Search

P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.

Peer reviewed: True, P elements were first discovered in the fruit fly Drosophila melanogaster as the causative agents of a syndrome of aberrant genetic traits called hybrid dysgenesis. This occurs when P element-carrying -males mate with females that lack P elements and results in progeny displaying sterility, mutations and chromosomal rearrangements. Since then numerous genetic, developmental, biochemical and structural studies have culminated in a deep understanding of P element transposition: from the cellular regulation and repression of transposition to the mechanistic details of the transposase nucleoprotein complex. Recent studies have revealed how piwi-interacting small RNA pathways can act to control splicing of the P element pre-mRNA to modulate transposase production in the germline. A recent cryo-electron microscopy structure of the P element transpososome reveals an unusual DNA architecture at the transposon termini and shows that the bound GTP cofactor functions to position the transposon ends within the transposase active site. Genome sequencing efforts have shown that there are P element transposase-homologous genes (called THAP9) in other animal genomes, including humans). This review highlights recent and previous studies, which together have led to new insights, and surveys our current understanding of the biology, biochemistry, mechanism and regulation of P element transposition.

In Drosophila , P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein–DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that we termed “Inverted Repeat Binding Protein (IRBP) 18” and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability.

Alternative pre-mRNA splicing (AS) is a critical regulatory mechanism that operates extensively in the nervous system to produce diverse protein isoforms. Fruitless AS isoforms have been shown to influence male courtship behavior, but the underlying mechanisms are unknown. Using genome-wide approaches and quantitative behavioral assays, we show that the P-element somatic inhibitor (PSI) and its interaction with the U1 small nuclear ribonucleoprotein complex (snRNP) control male courtship behavior. PSI mutants lacking the U1 snRNP-interacting domain (PSIΔAB mutant) exhibit extended but futile mating attempts. The PSIΔAB mutant results in significant changes in the AS patterns of ∼1,200 genes in the Drosophila brain, many of which have been implicated in the regulation of male courtship behavior. PSI directly regulates the AS of at least one-third of these transcripts, suggesting that PSI-U1 snRNP interactions coordinate the behavioral network underlying courtship behavior. Importantly, one of these direct targets is fruitless, the master regulator of courtship. Thus, PSI imposes a specific mode of regulatory control within the neuronal circuit controlling courtship, even though it is broadly expressed in the fly nervous system. This study reinforces the importance of AS in the control of gene activity in neurons and integrated neuronal circuits, and provides a surprising link between a pleiotropic pre-mRNA splicing pathway and the precise control of successful male mating behavior.

Alternative pre-mRNA splicing (AS) greatly diversifies metazoan transcriptomes and proteomes and is crucial for gene regulation. Current computational analysis methods of AS from Illumina RNA-sequencing data rely on preannotated libraries of known spliced transcripts, which hinders AS analysis with poorly annotated genomes and can further mask unknown AS patterns. To address this critical bioinformatics problem, we developed a method called the junction usage model (JUM) that uses a bottom-up approach to identify, analyze, and quantitate global AS profiles without any prior transcriptome annotations. JUM accurately reports global AS changes in terms of the five conventional AS patterns and an additional “composite” category composed of inseparable combinations of conventional patterns. JUM stringently classifies the difficult and disease-relevant pattern of intron retention (IR), reducing the false positive rate of IR detection commonly seen in other annotation-based methods to near-negligible rates. When analyzing AS in RNA samples derived from Drosophila heads, human tumors, and human cell lines bearing cancer-associated splicing factor mutations, JUM consistently identified approximately twice the number of novel AS events missed by other methods. Computational simulations showed JUM exhibits a 1.2 to 4.8 times higher true positive rate at a fixed cutoff of 5% false discovery rate. In summary, JUM provides a framework and improved method that removes the necessity for transcriptome annotations and enables the detection, analysis, and quantification of AS patterns in complex metazoan transcriptomes with superior accuracy.

Although alternative pre-mRNA splicing (AS) significantly diversifies the neuronal proteome, the extent of AS is still unknown due in part to the large number of diverse cell types in the brain. To address this complexity issue, we used an annotation-free computational method to analyze and compare the AS profiles between small specific groups of Drosophila circadian neurons. The method, the Junction Usage Model (JUM), allows the comprehensive profiling of both known and novel AS events from specific RNA-seq libraries. The results show that many diverse and novel pre-mRNA isoforms are preferentially expressed in one class of clock neuron and also absent from the more standard Drosophila head RNA preparation. These AS events are enriched in potassium channels important for neuronal firing, and there are also cycling isoforms with no detectable underlying transcriptional oscillations. The results suggest massive AS regulation in the brain that is also likely important for circadian regulation., eLife digest The life of nearly all creatures on Earth follows the rhythm of day and night. For example, in fruit flies, darkness and light dictate when the insects feed, rest, move or mate. This is possible thanks to the circadian clock, an internal program which is synchronized with the environment to tell cells in the body when to perform certain roles. In fruit flies, the structure that keeps the body clock ticking is formed of about 150 ‘circadian neurons’, which are divided into several subgroups. In these cells, a complex genetic programis at work, with networks of genes being ‘switched on’ in a cyclical way. To understand how this program works, scientists need to know which genes are turned on and when, as well as which proteins are created based on the information contained in these genes. This can be difficult because one gene does not necessarily code for only one protein. Indeed, when a gene is turned on, it gets copied into a pre-messenger RNA (pre-mRNA), which is formed of several modules. The pre-mRNA can then go through a process called alternative splicing that shuffles or removes the different modules. This means that one gene can give rise to different pre-mRNA molecules that will each serve as a template to build a distinct protein. Until now, there have been few studies that examine the different types of pre-mRNAs found in circadian neurons, and how these change with the time of day. Here, Wang, Abruzzi et al. extract three subgroups of circadian neurons, and one subgroup of non-circadian neurons, from the brain of fruit flies. The pre-mRNAs are isolated, and then a new computational method, called JUM, identifies, counts and categorizes the pre-mRNA molecules in the different groups of neurons. This analysis reveals hundreds of previously unknown pre-mRNA molecules, many of which differed extensively between the types of brain cells. When comparing circadian and non-circadian neurons, Wang, Abruzzi et al. show that the circadian cells had more pre-mRNAs that code for proteins that help the cell communicate with other neurons. Finally, many genes in the circadian neurons use alternative splicing to turn on different types of pre-mRNA molecules at different times of the day in a cyclical way; this suggests that these pre-mRNAs might be participating in the genetic circadian program. Many human disorders, such as certain forms of insomnia, emerge when the circadian clock is thrown off balance. The results reported by Wang, Abruzzi et al. show that alternative splicing may be an overlooked mechanism that shapes this complex program.

