Recognition of polypyrimidine (Py) tracts typically present between the branch point and the 3 splice site by the large subunit of the essential splicing factor U2AF is a key early step in pre-mRNA splicing. Diverse intronic sequence arrangements exist, however, including 3 splice sites lacking recognizable Py tracts, which raises the question of how general the requirement for U2AF is for various intron architectures. Our analysis of fission yeast introns in vivo has unexpectedly revealed that whereas introns lacking Py tracts altogether remain dependent on both subunits of U2AF, introns with long Py tracts, unconventionally positioned upstream of branch points, are unaffected by U2AF inactivation. Nevertheless, mutation of these Py tracts causes strong dependence on the large subunit U2AF 59 . We also find that Py tract diversity influences the requirement for the conserved C-terminal domain of U2AF 59 (RNA recognition motif 3), which has been implicated in protein-protein interactions with other splicing factors. Together, these results suggest that in addition to Py tract binding by U2AF, supplementary mechanisms of U2AF recruitment and 3 splice site identification exist to accommodate diverse intron architectures, which have gone unappreciated in biochemical studies of model pre-mRNAs.In animals, removal of introns from the majority (Ͼ80 to 90%) of nascent transcripts via pre-mRNA splicing is an important step for gene regulation (19). Alternative splicing serves important biological roles in diverse developmental contexts and provides an important mechanism to generate molecular diversity (7). The 5Ј splice site (SS), the branch point sequence (BPS), and the polypyrimidine (Py) tract 3Ј SS in the pre-mRNA are important splicing signals; they are recognized by the U1 snRNP, the U2 snRNP, and the U2 snRNP auxiliary factor U2AF, respectively, resulting in the formation of a dynamic RNA-protein complex called the spliceosome (23).In humans, the essential splicing factor U2AF is a heterodimer of a large protein subunit (U2AF 65 ) and a small protein subunit (U2AF 35 ). U2AF 65 binds to the Py tract (62), and U2AF35 recognizes the 3Ј SS (33,61,65). Both subunits of U2AF are essential for viability in many model organisms, such as the zebrafish, the fruit fly, the nematode worm, and fission yeast (U2AF 59 ) (14,24,36,43,56,66). However, in budding yeast the large subunit is dispensable (1) and the small subunit is absent. U2AF 65 interacts with other splicing factors such as BBP/SF1, UAP56, SAP155 (or SF3b155), and SRp54 (1, 10, 15, 39, 64). The branch point binding protein BBP/SF1 binds to the BPS and cooperates with U2AF 65 for RNA binding (2, 6). Detailed in vitro biochemical analyses using model splicing substrates in metazoans have significantly contributed to our mechanistic view of the role of U2AF 65 function in splicing. The N terminus of U2AF 65 harbors an arginine-serine-rich (RS) activation domain, and its C terminus contains three RNA recognition motifs (RRMs), each with a four-stranded antiparallel ...

Although mammalian polypyrimidine tract-binding (PTB) protein functions in most or all cell types to regulate a wide spectrum of transcripts, Drosophila PTB encodes an abundant male germlinespecific mRNA isoform (dmPTB) whose expression correlates with male fertility. The biological function of this isoform is unknown. Using selection-amplification, we show that mammalian and Drosophila PTB have similar RNA sequence preference, suggesting that cell-specific expression rather than unique RNA-binding properties account for the sex-specific function of dmPTB. We also show that the dmPTB protein isoform expressed in the male germline is by far the most abundant isoform, and reduction of its levels correlates with male sterility. Finally, we show that dmPTB expression is necessary for proper spermatid individualization, the terminal step necessary for production of motile sperm. Loss of dmPTB results in severe disruption of the actin cones of the spermatid individualization complex. This represents a cytological defect resulting from PTB loss. We discuss the basis for functional differences between mammalian and Drosophila PTB orthologs.individualization complex | spermatogenesis | male germline | selectionamplification | RNA binding protein R NA-binding proteins play an important role in posttranscriptional regulation. Among hundreds of known RNA-binding proteins, the biological function is known for only a few. RNAbinding proteins typically bind short, degenerate sequences, which occur frequently by chance throughout the genome and make functional analysis harder. The mammalian polypyrimidine tractbinding protein or heterogeneous nuclear ribonucleoprotein I (PTB/hnRNP I) is one of the well-studied RNA-binding proteins. The hnRNP proteins are ubiquitously expressed, associate with nascent transcripts, and play various roles in RNA metabolism. PTB is known to affect mRNA splicing, polyadenylation, translation, mRNA stability/degradation, and mRNA localization (reviewed in refs. 1 and 2).Mammalian PTB, which is considered a general splice site repressor, regulates the tissue-specific alternative splicing or localization of a wide spectrum of premRNAs (e.g., CT/CGRP α-tropomyosin, c-src, α-actinin, and fibronectin) (3-6) involving tissue-specific expression of corepressors or PTB antagonists (7). PTB contains four RNA recognition motifs (RRMs) and binds to pyrimidine-rich sequences with (short, degenerate) motifs, such as UCUUC and CUCUCU (8); RRM3 and RRM4 provide the major contribution to specific RNA recognition (9). Diverse mechanisms have been proposed for how PTB binding regulates splice site choice. These include direct competition with the splicing factor U2AF 65 (8, 10); multimerization across an exon to create a zone of silencing or cause exon looping (3, 9, 11-13); interference with exon definition (14); and competition with tissue-specific paralogs (nPTB/brPTB) (15) or with PTB antagonists (ETR-3, RBM4, and CELF) (16-18).Previously, we found that in Drosophila a major PTB transcript is male germline specifi...

