Dengue fever (DF) is the most frequent arthropod-borne viral disease of humans, with almost half of the world's population at risk of infection1. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make DF a global public health threat1, 2. Given their compact genomes, dengue viruses (DENV 1-4) and other flaviviruses likely require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors have been identified3-10. To discover insect host factors required for DENV-2 propagation, we carried out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 dsRNA library. This screen identified 116 candidate dengue virus host factors (DVHFs) (Supplementary Fig. 1). While some were previously associated with flaviviruses (e.g., V-ATPases and alpha-glucosidases)3-5, 7, 9, 10, most DVHFs were newly implicated in DENV propagation. The dipteran DVHFs had eighty-two readily recognizable human homologues and, using a targeted siRNA screen, we showed that forty-two of these are human DVHFs. This indicates remarkable conservation of required factors between dipteran and human hosts. This work suggests novel approaches to control infection in the insect vector and the mammalian host.
Flaviviruses cause a wide range of severe diseases ranging from encephalitis to hemorrhagic fever. Discovery of host factors that regulate the fate of flaviviruses in infected cells could provide insight into the molecular mechanisms of infection and therefore facilitate the development of anti-flaviviral drugs. We performed genome-scale siRNA screens to discover human host factors required for yellow fever virus (YFV) propagation. Using a 2×2 siRNA pool screening format and a duplicate of the screen, we identified a high confidence list of YFV host factors. To find commonalities between flaviviruses, these candidates were compared to host factors previously identified for West Nile virus (WNV) and dengue virus (DENV). This comparison highlighted a potential requirement for the G protein-coupled receptor kinase family, GRKs, for flaviviral infection. The YFV host candidate GRK2 (also known as ADRBK1) was validated both in siRNA-mediated knockdown HuH-7 cells and in GRK−/− mouse embryonic fibroblasts. Additionally, we showed that GRK2 was required for efficient propagation of DENV and Hepatitis C virus (HCV) indicating that GRK2 requirement is conserved throughout the Flaviviridae. Finally, we found that GRK2 participates in multiple distinct steps of the flavivirus life cycle by promoting both entry and RNA synthesis. Together, our findings identified GRK2 as a novel regulator of flavivirus infection and suggest that inhibition of GRK2 function may constitute a new approach for treatment of flavivirus associated diseases.
The Flavivirus genus includes several emergent and reemergent viruses, such as dengue and yellow fever viruses, which cause severe diseases in humans for which there is no approved treatment. Flaviviruses are transmitted to humans by arthropods and they rely on scores of vertebrate and invertebrate factors to replicate in these disparate hosts. Identifying the host factors involved in viral propagation is critical to understanding the molecular mechanisms of infection and the development of new therapeutics. To identify human host factors required for yellow fever virus propagation, we completed two genome-scale siRNA screens. Among the candidates discovered were the G protein-coupled receptor kinases GRK2 and GRK4. We focused on the protein GRK2, a kinase first identified for its role in cellular signal transduction. We found that GRK2 was a host factor needed for productive infection by yellow fever, dengue and hepatitis C viruses and was required for both viral entry and efficient replication of the viral genome. GRKs, which are considered druggable, may be used as targets to develop broadspectrum anti-flavivirals.
During cancer progression malignant cells undergo epithelial-mesenchymal transitions (EMTs) and mesenchymal-epithelial transitions (METs) as part of a broad invasion and metastasis program. We previously observed MET events among lung metastases in a preclinical model of prostate adenocarcinoma that suggested a relationship between epithelial plasticity and metastatic spread. We thus sought to translate these findings into clinical evidence by examining the existence of EMT in circulating tumor cells (CTCs) from patients with progressive metastatic solid tumors, with a focus on men with castration-resistant prostate cancer (CRPC) and women with metastatic breast cancer (BC). We show that the majority (>80%) of these CTCs in patients with metastatic CRPC co-express epithelial proteins such as EpCAM, CK, and E-cadherin, mesenchymal proteins, including vimentin, N-cadherin, and O-cadherin, and the stem cell marker CD133. Equally, we find that over 75% of CTCs from women with metastatic BC co-express cytokeratin, vimentin, and N-cadherin. The existence and high frequency of these CTCs co-expressing epithelial, mesenchymal, and stem-cell markers in patients with progressive metastases has important implications for the application and interpretation of approved methods to detect CTCs.
