High Throughput Sequencing Identifies Misregulated Genes in the Drosophila Polypyrimidine Tract-Binding Protein (hephaestus) Mutant Defective in Spermatogenesis.

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.

Drosophila polypyrimidine tract-binding protein (DmPTB) regulates dorso-ventral patterning genes in embryos.

The Drosophila polypyrimidine tract-binding protein (dmPTB or hephaestus) plays an important role during embryogenesis. A loss of function mutation, heph(03429), 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.

FUS binds the CTD of RNA polymerase II and regulates its phosphorylation at Ser2.

Mutations in the RNA-binding protein FUS (fused in sarcoma)/TLS have been shown to cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS), but the normal role of FUS is incompletely understood. We found that FUS binds the C-terminal domain (CTD) of RNA polymerase II (RNAP2) and prevents inappropriate hyperphosphorylation of Ser2 in the RNAP2 CTD at thousands of human genes. The loss of FUS leads to RNAP2 accumulation at the transcription start site and a shift in mRNA isoform expression toward early polyadenylation sites. Thus, in addition to its role in alternative RNA splicing, FUS has a general function in orchestrating CTD phosphorylation during RNAP2 transcription.

Distinct cardiac transcriptional profiles defining pregnancy and exercise.

BACKGROUND: Although the hypertrophic responses of the heart to pregnancy and exercise are both considered to be physiological processes, they occur in quite different hormonal and temporal settings. In this study, we have compared the global transcriptional profiles of left ventricular tissues at various time points during the progression of hypertrophy in exercise and pregnancy. METHODOLOGY/PRINCIPAL FINDINGS: The following groups of female mice were analyzed: non-pregnant diestrus cycle sedentary control, mid-pregnant, late-pregnant, and immediate-postpartum, and animals subjected to 7 and 21 days of voluntary wheel running. Hierarchical clustering analysis shows that while mid-pregnancy and both exercise groups share the closest relationship and similar gene ontology categories, late pregnancy and immediate post-partum are quite different with high representation of secreted/extracellular matrix-related genes. Moreover, pathway-oriented ontological analysis shows that metabolism regulated by cytochrome P450 and chemokine pathways are the most significant signaling pathways regulated in late pregnancy and immediate-postpartum, respectively. Finally, increases in expression of components of the proteasome observed in both mid-pregnancy and immediate-postpartum also result in enhanced proteasome activity. Interestingly, the gene expression profiles did not correlate with the degree of cardiac hypertrophy observed in the animal groups, suggesting that distinct pathways are employed to achieve similar amounts of cardiac hypertrophy. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that cardiac adaptation to the later stages of pregnancy is quite distinct from both mid-pregnancy and exercise. Furthermore, it is very dynamic since, by 12 hours post-partum, the heart has already initiated regression of cardiac growth, and 50 genes have changed expression significantly in the immediate-postpartum compared to late-pregnancy. Thus, pregnancy-induced cardiac hypertrophy is a more complex process than exercise-induced cardiac hypertrophy and our data suggest that the mechanisms underlying the two types of hypertrophy have limited overlap.

Genome-wide expression analysis identifies a modulator of ionizing radiation-induced p53-independent apoptosis in Drosophila melanogaster.

Tumor suppressor p53 plays a key role in DNA damage responses in metazoa, yet more than half of human tumors show p53 deficiencies. Therefore, understanding how therapeutic genotoxins such as ionizing radiation (IR) can elicit DNA damage responses in a p53-independent manner is of clinical importance. Drosophila has been a good model to study the effects of IR because DNA damage responses as well as underlying genes are conserved in this model, and because streamlined gene families make loss-of-function analyses feasible. Indeed, Drosophila is the only genetically tractable model for IR-induced, p53-independent apoptosis and for tissue regeneration and homeostasis after radiation damage. While these phenomenon occur only in the larvae, all genome-wide gene expression analyses after irradiation to date have been in embryos. We report here the first analysis of IR-induced, genome-wide gene expression changes in wild type and p53 mutant Drosophila larvae. Key data from microarrays were confirmed by quantitative RT-PCR. The results solidify the central role of p53 in IR-induced transcriptome changes, but also show that nearly all changes are made of both p53-dependent and p53-independent components. p53 is found to be necessary not just for the induction of but also for the repression of transcript levels for many genes in response to IR. Furthermore, Functional analysis of one of the top-changing genes, EF1a-100E, implicates it in repression of IR-induced p53-independent apoptosis. These and other results support the emerging notion that there is not a single dominant mechanism but that both positive and negative inputs collaborate to induce p53-independent apoptosis in response to IR in Drosophila larvae.

Genomic mRNA profiling reveals compensatory mechanisms for the requirement of the essential splicing factor U2AF.

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.

Whole transcriptome analysis of the fasting and fed Burmese python heart: insights into extreme physiological cardiac adaptation.

The infrequently feeding Burmese python (Python molurus) experiences significant and rapid postprandial cardiac hypertrophy followed by regression as digestion is completed. To begin to explore the molecular mechanisms of this response, we have sequenced and assembled the fasted and postfed Burmese python heart transcriptomes with Illumina technology using the chicken (Gallus gallus) genome as a reference. In addition, we have used RNA-seq analysis to identify differences in the expression of biological processes and signaling pathways between fasted, 1 day postfed (DPF), and 3 DPF hearts. Out of a combined transcriptome of ∼2,800 mRNAs, 464 genes were differentially expressed. Genes showing differential expression at 1 DPF compared with fasted were enriched for biological processes involved in metabolism and energetics, while genes showing differential expression at 3 DPF compared with fasted were enriched for processes involved in biogenesis, structural remodeling, and organization. Moreover, we present evidence for the activation of physiological and not pathological signaling pathways in this rapid, novel model of cardiac growth in pythons. Together, our data provide the first comprehensive gene expression profile for a reptile heart.