RNA sequence elements involved in the regulation of pre-mRNA splicing have previously been identified in vertebrate genomes by computational methods. Here, we apply such approaches to predict splicing regulatory elements in Drosophila melanogaster and compare them with elements previously found in the human, mouse, and pufferfish genomes. We identified 99 putative exonic splicing enhancers (ESEs) and 231 putative intronic splicing enhancers (ISEs) enriched near weak 5′ and 3′ splice sites of constitutively spliced introns, distinguishing between those found near short and long introns. We found that a significant proportion (58%) of fly enhancer sequences were previously reported in at least one of the vertebrates. Furthermore, 20% of putative fly ESEs were previously identified as ESEs in human, mouse, and pufferfish; while only two fly ISEs, CTCTCT and TTATAA, were identified as ISEs in all three vertebrate species. Several putative enhancer sequences are similar to characterized binding-site motifs for Drosophila and mammalian splicing regulators. To provide additional evidence for the function of putative ISEs, we separately identified 298 intronic hexamers significantly enriched within sequences phylogenetically conserved among 15 insect species. We found that 73 putative ISEs were among those enriched in conserved regions of the D. melanogaster genome. The functions of nine enhancer sequences were verified in a heterologous splicing reporter, demonstrating that these sequences are sufficient to enhance splicing in vivo. Taken together, these data identify a set of predicted positive-acting splicing regulatory motifs in the Drosophila genome and reveal regulatory sequences that are present in distant metazoan genomes.

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.

In Drosophila melanogaster, female-specific expression of Sex-lethal (SXL) and Transformer (TRA) proteins controls sex-specific alternative splicing and/or translation of a handful of regulatory genes responsible for sexual differentiation and behavior. Recent findings in 2009 by Telonis-Scott et al. document widespread sex-biased alternative splicing in fruitflies, including instances of tissue-restricted sex-specific splicing. Here we report results arguing that some of these novel sex-specific splicing events are regulated by mechanisms distinct from those established by female-specific expression of SXL and TRA. Bioinformatic analysis of SXL/TRA binding sites, experimental analysis of sex-specific splicing in S2 and Kc cells lines and of the effects of SXL knockdown in Kc cells indicate that SXL-dependent and SXL-independent regulatory mechanisms coexist within the same cell. Additional determinants of sex-specific splicing can be provided by sex-specific differences in the expression of RNA binding proteins, including Hrp40/Squid. We report that sex-specific alternative splicing of the gene hrp40/squid leads to sex-specific differences in the levels of this hnRNP protein. The significant overlap between sex-regulated alternative splicing changes and those induced by knockdown of hrp40/squid and the presence of related sequence motifs enriched near subsets of Hrp40/Squid-regulated and sex-regulated splice sites indicate that this protein contributes to sex-specific splicing regulation. A significant fraction of sex-specific splicing differences are absent in germline-less tudor mutant flies. Intriguingly, these include alternative splicing events that are differentially spliced in tissues distant from the germline. Collectively, our results reveal that distinct genetic programs control widespread sex-specific splicing in Drosophila melanogaster.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) have been traditionally seen as proteins packaging RNA nonspecifically into ribonucleoprotein particles (RNPs), but evidence suggests specific cellular functions on discrete target pre-mRNAs. Here we report genome-wide analysis of alternative splicing patterns regulated by four Drosophila homologues of the mammalian hnRNP A/B family (hrp36, hrp38, hrp40 and hrp48). Analysis of the global RNA binding distributions of each protein revealed both small and also extensively bound regions on target transcripts. A significant subset of RNAs were bound and regulated by more than one hnRNP protein, revealing a combinatorial network of interactions. In vitro RNA binding site selection experiments (SELEX) identified distinct binding motif specificities for each protein that were over-represented in their respective regulated and bound transcripts. These results indicate that individual heterogeneous ribonucleoproteins have specific affinities for overlapping, but distinct, populations of target pre-mRNAs controlling their patterns of RNA processing.

The Down syndrome cell adhesion molecule (Dscam) gene has essential roles in neural wiring and pathogen recognition in Drosophila melanogaster. Dscam encodes 38,016 distinct isoforms via extensive alternative splicing. The 95 alternative exons in Dscam are organized into clusters that are spliced in a mutually exclusive manner. The exon 6 cluster contains 48 variable exons and uses a complex system of competing RNA structures to ensure that only one variable exon is included. Here we show that the heterogeneous nuclear ribonucleoprotein hrp36 acts specifically within, and throughout, the exon 6 cluster to prevent the inclusion of multiple exons. Moreover, hrp36 prevents serine/arginine-rich proteins from promoting the ectopic inclusion of multiple exon 6 variants. Thus, the fidelity of mutually exclusive splicing in the exon 6 cluster is governed by an intricate combination of alternative RNA structures and a globally acting splicing repressor.