metastatic castration resistant prostate cancer (mCRPC); epithelial mesenchymal transition (EMT); mesenchymal epithelial transition (MET); circulating tumor cells (CTCs); metastatic breast cancer; N-Cadherin; CD133; O-Cadherin; EpCAM
Dengue virus (DENV) is a rapidly re-emerging flavivirus that causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), diseases for which there are no available therapies or vaccines. The DENV-2 positive-strand RNA genome contains 5′ and 3′ untranslated regions (UTRs) that have been shown to form secondary structures required for virus replication and interaction with host cell proteins. In order to comprehensively identify host cell factors that bind the DENV-2 UTRs, we performed RNA chromatography, using the DENV-2 5′ and 3′ UTRs as “bait”, combined with quantitative mass spectrometry. We identified several proteins, including DDX6, G3BP1, G3BP2, Caprin1 and USP10, implicated in P body (PB) and stress granule (SG) function, and not previously known to bind DENV RNAs. Indirect immunofluorescence microscopy showed these proteins to colocalize with the DENV replication complex. Moreover, DDX6 knockdown resulted in reduced amounts of infectious particles and viral RNA in tissue culture supernatants following DENV infection. DDX6 interacted with DENV RNA in vivo during infection and in vitro this interaction was mediated by the DB1 and DB2 structures in the 3′ UTR, possibly by formation of a pseudoknot structure. Additional experiments demonstrate that, in contrast to DDX6, the SG proteins G3BP1, G3BP2, Caprin1 and USP10 bind to the variable region (VR) in the 3′ UTR. These results suggest that the DENV-2 3′ UTR is a site for assembly of PB and SG proteins and, for DDX6, assembly on the 3′ UTR is required for DENV replication.
DDX6; dengue; host factors; RNA; stress granules
Dengue virus (DENV) is the etiologic agent for dengue fever, for which there is no approved vaccine or specific anti-viral drug. As a remedy for this, we explored the use of compounds that interfere with the action of required host factors and describe here the characterization of a kinase inhibitor (SFV785), which has selective effects on NTRK1 and MAPKAPK5 kinase activity, and anti-viral activity on Hepatitis C, DENV and yellow fever viruses. SFV785 inhibited DENV propagation without inhibiting DENV RNA synthesis or translation. The compound did not cause any changes in the cellular distribution of non-structural 3, a protein critical for DENV RNA synthesis, but altered the distribution of the structural envelope protein from a reticulate network to enlarged discrete vesicles, which altered the co-localization with the DENV replication complex. Ultrastructural electron microscopy analyses of DENV-infected SFV785-treated cells showed the presence of viral particles that were distinctly different from viable enveloped virions within enlarged ER cisternae. These viral particles were devoid of the dense nucleocapsid. The secretion of the viral particles was not inhibited by SFV785, however a reduction in the amount of secreted infectious virions, DENV RNA and capsid were observed. Collectively, these observations suggest that SFV785 inhibited the recruitment and assembly of the nucleocapsid in specific ER compartments during the DENV assembly process and hence the production of infectious DENV. SFV785 and derivative compounds could be useful biochemical probes to explore the DENV lifecycle and could also represent a new class of anti-virals.
RNA interference-based screening is a powerful new genomic technology which addresses gene function en masse. To evaluate factors influencing hit list composition and reproducibility, we performed two identically designed small interfering RNA (siRNA)-based, whole genome screens for host factors supporting yellow fever virus infection. These screens represent two separate experiments completed five months apart and allow the direct assessment of the reproducibility of a given siRNA technology when performed in the same environment. Candidate hit lists generated by sum rank, median absolute deviation, z-score, and strictly standardized mean difference were compared within and between whole genome screens. Application of these analysis methodologies within a single screening dataset using a fixed threshold equivalent to a p-value ≤ 0.001 resulted in hit lists ranging from 82 to 1,140 members and highlighted the tremendous impact analysis methodology has on hit list composition. Intra- and inter-screen reproducibility was significantly influenced by the analysis methodology and ranged from 32% to 99%. This study also highlighted the power of testing at least two independent siRNAs for each gene product in primary screens. To facilitate validation we conclude by suggesting methods to reduce false discovery at the primary screening stage.
