Drosophila melanogaster is one of the most well studied genetic model organisms, nonetheless its genome still contains unannotated coding and non-coding genes, transcripts, exons, and RNA editing sites. Full discovery and annotation are prerequisites for understanding how the regulation of transcription, splicing, and RNA editing directs development of this complex organism. We used RNA-Seq, tiling microarrays, and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. Together, these data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.

Long-range and highly accurate de novo assembly from short-read data is one of the most pressing challenges in genomics. Recently, it has been shown that read pairs generated by proximity ligation of DNA in chromatin of living tissue can address this problem, dramatically increasing the scaffold contiguity of assemblies. Here, we describe a simpler approach (“Chicago”) based on in vitro reconstituted chromatin. We generated two Chicago data sets with human DNA and developed a statistical model and a new software pipeline (“HiRise”) that can identify poor quality joins and produce accurate, long-range sequence scaffolds. We used these to construct a highly accurate de novo assembly and scaffolding of a human genome with scaffold N50 of 20 Mbp. We also demonstrated the utility of Chicago for improving existing assemblies by reassembling and scaffolding the genome of the American alligator. With a single library and one lane of Illumina HiSeq sequencing, we increased the scaffold N50 of the American alligator from 508 kbp to 10 Mbp.

The RNA-binding protein hnRNP A1 is a splicing regulator produced by exclusion of alternative exon 7B from the A1 pre-mRNA. Each intron flanking exon 7B contains a high-affinity A1-binding site. The A1-binding elements promote exon skipping in vivo, activate distal 5Ј splice site selection in vitro and improve the responsiveness of pre-mRNAs to increases in the concentration of A1. Whereas the glycine-rich C-terminal domain of A1 is not required for binding, it is essential to activate the distal 5Ј splice site. Because A1 complexes can interact simultaneously with two A1-binding sites, we propose that an interaction between bound A1 proteins facilitates the pairing of distant splice sites. Based on the distribution of putative A1-binding sites in various pre-mRNAs, an A1-mediated change in pre-mRNA conformation may help define the borders of mammalian introns. We also identify an intron element which represses the 3Ј splice site of exon 7B. The activity of this element is mediated by a factor distinct from A1. Our results suggest that exon 7B skipping results from the concerted action of several intron elements that modulate splice site recognition and pairing.

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

SR proteins are a well-conserved class of RNA-binding proteins that are essential for regulation of splice-site selection, and have also been implicated as key regulators during other stages of RNA metabolism. For many SR proteins, the complexity of the RNA targets and specificity of RNA-binding location are poorly understood. It is also unclear if general rules governing SR protein alternative pre-mRNA splicing (AS) regulation uncovered for individual SR proteins on few model genes, apply to the activity of all SR proteins on endogenous targets. Using RNA-seq, we characterize the global AS regulation of the eight Drosophila SR protein family members. We find that a majority of AS events are regulated by multiple SR proteins, and that all SR proteins can promote exon inclusion, but also exon skipping. Most coregulated targets exhibit cooperative regulation, but some AS events are antagonistically regulated. Additionally, we found that SR protein levels can affect alternative promoter choices and polyadenylation site selection, as well as overall transcript levels. Cross-linking and immunoprecipitation coupled with highthroughput sequencing (iCLIP-seq), reveals that SR proteins bind a distinct and functionally diverse class of RNAs, which includes several classes of noncoding RNAs, uncovering possible novel functions of the SR protein family. Finally, we find that SR proteins exhibit positional RNA binding around regulated AS events. Therefore, regulation of AS by the SR proteins is the result of combinatorial regulation by multiple SR protein family members on most endogenous targets, and SR proteins have a broader role in integrating multiple layers of gene expression regulation.