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.

Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites.

Faithful propagation of eukaryotic chromosomes usually requires that no DNA segment be replicated more than once during one cell cycle. Cyclin-dependent kinases (Cdks) are critical for the re-replication controls that inhibit the activities of components of the pre-replication complexes (pre-RCs) following origin activation. The origin recognition complex (ORC) initiates the assembly of pre-RCs at origins of replication and Cdk phosphorylation of ORC is important for the prevention of re-initiation. Here we show that Drosophila melanogaster ORC (DmORC) is phosphorylated in vivo and is a substrate for Cdks in vitro. Cdk phosphorylation of DmORC subunits DmOrc1p and DmOrc2p inhibits the intrinsic ATPase activity of DmORC without affecting ATP binding to DmOrc1p. Moreover, Cdk phosphorylation inhibits the ATP-dependent DNA-binding activity of DmORC in vitro, thus identifying a novel determinant for DmORC-DNA interaction. DmORC is a substrate for both Cdk2·cyclin E and Cdk1·cyclin B in vitro. Such phosphorylation of DmORC by Cdk2·cyclin E, but not by Cdk1·cyclin B, requires an “RXL” motif in DmOrc1p. We also identify casein kinase 2 (CK2) as a kinase activity in embryonic extracts targeting DmORC for modification. CK2 phosphorylation does not affect ATP hydrolysis by DmORC but modulates the ATP-dependent DNA-binding activity of DmORC. These results suggest molecular mechanisms by which Cdks may inhibit ORC function as part of re-replication control and show that DmORC activity may be modulated in response to phosphorylation by multiple kinases.

Distinct isoforms of spliceosomal RNAs may be involved in regulating pre-messenger RNA splicing in eukaryotic cells. During a large-scale effort to identify small noncoding RNAs in Drosophila, we isolated a U5 snRNA-like molecule containing a 5′ segment identical to that of the canonical (major) U5 snRNA but with a variant Sm binding site and a distinct 3′ hairpin sequence. Based on this finding, another six similar U5 snRNA-like sequences were identified within the Drosophila genome by sequence similarity to the invariant loop in the 5′ half of U5. Interestingly, although all of these variants are expressed in vivo, each shows a distinct temporal expression profile during Drosophila development, and one is expressed primarily in fly heads. The presence of these U5 snRNA variants within RNP particles suggests their role in splicing and implies a possible connection to regulation of developmental and tissue-specific gene expression.

The protein factor U2AF is an essential component required for pre-mRNA splicing. Mutations identified in the S. pombe large U2AF subunit were used to engineer transgenic Drosophila carrying temperature-sensitive U2AF large subunit alleles. Mutant recombinant U2AF heterodimers showed reduced polypyrimidine tract RNA binding at elevated temperatures. Genome-wide RNA profiling comparing wild-type and mutant strains identified more than 400 genes differentially expressed in the dU2AF50 mutant flies grown at the restrictive temperature. Surprisingly, almost 40% of the downregulated genes lack introns. Microarray analyses revealed that nuclear export of a large number of intronless mRNAs is impaired in Drosophila-cultured cells RNAi knocked down for dU2AF50. Immunopurification of nuclear RNP complexes showed that dU2AF50 associates with intronless mRNAs. These results reveal an unexpected role for the splicing factor dU2AF50 in the nuclear export of intronless mRNAs.

We have used genetic and microarray analysis to determine how ionizing radiation (IR) induces p53-dependent transcription and apoptosis in Drosophila melanogaster. IR induces MNK/Chk2-dependent phosphorylation of p53 without changing p53 protein levels, indicating that p53 activity can be regulated without an Mdm2-like activity. In a genome-wide analysis of IR-induced transcription in wild-type and mutant embryos, all IR-induced increases in transcript levels required both p53 and the Drosophila Chk2 homolog MNK. Proapoptotic targets of p53 include hid, reaper, sickle, and the tumor necrosis factor family member EIGER: Overexpression of Eiger is sufficient to induce apoptosis, but mutations in Eiger do not block IR-induced apoptosis. Animals heterozygous for deletions that span the reaper, sickle, and hid genes exhibited reduced IR-dependent apoptosis, indicating that this gene complex is haploinsufficient for induction of apoptosis. Among the genes in this region, hid plays a central, dosage-sensitive role in IR-induced apoptosis. p53 and MNK/Chk2 also regulate DNA repair genes, including two components of the nonhomologous end-joining repair pathway, Ku70 and Ku80. Our results indicate that MNK/Chk2-dependent modification of Drosophila p53 activates a global transcriptional response to DNA damage that induces error-prone DNA repair as well as intrinsic and extrinsic apoptosis pathways.

Precursor messenger RNA (pre-mRNA) splicing is a critical step in the posttranscriptional regulation of gene expression, providing significant expansion of the functional proteome of eukaryotic organisms with limited gene numbers. Split eukaryotic genes contain intervening sequences or introns disrupting protein-coding exons, and intron removal occurs by repeated assembly of a large and highly dynamic ribonucleoprotein complex termed the spliceosome, which is composed of five small nuclear ribonucleoprotein particles, U1, U2, U4/U6, and U5. Biochemical studies over the past 10 years have allowed the isolation as well as compositional, functional, and structural analysis of splicing complexes at distinct stages along the spliceosome cycle. The average human gene contains eight exons and seven introns, producing an average of three or more alternatively spliced mRNA isoforms. Recent high-throughput sequencing studies indicate that 100% of human genes produce at least two alternative mRNA isoforms. Mechanisms of alternative splicing include RNA–protein interactions of splicing factors with regulatory sites termed silencers or enhancers, RNA–RNA base-pairing interactions, or chromatin-based effects that can change or determine splicing patterns. Disease-causing mutations can often occur in splice sites near intron borders or in exonic or intronic RNA regulatory silencer or enhancer elements, as well as in genes that encode splicing factors. Together, these studies provide mechanistic insights into how spliceosome assembly, dynamics, and catalysis occur; how alternative splicing is regulated and evolves; and how splicing can be disrupted by cis- and trans-acting mutations leading to disease states. These findings make the spliceosome an attractive new target for small-molecule, antisense, and genome-editing therapeutic interventions.