In this study we present the first comprehensive comparison of multiple analysis strategies, and demonstrate the impact of the analysis methodology on the composition of the “hit list”. Therefore, we propose that the entire dataset derived from functional genome-scale screens, especially if publicly funded, should be made available as is done with data derived from gene expression and genome-wide association studies.
RNA interference; analysis; RNAi screen analysis; siRNA; RNAi; siRNA screening; sum rank; median absolute deviation; strictly standardized mean difference; genome-wide; whole-genome; comparison; overlap; hit list
Alternative splicing is a general mechanism for regulating gene expression that affects the RNA products of more than 90% of human genes. Not surprisingly, alternative splicing is observed among gene products of metazoan immune systems, which have evolved to efficiently recognize pathogens and discriminate between “self” and “non-self”, and thus need to be both diverse and flexible. In this review we focus on the specific interface between alternative splicing and autoimmune diseases, which result from a malfunctioning of the immune system and are characterized by the inappropriate reaction to self-antigens. Despite the widespread recognition of alternative splicing as one of the major regulators of gene expression, the connections between alternative splicing and autoimmunity have not been apparent. We summarize recent findings connecting splicing and autoimmune disease, and attempt to find common patterns of splicing regulation that may advance our understanding of autoimmune diseases and open new avenues for therapy.
alternative splicing; autoimmune disease; SNP; multiple sclerosis; interleukin 7 receptor alpha chain
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 en masse 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.
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 in vivo and in vitro 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 in vitro 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 EGFR 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.
Using SplicerAV, we identified several hundred previously uncharacterized isoform changes induced by in vitro 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 en masse 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.
Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the MAPT gene, encoding the tau protein that accumulates in intraneuronal lesions in a number of neurodegenerative diseases. Several FTDP-17 mutations affect alternative splicing and result in excess exon 10 (E10) inclusion in tau mRNA. RNA reprogramming using spliceosome-mediated RNA trans-splicing (SMaRT) could be a method of choice to correct aberrant E10 splicing resulting from FTDP-17 mutations. SMaRT creates a hybrid mRNA through a trans-splicing reaction between an endogenous target pre-mRNA and a pre-trans-splicing RNA molecule (PTM). However, FTDP-17 mutations affect the strength of cis-splicing elements and could favor cis-splicing over trans-splicing. Excess E10 inclusion in FTDP-17 can be caused by intronic mutations destabilizing a stem-loop protecting the 5′ splice site at the E10/intron 10 junction. COS cells transfected with a minigene containing the intronic +14 mutation produce exclusively E10+ RNA. Generation of E10− RNA was restored after co-transfection with a PTM designed to exclude E10. Similar results were obtained with a target containing the exonic N279K mutation which strengthens a splicing enhancer within E10. Conversely, increase or decrease in E10 content was achieved by trans-splicing from a target carrying the Δ280K mutation, which weakens the same splicing enhancer. Thus E10 inclusion can be modulated by trans-splicing irrespective of the strength of the cis-splicing elements affected by FTDP-17 mutations. In conclusion, RNA trans-splicing could provide the basis of therapeutic strategies for impaired alternative splicing caused by pathogenic mutations in cis-acting splicing elements.
HIV-1 relies on several host proteins for productive viral transcription. HIV-1 Tat-specific factor 1 (Tat-SF1) is among these cofactors that were identified by in vitro reconstituted transcription reactions with immunodepleted nuclear extracts. At the onset of this work, the prevailing hypothesis was that Tat-SF1 was a required cofactor for the viral regulatory protein, Tat; however, this had not previously been formally tested in vivo.
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.
