P-element transposase required both 5′ and 3′ P-element termini for efficient DNA cleavage to occur, suggesting that a synaptic complex forms prior to cleavage. Transposase made a staggered cleavage at the P-element termini that is novel for all known site-specific endonucleases: the 3′ cleavage site is at the end of the P-element, whereas the 5′ cleavage site is 17 bp within the P-element 31-bp inverted repeats. The P-element termini were protected from exonucleolytic degradation following the cleavage reaction, suggesting that a stable protein complex remains bound to the element termini after cleavage. These data are consistent with a cut-and-paste mechanism for P-element transposition and may explain why P elements predominantly excise imprecisely in vivo.

Sex determination and X chromosome dosage compensation in Drosophila melanogaster are directed by the Sex-lethal (Sxl) protein. In part, Sxl functions by regulating the splicing of the transformer pre-mRNA by binding to a 3' splice site polypyrimidine tract. Polypyrimidine tracts are essential for splicing of metazoan pre-mRNAs. To unravel the mechanism of splicing regulation at polypyrimidine tracts we analyzed the interaction of Sxl with RNA. The RNA binding activity of Sxl was mapped to the two ribonucleoprotein consensus sequence domains of the protein. Quantitation of binding showed that both RNA binding domains (RBDs) were required in cis for site-specific RNA binding. Individual RBDs interacted with RNA more weakly and had lost the ability to discriminate between wild-type and mutant transformer polypyrimidine tracts. Structural elements in one of the RBDs that are likely to interact with a polypyrimidine tract were identified using nuclear magnetic resonance techniques. In addition, our data suggest that multiple imino protons of the transformer polypyrimidine tract were involved in hydrogen bonding. Interestingly, in vitro Sxl bound with equal affinity to polypyrimidine tracts of pre-mRNAs that it does not regulate in vivo. We discuss the implications of this finding for the mechanism through which Sxl may gain selectivity for particular polypyrimidine tracts in vivo.

An intron is an extended genomic feature whose function requires multiple constrained positions—donor and acceptor splice sites, a branch point, a polypyrimidine tract and suitable splicing enhancers—that may be distributed over hundreds or thousands of nucleotides. New introns are therefore unlikely to emerge by incremental accumulation of functional sub-elements. Here we demonstrate that a functional intron can be created de novo in a single step by a segmental genomic duplication. This experiment recapitulates in vivo the birth of an intron that arose in the ancestral jawed vertebrate lineage nearly half-a-billion years ago., The appearance of a new intron that splits an exon without disrupting the corresponding peptide sequence is a rare event in vertebrate genomes. Hellsten et al. demonstrate that, under certain circumstances, a functional intron can be produced in a single step by segmental genomic duplication.

We describe experiments which demonstrated that the simian virus 40 (SV40) enhancer affects certain transcriptional units differently. We also found that a specific enhancer-transcriptional unit interaction can be regulated by trans-acting factors. Using transient assays, we examined the effects of the SV40 enhancer on herpesvirus thymidine kinase (tk) RNA levels when transcription was initiated either by the herpesvirus tk promoter or by an SV40 early promoter-tk fusion. We were unable to detect any effect of the enhancer on transcription from the tk promoter in CV-1 or HeLa cells. However, we found that the addition of T-antigen in trans allowed the enhancer to stimulate expression from the tk promoter. This induction by T-antigen did not require T-antigen-binding sites in cis and appeared to be an indirect effect. In contrast, tk expression from the SV40 early promoter fusion was greatly stimulated by the enhancer in CV-1 cells. Furthermore, in 293 cells the SV40 enhancer had only a marginal effect on the SV40 promoter-tk fusion, whereas it strongly stimulated tk expression from the tk promoter. Our results raise the possibility that the enhancer function may not show cell specificity per se; rather, the interaction between the enhancer and a specific gene may be responsible for cell specificity. We discuss these observations in terms of the SV40 early gene-to-late gene switch that occurs during SV40 lytic growth.

A genome-wide analysis of the response to insulin and wingless activation using splicing-sensitive microarrays shows distinct but overlapping programs of transcriptional and posttranscriptional regulation., Background Despite the prevalence and biological relevance of both signaling pathways and alternative pre-mRNA splicing, our knowledge of how intracellular signaling impacts on alternative splicing regulation remains fragmentary. We report a genome-wide analysis using splicing-sensitive microarrays of changes in alternative splicing induced by activation of two distinct signaling pathways, insulin and wingless, in Drosophila cells in culture. Results Alternative splicing changes induced by insulin affect more than 150 genes and more than 50 genes are regulated by wingless activation. About 40% of the genes showing changes in alternative splicing also show regulation of mRNA levels, suggesting distinct but also significantly overlapping programs of transcriptional and post-transcriptional regulation. Distinct functional sets of genes are regulated by each pathway and, remarkably, a significant overlap is observed between functional categories of genes regulated transcriptionally and at the level of alternative splicing. Functions related to carbohydrate metabolism and cellular signaling are enriched among genes regulated by insulin and wingless, respectively. Computational searches identify pathway-specific sequence motifs enriched near regulated 5' splice sites. Conclusions Taken together, our data indicate that signaling cascades trigger pathway-specific and biologically coherent regulatory programs of alternative splicing regulation. They also reveal that alternative splicing can provide a novel molecular mechanism for crosstalk between different signaling pathways.