The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5 splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5 splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5 splice site. RNase T 1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5 splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5 splice site. In support of a direct role of the A1-CE1 interaction in 5-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5 splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5 splice sites.Most mammalian pre-mRNAs contain introns that must be removed through RNA splicing. Pre-mRNA splicing takes place in the spliceosome, which is a large, multicomponent complex that includes a set of the four snRNPs, U1, U2, U4/ U6, and U5, and a number of non-snRNP proteins (reviewed in references 28, 29, and 46). Multiple snRNP-snRNP and snRNP-pre-mRNA interactions are crucial in specifying the efficiency and the precision of splicing (reviewed in references 49 and 54). One challenging aspect of RNA splicing is to understand how appropriate pairs of 5Ј and 3Ј splice sites are selected in pre-mRNAs containing multiple introns. Alternative splicing brings another dimension to the issue of splice site selection as it involves modulating the pairing of splices sites during development and differentiation.The selection of splice sites is determined by several parameters including the proximity and strength of splicing signals (reviewed in reference 5). Although mutations in splicing signals often affect splice site selection, the fit of a splice site to consensus sequences is not sufficient to predict splice site utilization. Sequences lying outside splice sites appear to provide a context that influences splice site recognition. This notion of context has remained ill defined, but recent progress has revealed that the recognition of a weak 3Ј splice site is improved by a downstream 5Ј splice site through exon-brid...

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 premRNA 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.

Transcription and splicing are coordinated processes in mammalian cells. We have used affinity chromatography with immobilized transcription elongation factor SII to purify a protein complex that contains core RNA polymerase II (RNA Pol II), the general transcription initiation factors, and several splicing factors, including the U1, U2, and U4 small nuclear RNPs, the U2AF 65 , and serine/arginine-rich proteins. The splicing factors and the transcription machinery co-purify through a gel filtration column and co-immunoprecipitate in experiments using an anti-U2AF 65 antibody, indicating that they are part of a unique complex. Although the RNA Pol II-containing complex does not possess splicing activity, it can complement small nuclear RNP-inactivated extracts and can promote the formation of a pre-spliceosome complex. Because interactions between components of the splicing and transcription machineries occur in the context of a complex containing a hypophosphorylated RNA Pol II capable of initiating transcription, our results suggest that the coupling between transcription and splicing begins before transcription initiation.The splicing of pre-mRNA in mammalian cells is the nuclear process during which introns are removed from primary transcripts synthesized by RNA polymerase II (RNA Pol II).1 Splicing is achieved by the spliceosome, a large macromolecular complex, composed of small nuclear ribonucleoprotein (snRNP) particles and additional proteins including members of the serine/arginine-rich (SR) protein family (reviewed in Refs. 1 and 2). Cytological studies have revealed that splicing can occur in a co-transcriptional manner (see Refs. 3 and 4 for examples) and that factors necessary for splicing are recruited to sites of transcription (5-7). A growing body of data indicates that coupling between transcription and a variety of RNA maturation events may be achieved through the C-terminal domain (CTD) of the RPB1 subunit of RNA Pol II (reviewed in Refs. 8 -11). The CTD is a seven-amino acid motif tandemly repeated 52 times in humans and 26 -27 times in yeast. It is highly conserved among eukaryotic organisms and is subject to reversible phosphorylation during the transcription cycle (reviewed in Refs. 12 and 13). Two isoforms of RNA Pol II exist in vivo, namely RNA Pol IIA and IIO. RNA Pol IIA possesses a hypophosphorylated CTD and preferentially enters the preinitiation complex, whereas RNA Pol IIO harbors an extensively phosphorylated CTD and is found in the elongation complex (13,14). Biochemical studies indicate that RNA Pol II physically interacts with components involved in capping (15-18), polyadenylation (19), and splicing (20 -24). Whereas a phosphorylated CTD is essential for the interaction with capping enzymes, the polyadenylation factor CPSF is brought to the transcription complex through an interaction with TFIID and transferred to the phosphorylated CTD after transcription initiation (19,25). The phosphorylated RNA Pol IIO isoform has been found to associate with splicing factors and has been i...