The heterogeneous nuclear ribonucleoprotein H (hnRNP) family of proteins has been shown to activate exon inclusion by binding intronic G triplets. Much less is known, however, about how hnRNP H and hnRNP F silence exons. In this study, we identify hnRNP H and hnRNP F proteins as being novel silencers of fibroblast growth factor receptor 2 exon IIIc. In cells that normally include this exon, we show that the overexpression of either hnRNP H1 or hnRNP F resulted in the dramatic silencing of exon IIIc. In cells that normally skip exon IIIc, skipping was disrupted when RNA interference was used to knock down both hnRNP H and hnRNP F. We show that an exonic GGG motif overlapped a critical exonic splicing enhancer, which was predicted to bind the SR protein ASF/SF2. Furthermore, the expression of ASF/SF2 reversed the silencing of exon IIIc caused by the expression of hnRNP H1. We show that hnRNP H and hnRNP F proteins are present in a complex with Fox2 and that the presence of Fox allows hnRNP H1 to better compete with ASF/SF2 for binding to exon IIIc. These results establish hnRNP H and hnRNP F as being repressors of exon inclusion and suggest that Fox proteins enhance their ability to antagonize ASF/SF2.
Using alternative splicing reporters we have previously observed mesenchymal epithelial transitions in Dunning AT3 rat prostate tumors. We demonstrate here that the Dunning DT and AT3 cells, which express epithelial and mesenchymal markers, respectively, represent an excellent model to study epithelial transitions since these cells recapitulate gene expression profiles observed during human prostate cancer progression. In this manuscript we also present the development of two new tools to study the epithelial transitions by imaging alternative splicing decisions: a bichromatic fluorescence reporter to evaluate epithelial transitions in culture and in vivo, and a luciferase reporter to visualize the distribution of mesenchymal epithelial transitions in vivo.
Electronic supplementary material
The online version of this article (doi:10.1007/s10585-008-9186-y) contains supplementary material, which is available to authorized users.
DT; AT3; Dunning; FGFR2; Alternative splicing; EMT; MET; Imaging
The replication of human immunodeficiency virus type 1 (HIV-1) can be inhibited by noncytolytic CD8+ T cell mediated suppression, an immune response that specifically targets HIV-1 gene expression. Clinical studies demonstrate that this immune response may play an important role in the host defense against HIV infection. In this study, we examined the distinct steps in viral gene expression for inhibition by noncytolytic CD8+ T cells. A primary HIV-1 infection system of CD4+ enriched peripheral blood mononuclear cells was utilized to examine the HIV-1 life cycle as a relevant ex vivo system. Established CD8+ T cell lines from two HIV+ long-term nonprogressors were used to examine differences at the level of transcriptional initiation and elongation of the HIV genome. This infection system coupled with the results from real-time measurement of newly transcribed RNA transcripts determined that there was a significant decrease (5-8 fold) in short intracellular viral RNA transcripts. These data strongly favor a role for the initiation of virus transcription in noncytolytic CD8+ T cell mediated suppression.
The human transcription elongation factor CA150 contains three N-terminal WW domains and six consecutive FF domains. WW and FF domains, versatile modules that mediate protein-protein interactions, are found in nuclear proteins involved in transcription and splicing. CA150 interacts with the splicing factor SF1 and with the phosphorylated C-terminal repeat domain (CTD) of RNA polymerase II (RNAPII) through its WW and FF domains, respectively. WW and FF domains may, therefore, serve to link transcription and splicing components and play a role in coupling transcription and splicing in vivo. In the study presented here, we investigated the subcellular localization and association of CA150 with factors involved in pre-mRNA transcriptional elongation and splicing. Endogenous CA150 colocalized with nuclear speckles, and this was not affected either by inhibition of cellular transcription or by RNAPII CTD phosphorylation. FF domains are essential for the colocalization to speckles, while WW domains are not required for colocalization. We also performed biochemical assays to understand the role of WW and FF domains in mediating the assembly of transcription and splicing components into higher-order complexes. Transcription and splicing components bound to a region in the amino-terminal part of CA150 that contains the three WW domains; however, we identified a region of the C-terminal FF domains that was also critical. Our results suggest that sequences located at both the amino and carboxyl regions of CA150 are required to assemble transcription/splicing complexes, which may be involved in the coupling of those processes.
En masse analysis of gene structure and function by array technologies will have a lasting and profound effect on biology and medicine. This impact can be compromised by low quality of probes within arrays, which we show can be caused by incomplete removal of chemical protecting groups. To solve this quality control problem, we present a sensitive, specific and facile method to detect these groups in situ on arrays using monoclonal antibodies and existing instrumentation. Screening of microarrays with these monoclonal antibodies should guide the consideration given to data derived from these and should enhance the accuracy of the results obtained.
Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts occurs in a cell-type-specific manner leading to the mutually exclusive use of exon IIIb in epithelia or exon IIIc in mesenchyme. Epithelial cell-specific exon choice is dependent on (U)GCAUG elements, which have been shown to bind Fox protein family members. In this paper we show that FGFR2 exon choice is regulated by (U)GCAUG elements and Fox protein family members. Fox-2 isoforms are differentially expressed in IIIb+ cells in comparison to IIIc+ cells, and expression of Fox-1 or Fox-2 in the latter led to a striking alteration in FGFR2 splice choice from IIIc to IIIb. This switch was absolutely dependent on the (U)GCAUG elements present in the FGFR2 pre-mRNA and required critical residues in the C-terminal region of Fox-2. Interestingly, Fox-2 expression led to skipping of exon 6 among endogenous Fox-2 transcripts and formation of an inactive Fox-2 isoform, which suggests that Fox-2 can regulate its own activity. Moreover, the repression of exon IIIc in IIIb+ cells was abrogated by interfering RNA-mediated knockdown of Fox-2. We also show that Fox-2 is critical for the FGFR2(IIIb)-to-FGFR2(IIIc) switch observed in T Rex-293 cells grown to overconfluency. Overconfluent T Rex-293 cells show molecular and morphological changes consistent with a mesenchymal-to-epithelial transition. If overconfluent cells are depleted of Fox-2, the switch from IIIc to IIIb is abrogated. The data in this paper place Fox-2 among critical regulators of gene expression during mesenchymal-epithelial transitions and demonstrate that this action of Fox-2 is mediated by mechanisms distinct from those described for other cases of Fox activity.
Mammalian host factors required for efficient viral gene expression and propagation have been often recalcitrant to genetic analysis. A case in point is the function of cellular factors that trans-activate internal ribosomal entry site (IRES)-driven translation, which is operative in many positive-stranded RNA viruses, including all picornaviruses. These IRES trans-acting factors have been elegantly studied in vitro, but their in vivo importance for viral gene expression and propagation has not been widely confirmed experimentally. Here we use RNA interference to deplete mammalian cells of one such factor, the polypyrimidine tract binding protein, and test its requirement in picornavirus gene expression and propagation. Depletion of the polypyrimidine tract binding protein resulted in a marked delay of particle propagation and significantly decreased synthesis and accumulation of viral proteins of poliovirus and encephalomyocarditis virus. These effects could be partially restored by expression of an RNA interference-resistant exogenous polypyrimidine tract binding protein. These data indicate a critical role for the polypyrimidine tract binding protein in picornavirus gene expression and strongly suggest a requirement for efficient IRES-dependent translation.
Alternative splicing of fibroblast growth factor receptor 2 (FGFR2) occurs in a cell-type-specific manner with the mutually exclusive use of exon IIIb or exon IIIc. Specific inclusion of exon IIIb is observed in epithelial cells, whereas exon IIIc inclusion is seen in mesenchymal cells. Epithelium-specific activation of exon IIIb and repression of exon IIIc are coordinately regulated by intronic activating sequence 2 (IAS2) and intronic splicing activator and repressor (ISAR) elements in FGFR2 pre-mRNA. Previously, it has been suggested that IAS2 and a 20-nucleotide core sequence of ISAR form a stem structure that allows for the proper regulation of FGFR2 alternative splicing. Replacement of IAS2 and the ISAR core with random sequences capable of stem formation resulted in the proper activation of exon IIIb and repression of exon IIIc in epithelial cells. Given the high degree of phylogenetic conservation of the IAS2-ISAR core structure and the fact that unrelated stem-forming sequences could functionally substitute for IAS2 and ISAR elements, we postulated that the stem structure facilitated the approximation of intronic control elements. Indeed, deletion of the entire stem-loop region and juxtaposition of sequences immediately upstream of IAS2 with sequences immediately downstream of the ISAR core maintained proper cell-type-specific inclusion of exon IIIb. These data demonstrate that IAS2 and the ISAR core are dispensable for the cell-type-specific activation of exon IIIb; thus, the major, if not the sole, role of the IAS2-ISAR stem in exon IIIb activation is to approximate sequences upstream of IAS2 with sequences downstream of the ISAR core. The downstream sequence is very likely a highly conserved GCAUG element, which we show was required for efficient exon IIIb activation.