This protocol describes how to generate and analyze products and intermediates in a pre-mRNA splicing reaction. The reaction relies on the use of labeled, capped, synthetic pre-mRNAs, prepared by in vitro transcription, and Drosophila Kc cell culture nuclear extracts. The pre-mRNA substrate is incubated in the nuclear extract under splicing conditions for 1–2 h. The products of the reaction are purified by phenol:chloroform extraction and precipitation with ethanol, and then loaded directly onto a denaturing urea–acrylamide gel. Visualization of the splicing reactions will reveal the pre-mRNA, the spliced mRNA, and the intermediates generated by the first step of splicing. For inefficient reactions, a more sensitive detection method, such as RNase protection, primer extension, or RT-PCR (reverse transcription–polymerase chain reaction), may be required.

This protocol describes how to prepare nuclear extracts from cultured Drosophila cells. It uses the Kc cell line, which grows without fetal bovine serum and so is suitable for large-scale suspension cultures. The suspension cells are harvested by centrifugation, washed to remove media components, and resuspended in a low-salt (hypotonic) buffer that makes the cells swell in size and easy to homogenize. Usually, nuclear extract preparations are performed on 4–32 L of cells at ∼3.5 × 106 cells/mL.

It is of interest to be able to define sets of cellular RNAs associated with specific RNA-binding proteins. This “guilt by association” can lead to new insights into how RNA-binding proteins modulate posttranscriptional gene expression of specific target RNAs. To identify these RNAs, antibodies against RNA-binding proteins can be used to immunopurify endogenous RNA–protein complexes from cells, and then the associated RNAs can be characterized. The method described here was developed to identify binding regions on nuclear transcripts for Drosophila heterogeneous nuclear ribonucleoproteins (hnRNPs). An antibody is added to an RNP extract and incubated to allow antigen–antibody complexes to form. The antibody–antigen complexes are then retrieved by binding of the antibody constant region to staphylococcal Protein A immobilized on Sepharose beads. The bead-immobilized complexes are then washed and RNA is prepared. The RNA is used to generate random-primed cDNA, cRNA, and biotinlyated cDNA probes for use on Affymetrix whole-genome Drosophila tiling arrays.

P element somatic inhibitor (PSI) is a 97-kDa RNA-binding protein with four KH motifs that is involved in the inhibition of splicing of the Drosophila P element third intron (IVS3) in somatic cells. PSI interacts with a negative regulatory element in the IVS3 5' exon. This element contains two pseudo-5' splice sites, termed F1 and F2. To identify high affinity binding sites for the PSI protein, in vitro selection (SELEX) was performed using a random RNA oligonucleotide pool. Alignment of high affinity PSI-binding RNAs revealed a degenerate consensus sequence consisting of a short core motif of CUU flanked by alternative purines and pyrimidines. Interestingly, this sequence resembles the F2 pseudo-5' splice site in the P element negative regulatory element. Additionally, a negative in vitro selection of PCR-mutagenized P element 5' exon regulatory element RNAs identified two U residues in the F1 and F2 pseudo-5' splice sites as important nucleotides for PSI binding and the U residue in the F2 region is a nearly invariant nucleotide in the consensus SELEX motif. The high affinity PSI SELEX sequence acted as a splicing inhibitor when placed in the context of a P element splicing pre-mRNA in vitro. Data from in vitro splicing assays, UV crosslinking and RNA-binding competition experiments indicates a strong correlation between the binding affinities of PSI for the SELEX sequences and their ability to modulate splicing of P element IVS3 in vitro.

By binding specific RNA transcripts, the Sex-lethal protein (SXL) governs sexual differentiation and dosage compensation in Drosophila melanogaster . To investigate the basis for RNA binding specificity, we determined the crystal structure of the tandem RNA recognition motifs (RRMs) of SXL. Both RRMs adopt the canonical RRM fold, and the 10-residue, interdomain linker shows significant disorder. In contrast to the previously determined structure of the two-RRM fragment of heterogeneous nuclear ribonucleoprotein Al, SXL displays no interdomain contacts between RRMs. These results suggest that the SXL RRMs are flexibly tethered in solution, and RNA binding restricts the orientation of RRMs. Therefore, the observed specificity for single-stranded, U-rich sequences does not arise from a predefined, rigid architecture of the isolated SXL RRMs.

