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.
BackgroundAlthough 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 FindingsThe 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/SignificanceOur 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.
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...
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.
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.
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