In the human genome, the majority of protein-encoding genes are interrupted by introns, which are removed from primary transcripts by a macromolecular enzyme known as the spliceosome. Spliceosomes can constitutively remove all the introns in a primary transcript to yield a fully spliced mRNA or alternatively splice primary transcripts leading to the production of many different mRNAs from one gene. This review examines how spliceosomes can recombine two primary transcripts in trans to reprogram messenger RNAs.
CA150 represses RNA polymerase II (RNAPII) transcription by inhibiting the elongation of transcripts. The FF repeat domains of CA150 bind directly to the phosphorylated carboxyl-terminal domain of the largest subunit of RNAPII. We determined that this interaction is required for efficient CA150-mediated repression of transcription from the α4-integrin promoter. Additional functional determinants, namely, the WW1 and WW2 domains of CA150, were also required for efficient repression. A protein that interacted directly with CA150 WW1 and WW2 was identified as the splicing-transcription factor SF1. Previous studies have demonstrated a role for SF1 in transcription repression, and we found that binding of the CA150 WW1 and WW2 domains to SF1 correlated exactly with the functional contribution of these domains for repression. The binding specificity of the CA150 WW domains was found to be unique in comparison to known classes of WW domains. Furthermore, the CA150 binding site, within the carboxyl-terminal half of SF1, contains a novel type of proline-rich motif that may be recognized by the CA150 WW1 and WW2 domains. These results support a model for the recruitment of CA150 to repress transcription elongation. In this model, CA150 binds to the phosphorylated CTD of elongating RNAPII and SF1 targets the nascent transcript.
Alternative splicing of fibroblast growth factor receptor 2 (FGF-R2) transcripts involves the mutually exclusive usage of exons IIIb and IIIc to produce two different receptor isoforms. Appropriate splicing of exon IIIb in rat prostate cancer DT3 cells requires a previously described cis element (ISAR, for “intronic splicing activator and repressor”) which represses the splicing of exon IIIc and activates the splicing of exon IIIb. This element is nonfunctional in rat prostate AT3 cells, which repress exon IIIb inclusion and splice to exon IIIc. We have now identified an intronic element upstream of exon IIIb that causes repression of exon IIIb splicing. Deletion of this element abrogates the requirement for ISAR in order for exon IIIb to be spliced in DT3 cells and causes inappropriate inclusion of exon IIIb in AT3 cells. This element consists of two intronic splicing silencer (ISS) sequences, ISS1 and ISS2. The ISS1 sequence is pyrimidine rich, and in vitro cross-linking studies demonstrate binding of polypyrimidine tract binding protein (PTB) to this element. Competition studies demonstrate that mutations within ISS1 that abolish PTB binding in vitro alleviate splicing repression in vivo. Cotransfection of a PTB-1 expression vector with a minigene containing exon IIIb and the intronic splicing silencer element demonstrate PTB-mediated repression of exon IIIb splicing. Furthermore, all described PTB isoforms were equally capable of mediating this effect. Our results support a model of splicing regulation in which exon IIIc splicing does not represent a default splicing pathway but rather one in which active repression of exon IIIb splicing occurs in both cells and in which DT3 cells are able to overcome this repression in order to splice exon IIIb.
Tat protein strongly activates transcription from the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by enhancing the elongation efficiency of RNA polymerase II complexes. Tat-mediated transcriptional activation requires cellular cofactors and specific cis-acting elements within the HIV-1 promoter, among them a functional TATA box. Here, we have investigated the mechanism by which one of these cofactors, termed CA150, regulates HIV-1 transcription in vivo. We present a series of functional assays that demonstrate that the regulation of the HIV-1 LTR by CA150 has the same functional requirements as the activation by Tat. We found that CA150 affects elongation of transcription complexes assembled on the HIV-1 promoter in a TATA-box-dependent manner. We discuss the data in terms of the involvement of CA150 in the regulation of Tat-activated HIV-1 gene expression. In addition, we also provide evidence suggesting a role for CA150 in the regulation of cellular transcriptional processes.