The generation of functional mRNA in eukaryotes requires the accurate removal of noncoding regions (introns) from pre-mRNA by a process termed pre-mRNA splicing (14, 25). Splicing takes place in the spliceosome, a dynamic RNA-protein complex that assembles in a stepwise manner on the pre-mRNA (10, 14). The spliceosome is composed of small nuclear ribonucleoprotein particles (snRNPs) and extrinsic (non-snRNP) factors. The recognition of exon/intron boundaries, the splice sites, by the splicing apparatus is a critical step in processing of both constitutively and alternatively spliced pre-mRNAs. U1 snRNP defines the 5′ splice site, and U2 snRNP defines the branchpoint sequence (3, 10, 14, 17). Since in most cases the first AG dinucleotide downstream of the branchpoint is used as the 3′ splice site, by defining the branchpoint, U2 snRNP defines the 3′ splice site (18, 27). Targeting of U2 snRNP to the branch site requires the extrinsic splicing factor U2 snRNP auxiliary factor (U2AF). U2AF binds site specifically to the intron pyrimidine tract between the branchpoint sequence and 3′ splice site at an early step in spliceosome assembly and recruits U2 snRNP to the branch site (22, 29, 42). Regulation of 3′ splice site choice, both positive and negative, can be realized by influencing the pyrimidine tract binding of U2AF (33, 35, 45). Because U2AF is a major determinant in 3′ splice site selection, it has been the subject of extensive biochemical and genetic investigation. Human U2AF is a heterodimer composed of a 65-kDa large subunit (hU2AF65) and a 35-kDa small subunit (hU2AF35) (41). Both subunits are conserved in other organisms (40), and U2AF homologs have been identified in Drosophila melanogaster (9, 21), Schizosaccharomyces pombe (16, 36), and Caenorhabditis elegans (3a, 39). The Drosophila U2AF large (dU2AF50)- and small (dU2AF38)-subunit homologs are 50 and 38 kDa, respectively (9, 21). The U2AF large subunit contains three RNA recognition motifs (RRMs) and an amino-terminal arginine-serine-rich (RS) domain (42). The small subunit contains a highly degenerate RRM (pseudo-RRM) (2), two Zn2+ binding motifs (37), and a carboxyl-terminal RS domain and glycine-rich region (43). Both U2AF subunits are involved in recognition of the intron pyrimidine tract. The large subunit (hU2AF65 and dU2AF50) is required for site-specific pyrimidine tract binding (9, 42). The small subunit acts as a cofactor to stabilize the large subunit on the pyrimidine tract, apparently through protein-protein interactions with constitutive and alternative splicing factors (38, 45). While it has been firmly established that all three RRMs on hU2AF65 are necessary for high-affinity RNA binding, a role for the large-subunit RS domain in RNA binding remains unresolved (11, 42). In one study removal of the hU2AF65 RS domain had a modest effect on RNA binding (42). In a second study, the RS domain was found to be absolutely required for RNA binding (11). In vitro splicing assays using U2AF-depleted extracts prepared by two independent methods have identified independent and essential roles for the two U2AF RS domains: the large-subunit RS domain is required to target U2 snRNP to the branch site (34, 42), and in the immunodepleted extracts under certain conditions, the small-subunit RS domain is apparently necessary for protein-protein interactions with constitutive and alternative splicing factors to stabilize hU2AF65 on the pyrimidine tract (38, 45). In contrast to the essential roles assigned to the two U2AF RS domains in vitro, molecular genetic analysis of the Drosophila U2AF RS domains indicates that either one of the RS domains is dispensable in vivo (19). Importantly, at least one RS domain on U2AF is essential for viability (19). The observation that the dU2AF38 RS domain is not essential in vivo (19) refocused our attention on domains present in the U2AF small subunit that are phylogenetically conserved. In an exhaustive database search for proteins containing RRMs, some of the signature sequences of this motif were identified in hU2AF35 (2). These sequences are also present in the Drosophila and S. pombe small-subunit homologs (21, 36). Although some of the most conserved residues in the RNA recognition motif are present in the U2AF small subunits, the RNP-1 octamer is highly degenerate and the RNP-2 hexamer is absent. Since these defining submotifs and other conserved residues are not present in the U2AF small-subunit RRM, it was termed a degenerate RRM or pseudo-RRM (2). Degenerate RRMs have been identified in a collection of RNA binding proteins, including several of the SR proteins, the pyrimidine tract binding protein, and the large subunit of U2AF (10). The degenerate RRMs in SRp30a (ASF/SF2) (4, 46), pyrimidine tract-binding protein (15), and hU2AF65 (42) were all found to be required for high-affinity RNA binding. Two putative Zn2+ binding domains, one on either side of the pseudo-RRM, were recently identified in hU2AF35 in a database search (37). These Cys3His Zn2+ binding motifs are conserved in all three small subunit homologs. Though sequence-specific RNA binding has not been described for proteins that contain this type of Zn2+ binding motif, several proteins that have this domain are involved in RNA metabolism (37). The evolutionary conservation of the pseudo-RRM and the two Zn2+ binding motifs in all three U2AF small subunit homologs suggested to us that these domains are important for function. The lack of requirement for the dU2AF38 RS domain in vivo prompted us to search for novel biochemical activities associated with the small subunit. The phylogenetically conserved, degenerate RRM (2) and two Zn2+ binding motifs (37) in the small subunit suggested that it might participate in RNA binding. While we detected weak RNA binding activity for the Drosophila small subunit on its own, we found that when complexed with the large subunit, dU2AF pyrimidine tract binding affinity increased 20-fold. This increase in RNA binding activity was not specific to Drosophila U2AF; the human U2AF heterodimer bound RNA with 15-fold-higher affinity than hU2AF65. Surprisingly, removal of the dU2AF38 RS domain abolished the increase in binding activity of the dU2AF heterodimer, indicating that the RS domain is necessary for high-affinity binding. Deletion of the dU2AF50 RS domain (dU2AF50ΔRS) dramatically reduced RNA binding activity of the large-subunit monomer. High-affinity binding was restored when the dU2AF38 RS domain was supplied in trans to dU2AF50ΔRS. These data suggest that high-affinity RNA binding activity requires at least one RS domain on U2AF, which is consistent with the requirement for at least one RS domain in vivo.

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.