The large subunit of the U2 auxiliary factor (U2AF) recognizes the polypyrimidine tract (Py-tract) located adjacent to the 3 splice site to facilitate U2 snRNP recruitment. While U2AF is considered essential for pre-mRNA splicing, its requirement for splicing on a genome-wide level has not been analyzed. Using Solexa sequencing, we performed mRNA profiling for splicing in the Schizosaccharomyces pombe U2AF 59 (prp2.1) temperature-sensitive mutant. Surprisingly, our analysis revealed that introns show a range of splicing defects in the mutant strain. While U2AF 59 inactivation (nonpermissive) conditions inhibit splicing of some introns, others are spliced apparently normally. Bioinformatics analysis indicated that U2AF 59 -insensitive introns have stronger 5 splice sites and higher A/U content. Most importantly, features that contribute to U2AF 59 insensitivity of an intron unexpectedly reside in its 5-most 30 nucleotides. These include the 5 splice site, a guanosine at position 7, and the 5 splice site-to-branch point sequence context. A differential requirement (similar to U2AF 59 ) for introns may also apply to other general splicing factors (e.g., prp10). Our combined results indicate that U2AF insensitivity is a common phenomenon and that varied intron features support the existence of unrecognized aspects of spliceosome assembly.Pre-mRNA splicing plays a major role in gene regulation (23). Furthermore, alternative splicing serves as an important mechanism to generate molecular diversity (5). In the initial stages of splicing, the U1 snRNP recognizes the 5Ј splice site (5Ј SS), and the U2 snRNP auxiliary factor (U2AF) recognizes the polypyrimidine tract (Py-tract)/3Ј splice site (3Ј SS), leading to U2 snRNP recruitment to the branch point sequence (BPS) (27).In humans, the essential splicing factor U2AF, which has been extensively studied, is a heterodimeric protein containing a large subunit (U2AF65) and a small subunit (U2AF35). These subunits bind to the Py-tract and the 3Ј SS, respectively (37,65,68,71). Both subunits of U2AF are essential for viability in many model organisms, such as zebrafish, the fruit fly, the nematode worm, and fission yeast Schizosaccharomyces pombe (U2AF59) (17,28,42,46,47,59,72). However, in the budding yeast Saccharomyces cerevisiae, the large subunit is dispensable and the small subunit is absent (1). In humans, U2AF65 interacts with several splicing factors (BBP/SF1, UAP56, SAP155 [or SF3b155], and p54) (1,14,19,43,45,70) and facilitates branch point recognition (2, 4). The mammalian U2AF65 is found to be dispensable for splicing of some introns in vitro in the presence of the SR protein SC35 (36). Microarray analyses revealed an unexpected role of the Drosophila melanogaster large subunit (dU2AF50) in the nuclear export of intronless mRNAs (8). Several splicing regulators, such as SXL, PTB, hnRNP A1, ASF/SF2, SC35, and TRA, can facilitate or antagonize U2AF activity for splicing regulation (5, 53).Biochemical and structural studies indicate that the essential splicing factor...

The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during embryogenesis. A loss of function mutation, heph03429, results in varied defects in embryonic developmental processes, leading to embryonic lethality. However, the suite of molecular functions that are disrupted in the mutant remains unknown. We have used an unbiased high throughput sequencing approach to identify transcripts that are misregulated in this mutant. Misregulated transcripts show evidence of significantly altered patterns of splicing (exon skipping, 5′ and 3′ splice site switching), alternative 5′ ends, and mRNA level changes (up and down regulation). These findings are independently supported by reverse-transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridization. We show that a group of genes, such as Zerknüllt, z600 and screw are among the most upregulated in the mutant and have been functionally linked to dorso-ventral patterning and/or dorsal closure processes. Thus, loss of dmPTB function results in specific misregulated transcripts, including those that provide the missing link between the loss of dmPTB function and observed developmental defects in embryogenesis. This study provides the first comprehensive repertoire of genes affected in vivo in the heph mutant in Drosophila and offers insight into the role of dmPTB during embryonic development.

The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during spermatogenesis. The heph2 mutation in this gene results in a specific defect in spermatogenesis, causing aberrant spermatid individualization and male sterility. However, the array of molecular defects in the mutant remains uncharacterized. Using an unbiased high throughput sequencing approach, we have identified transcripts that are misregulated in this mutant. Aberrant transcripts show altered expression levels, exon skipping, and alternative 5’ ends. We independently verified these findings by reverse-transcription and polymerase chain reaction (RT-PCR) analysis. Our analysis shows misregulation of transcripts that have been connected to spermatogenesis, including components of the actomyosin cytoskeletal apparatus. We show, for example, that the Myosin light chain 1 (Mlc1) transcript is aberrantly spliced. Furthermore, bioinformatics analysis reveals that Mlc1 contains a high affinity binding site(s) for dmPTB and that the site is conserved in many Drosophila species. We discuss that Mlc1 and other components of the actomyosin cytoskeletal apparatus offer important molecular links between the loss of dmPTB function and the observed developmental defect in spermatogenesis. This study provides the first comprehensive list of genes misregulated in vivo in the heph2 mutant in Drosophila and offers insight into the role of dmPTB during spermatogenesis.