Canine Cyclin T1 Rescues Equine Infectious Anemia Virus Tat Trans-Activation in Human Cells

AbstractHuman immunodeficiency virus-1 Tat protein and human Cyclin T1 mediate transcriptional activation by enhancing the elongation efficiency of RNA polymerase II. Activation of transcription of the related equine infectious anemia virus (EIAV) requires a similar protein known as eTat, which does not function in human cells. Expression of equine Cyclin T1 in human cells rescues eTat function, suggesting a general mechanism of transcription activation among lentiviruses. Here we present the cloning of Cyclin T1 from canine D17 osteosarcoma cells, which support EIAV transactivation, and show that canine Cyclin T1 confers eTat transactivation to human cells. A two-amino-acid change, from 79–proline–glycine–80 to 79–histidine–arginine–80, confers on the human Cyclin T1 the ability to cooperate with eTat in transcriptional activation. These findings suggested that the regions of Cyclin T1 that interact with lentiviral Tat proteins and TAR RNA elements form an extended domain, which very likely has a conserved fold

Tat-SF1 Is Not Required for Tat Transactivation but Does Regulate the Relative Levels of Unspliced and Spliced HIV-1 RNAs

.To directly address the involvement of Tat-SF1 in HIV-1 gene expression, we depleted Tat-SF1 in HeLa cells by conventional expression of shRNAs and in T- Rex -293 cells containing tetracycline-inducible shRNAs targeting Tat-SF1. We achieved efficient depletion of Tat-SF1 and demonstrated that this did not affect cell viability. HIV-1 infectivity decreased in Tat-SF1-depleted cells, but only when multiple rounds of infection occurred. Neither Tat-dependent nor basal transcription from the HIV-1 LTR was affected by Tat-SF1 depletion, suggesting that the decrease in infectivity was due to a deficiency at a later step in the viral lifecycle. Finally, Tat-SF1 depletion resulted in an increase in the ratio of unspliced to spliced viral transcripts.Tat-SF1 is not required for regulating HIV-1 transcription, but is required for maintaining the ratios of different classes of HIV-1 transcripts. These new findings highlight a novel, post-transcriptional role for Tat-SF1 in the HIV-1 life cycle

SplicerAV: a tool for mining microarray expression data for changes in RNA processing

<p>Abstract</p> <p>Background</p> <p>Over the past two decades more than fifty thousand unique clinical and biological samples have been assayed using the Affymetrix HG-U133 and HG-U95 GeneChip microarray platforms. This substantial repository has been used extensively to characterize changes in gene expression between biological samples, but has not been previously mined <it>en masse </it>for changes in mRNA processing. We explored the possibility of using HG-U133 microarray data to identify changes in alternative mRNA processing in several available archival datasets.</p> <p>Results</p> <p>Data from these and other gene expression microarrays can now be mined for changes in transcript isoform abundance using a program described here, SplicerAV. Using <it>in vivo </it>and <it>in vitro </it>breast cancer microarray datasets, SplicerAV was able to perform both gene and isoform specific expression profiling within the same microarray dataset. Our reanalysis of Affymetrix U133 plus 2.0 data generated by <it>in vitro </it>over-expression of HRAS, E2F3, beta-catenin (CTNNB1), SRC, and MYC identified several hundred oncogene-induced mRNA isoform changes, one of which recognized a previously unknown mechanism of <it>EGFR </it>family activation. Using clinical data, SplicerAV predicted 241 isoform changes between low and high grade breast tumors; with changes enriched among genes coding for guanyl-nucleotide exchange factors, metalloprotease inhibitors, and mRNA processing factors. Isoform changes in 15 genes were associated with aggressive cancer across the three breast cancer datasets.</p> <p>Conclusions</p> <p>Using SplicerAV, we identified several hundred previously uncharacterized isoform changes induced by <it>in vitro </it>oncogene over-expression and revealed a previously unknown mechanism of EGFR activation in human mammary epithelial cells. We analyzed Affymetrix GeneChip data from over 400 human breast tumors in three independent studies, making this the largest clinical dataset analyzed for <it>en masse </it>changes in alternative mRNA processing. The capacity to detect RNA isoform changes in archival microarray data using SplicerAV allowed us to carry out the first analysis of isoform specific mRNA changes directly associated with cancer survival.</p

An Intronic Sequence Element Mediates Both Activation and Repression of Rat Fibroblast Growth Factor Receptor 2 Pre-mRNA Splicing

Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) is an example of highly regulated alternative splicing in which exons IIIb and IIIc are utilized in a mutually exclusive manner in different cell types. The importance of this splicing choice is highlighted by studies which indicate that deregulation of the FGF-R2 splicing is associated with progression of prostate cancer. Loss of expression of a IIIb exon-containing isoform of FGF-R2 [FGF-R2 (IIIb)] accompanies the transition of a well-differentiated, androgen-dependent rat prostate cancer cell line, DT3, to the more aggressive, androgen-independent AT3 cell line. We have used transfection of rat FGF-R2 minigenes into DT3 and AT3 cancer cell lines to study the mechanisms that control alternative splicing of rat FGF-R2. Our results support a model in which an important cis-acting element located in the intron between these alternative exons mediates activation of splicing using the upstream IIIb exon and repression of the downstream IIIc exon in DT3 cells. This element consists of 57 nucleotides (nt) beginning 917 nt downstream of the IIIb exon. Analysis of mutants further demonstrates that an 18-nt “core sequence” within this element is most crucial for its function. Based on our observations, we have termed this sequence element ISAR (for intronic splicing activator and repressor), and we suggest that factors which bind this sequence are required for maintenance of expression of the FGF-R2 (IIIb) isoform