The Drosophila melanogaster hnRNP protein, hrp48, is an abundant heterogeneous nuclear RNA-associated protein. Previous biochemical studies have implicated hrp48 as a component of a ribonucleoprotein complex involved in the regulation of the tissue-specific alternative splicing of the P-element third intron (IVS3). We have taken a genetic approach to analyzing the role of hrp48. Mutations in the hrp48 gene were identified and characterized. hrp48 is an essential gene. Hypomorphic mutations which reduce the level of hrp48 protein display developmental defects, including reduced numbers of ommatidia in the eye and morphological bristle abnormalities. Using a P-element third-intron reporter transgene, we found that reduced levels of hrp48 partially relieve IVS3 splicing inhibition in somatic cells. This is the first direct evidence that hrp48 plays a functional role in IVS3 splicing inhibition.

In mammalian cells, the Ku heterodimer is involved in DNA double-strand-break recognition and repair. We have established in yeast a connection between Ku activity and DNA double-strand-break damage repair, and a connection between Ku activity and commitment to DNA replication. We generated double-stranded DNA breaks in yeast cells in vivo by expressing a restriction endonuclease and have shown that yeast mutants lacking Ku p70 activity died while isogenic wild-type cells did not. Moreover, we have discovered that DNA damage occurs spontaneously during normal yeast mitotic growth, and that Ku functions in repair of this damage. We also observed that mitotically growing Ku p70 mutants have an anomalously high DNA content, suggesting a role for Ku in regulation of DNA synthesis. Finally, we present evidence that Ku p70 function is conserved between yeast, Drosophila , and humans.

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.

Drosophila P elements are mobile DNA elements that encode an 87-kDa transposase enzyme and transpositional repressor proteins. One of these repressor proteins is the 207-amino-acid KP protein which is encoded by a naturally occurring P element with an internal deletion. To study the molecular mechanisms by which KP represses transposition, the protein was expressed, purified, and characterized. We show that the KP protein binds to multiple sites on the ends of P-element DNA, unlike the full-length transposase protein. These sites include the high-affinity transposase binding site, an 11-bp transpositional enhancer, and, at the highest concentrations tested, the terminal 31-hp inverted repeats. The DNA binding domain was localized to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal binding motif. We also demonstrate that the KP repressor protein can dimerize and contains two protein-protein interaction regions and that this dimerization is essential for high-affinity DNA binding.

Mobilizing Transposable Elements Transposable elements are parasitic DNA elements that can jump around the genome. They make up large portions of eukaryotic genomes (up to 50% of the human genome). In the short term, transposable elements can generate mutations and cause large-scale damage; but, in the long term, they can drive evolution. Majumdar et al. (p. 446 ) show that the human THAP9 gene (hTh9), which contains a THAP DNA binding domain also found in the Drosophila P -element transposase, is capable of mobilizing P -elements in Drosophila cells. The hTH9 gene also caused the excision of plasmid-borne Drosophila P -elements in human cells. Furthermore, hTh9 is able to nick P -element DNA and direct its transposition into the human genome.

Alternative splicing of pre-mRNA is a highly regulated process that allows cells to change their genetic informational output. These changes are mediated by protein factors that directly bind specific pre-mRNA sequences. Although much is known about how these splicing factors regulate pre-mRNA splicing events, comparatively little is known about the regulation of the splicing factors themselves. Here, we show that the Drosophila splicing factor P element Somatic Inhibitor (PSI) is phosphorylated at at least two different sites by at minimum two different kinases, casein kinase II (CK II) and tousled-like kinase (tlk). These phosphorylation events may be important for regulating protein-protein interactions involving PSI. Additionally, we show that PSI interacts with several proteins in Drosophila S2 tissue culture cells, the majority of which are splicing factors.

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.

The RNA-binding protein Sex-lethal (Sxl) is a critical regulator of sexual differentiation and dosage compensation in Drosophila. This regulatory activity is a consequence of the ability of Sxl to bind uridine-rich RNA tracts involved in pre-mRNA splicing. Sxl contains two RNP consensus-type RNA-binding domains (RBDs). A structural study of a portion of Sxl (amino acids 199-294) containing the second RNA-binding domain (RBD-2) using multidimensional heteronuclear NMR is presented here. Nearly complete 1H, 13C, and 15N resonance assignments have been obtained from 15N- and 13C/15N-uniformly labeled protein. These assignments were used to analyze 3D 15N-separated NOESY and 13C/13C-separated 4D NOESY spectra which produced 494 total and 169 long-range NOE-derived distance restraints. Along with 41 backbone dihedral restraints, these distance restraints were employed to generate an intermediate-resolution family of calculated structures, which exhibits the beta alpha beta-beta alpha beta tertiary fold found in other RBD-containing proteins. The RMSD to the average structure for the backbone atoms of residues 11-93 is 1.55 +/- 0.30 A, while the RMSD for backbone atoms involved in secondary structure is 0.76 +/- 0.14 A. A capping box [Harper, E.T., & Rose, G.D. (1993) Biochemistry 32, 7605-7609] was identified at the N-terminus of the first helix and has been characterized by short- and medium-range NOEs. Finally, significant structural similarities and differences between Sxl RBD-2 and other RBD-containing proteins are discussed.

The large subunit of the human pre-messenger RNA splicing factor U2 small nuclear ribonucleoprotein auxiliary factor (hU2AF65) is required for spliceosome assembly in vitro. A complementary DNA clone encoding the large subunit of Drosophila U2AF (dU2AF50) has been isolated. The dU2AF50 protein is closely related to its mammalian counterpart and contains three carboxyl-terminal ribonucleoprotein consensus sequence RNA binding domains and an amino-terminal arginine- and serine-rich (R/S) domain. Recombinant dU2AF50 protein complements mammalian splicing extracts depleted of U2AF activity. Germline transformation of Drosophila with the dU2AF50 complementary DNA rescues a lethal mutation, establishing that the dU2AF50 gene is essential for viability. R/S domains have been found in numerous metazoan splicing factors, but their function is unknown. The mutation in Drosophila U2AF will allow in vivo analysis of a conserved R/S domain-containing general splicing factor.

Over the past year, significant progress has been made in the understanding of how RNA-binding factors may facilitate splice-site selection and spliceosome assembly, and confer fidelity to the pre-mRNA splicing reaction. In addition, a number of studies have revealed a complex network of RNA-RNA interactions in the spliceosome, strengthening the structural and functional parallels between nuclear pre-mRNA splicing and the self-splicing group I and group II introns. These new data further support the idea that pre-mRNA splicing occurs by RNA-mediated catalysis and illustrate quite dramatically the dynamic nature of conformational changes in the spliceosome cycle. With respect to tissue-specific pre-mRNA splicing, a number of studies have begun to illuminate mechanisms underlying control of splice-site selection and how so-called 'general' RNA-binding proteins, such as heterogeneous nuclear ribonucleoproteins, may be involved in determining different splicing patterns. Finally, an emerging theme involving the role of splicing in development is that differential transcriptional programs can be triggered in different cell types by alternative splicing patterns that generate transcription factor isoforms with different activities or DNA-binding specificities.

Perhaps the most important and certainly the most often used technique in RNA analysis is gel electrophoresis. This technique is generally applicable for RNA detection, quantification, purification by size, and quality assessment. Because RNAs are negatively charged, they migrate toward the anode in the presence of electric current. The gel acts as a sieve to selectively impede the migration of the RNA in proportion to its mass, given that its mass is generally proportional to its charge. Because mass is approximately related to chain length, the length of an RNA is more generally determined by its migration. In addition, topology (i.e., circularity) can affect migration, making RNAs appear longer on the gel than they actually are. Gels are used in a wide variety of techniques, including Northern blotting, primer extension, footprinting, and analyzing processing reactions. They are invaluable as preparative and fractionating tools. There are two common types of gel: polyacrylamide and agarose. For most applications, denaturing acrylamide gels are most appropriate. These gels are extremely versatile and can resolve RNAs from ~600 to/=20 nucleotides (nt). In certain circumstances, e.g., resolving different conformers of RNAs or RNA-protein complexes, native gels are appropriate. The only disadvantage to acrylamide gels is that they are not suitable for analyzing large RNAs (or =600 nt); for such applications, agarose gels are preferred. This protocol describes how to prepare, load, and run polyacrylamide gels for RNA analysis.

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.

We have developed an in vitro reaction system for Drosophila P element transposition. Transposition products were recovered by selection in E. coli, and contained simple P element insertions flanked by 8 bp target site duplications as observed in vivo. Transposition required Mg +2 and partially purified P element transposase. Unlike other DNA rearrangement reactions, P element transposition in vitro used GTP as a cofactor; deoxyGTP, dideoxyGTP, or the nonhydrolyzable GTP analogs GMP-PNP or GMP-PCP were also used. Transposon DNA molecules cleaved at the P element termini were able to transpose, but those lacking 3′-hydroxyl groups were inactive. These biochemical data are consistent with genetic data suggesting that P element transposition occurs via a "cut-and-paste" mechanism.

THAP-family C(2)CH zinc-coordinating DNA-binding proteins function in diverse eukaryotic cellular processes, such as transposition, transcriptional repression, stem-cell pluripotency, angiogenesis and neurological function. To determine the molecular basis for sequence-specific DNA recognition by THAP proteins, we solved the crystal structure of the Drosophila melanogaster P element transposase THAP domain (DmTHAP) in complex with a natural 10-base-pair site. In contrast to C(2)H(2) zinc fingers, DmTHAP docks a conserved beta-sheet into the major groove and a basic C-terminal loop into the adjacent minor groove. We confirmed specific protein-DNA interactions by mutagenesis and DNA-binding assays. Sequence analysis of natural and in vitro-selected binding sites suggests that several THAPs (DmTHAP and human THAP1 and THAP9) recognize a bipartite TXXGGGX(A/T) consensus motif; homology suggests THAP proteins bind DNA through a bipartite interaction. These findings reveal the conserved mechanisms by which THAP-family proteins engage specific chromosomal target elements.

Alternative mRNA splicing adds a layer of regulation to the expression of thousands of genes in Drosophila melanogaster. Not all alternative splicing results in functional protein; it can also yield mRNA isoforms with premature stop codons that are degraded by the nonsense-mediated mRNA decay (NMD) pathway. This coupling of alternative splicing and NMD provides a mechanism for gene regulation that is highly conserved in mammals. NMD is also active in Drosophila, but its effect on the repertoire of alternative splice forms has been unknown, as has the mechanism by which it recognizes targets. Here, we have employed a custom splicing-sensitive microarray to globally measure the effect of alternative mRNA processing and NMD on Drosophila gene expression. We have developed a new algorithm to infer the expression change of each mRNA isoform of a gene based on the microarray measurements. This method is of general utility for interpreting splicing-sensitive microarrays and high-throughput sequence data. Using this approach, we have identified a high-confidence set of 45 genes where NMD has a differential effect on distinct alternative isoforms, including numerous RNA–binding and ribosomal proteins. Coupled alternative splicing and NMD decrease expression of these genes, which may in turn have a downstream effect on expression of other genes. The NMD–affected genes are enriched for roles in translation and mitosis, perhaps underlying the previously observed role of NMD factors in cell cycle progression. Our results have general implications for understanding the NMD mechanism in fly. Most notably, we found that the NMD–target mRNAs had significantly longer 3′ untranslated regions (UTRs) than the nontarget isoforms of the same genes, supporting a role for 3′ UTR length in the recognition of NMD targets in fly., Author Summary A gene can be processed into multiple mRNAs through alternative splicing. Alternative splicing increases the number of proteins encoded by the genome, but not all alternative mRNAs produce protein. Instead, some are degraded by nonsense-mediated mRNA decay (NMD), a surveillance system that was originally identified as a means of clearing the cell of mRNAs with nonsense, or stop codon, mutations. Alternative splicing that introduces early stop codons will lead to NMD, offering a way for the cell to down-regulate gene expression after a gene has been transcribed. In this paper, we have developed a new analysis method to study the combined effect of alternative splicing and degradation in the fruit fly Drosophila melanogaster using microarrays. We have found a stringently defined set of 45 genes that can be spliced either into an mRNA that encodes a protein or into an mRNA that is degraded by NMD, down-regulating the overall gene expression. The affected genes include a number that are central to the cell's regulatory processes, including translation, RNA splicing, and cell cycle progression. Our results also help shed light on how NMD determines whether a stop codon is premature, and thus whether to target an mRNA for degradation.

Mobility of P transposable elements in Drosophila melanogaster depends on the 87-kDa transposase protein encoded by the P element. Transposase recognizes a 10-base-pair DNA sequence that overlaps an A + T-rich region essential for transcription from the P-element promoter. We report here that transposase represses transcription from the P-element promoter in vitro. This transcriptional repression is blocked by prior formation of an RNA polymerase II transcription complex on the template DNA. Binding of transposase on the P-element promoter is blocked by prior binding of either the Drosophila RNA polymerase II complex or the yeast transcription factor TFIID. These data suggest that transposase represses transcription by preventing assembly of an RNA polymerase II complex at the P-element promoter.

Drosophila P transposable elements are the best-studied family of eukaryotic non-retroviral transposons. P element transposition is regulated in several different ways and has thus provided a unique system with which to study the control of DNA rearrangements and gene expression in metazoans. Recent genetic and biochemical experiments have begun to shed light on the mechanism of P element transposition and the mechanisms controlling the temporal and spatial patterns of transposition.

Drosophila P transposable elements encode two proteins, an 87 kd transposase protein and a 66 kd protein that has been hypothesized to repress transposition. We have made germline transformants carrying modified P element derivatives that encode only the 66 kd protein and shown that these elements repress transposase activity in both the germline and the soma. The position of these elements in the genome quantitatively affected their ability to negatively regulate transposase and to express the 66 kd protein. Single 66 kd element-containing strains did not exhibit the maternal inheritance of P cytotype characteristic of P strains. However, we demonstrated that a true P strain produced higher levels of the 66 kd protein during oogenesis than single 66 kd P elements. Thus, the expression of the 66 kd repressor during oogenesis may be a major determinant of the maternal effect of P cytotype.

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 members of a larger class of mobile elements that move using a cut-and-paste mechanism. P-element transposase uses guanosine triphosphate (GTP) as a cofactor for transposition. Here, we use atomic force microscopy (AFM) to visualize protein-DNA complexes formed during the initial stages of P-element transposition. These studies reveal that GTP acts to promote assembly of the first detectable noncovalent precleavage synaptic complex. This initial complex then randomly and independently cleaves each P-element end. These data show that GTP acts to promote protein-DNA assembly, and may explain why P-element excision often leads to unidirectional deletions.

Pre-mRNA adenosine deaminase (ADAR) is involved in many physiological processes by either directly converting adenosine to inosine in certain pre-mRNAs or indirectly regulating expression of certain genes. Mutations of Drosophila ADAR (dADAR) results in neuronal dysfunction and hypersensitivity to oxygen deprivation. Recently, we found that the mutant flies were very resistant to paraquat, a compound that generates free radicals. In order to further characterize the neuronal role of dADAR and understand the basis for the resistance to the oxidative stress, we investigated the effect of dADAR on the expression of genes encoding scavengers of cellular reactive oxygen species (ROS) in both dADAR mutant and overexpression flies. Our data show that the expression of the genes encoding known ROS scavengers [superoxide dismutase (SOD) and catalase] is not regulated by dADAR. However, the transcripts of genes encoding two potential ROS scavengers (dhd and Cyp4g1) were robustly increased in dADAR mutant flies, and conversely both were significantly decreased in dADAR overexpressing flies. Using dhd [encoding a Drosophila homolog of the mammalian protein thioredoxin (Trx)] transgenic flies, we confirmed that the resistance of dADAR mutant flies to paraquat resulted, at least partially, from the up-regulation of dhd gene in dADAR mutant flies. Our data not only confirm the importance of ADAR in maintenance of neuronal function but also reveal its regulatory role in the expression of genes encoding ROS scavengers.

P element excision generates a DNA double-strand break at the transposon donor site. Genetic studies have demonstrated a strong bias toward repair of P element-induced DNA breaks by homologous recombination with the sister chromatid, suggesting that P element excision occurs after DNA replication, in G2 of the cell cycle. We developed methods to arrest Drosophila tissue culture cells and assay P element excision in either G1- or G2-arrested cells. Dacapo or tribbles transgene expression arrests cells in either G2 or G2, respectively. RNA-mediated gene interference (RNAi) directed against cyclin E or cyclin A arrests cells in G1 or G2, respectively. P element excision occurs efficiently in both G1- and G2-arrested cells, suggesting that cell cycle regulation of P element transposase does not occur in our somatic cell system. DNA double-strand break repair occurs by two predominant mechanisms: homologous recombination (HR) and non-homologous end joining (NHEJ). HR is thought to be restricted to the post-replicative, G2, phase of the cell cycle, while NHEJ may occur throughout the cell cycle. Our results indicate that NHEJ repair of an extrachromasomal plasmid substrate occurs at least as efficiently in G2-arrested cells as in asynchronous cells or in G1-arrested cells.

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.