The transcriptional transactivator Tat selectively regulates viral splicing

Basic Science Department, Florida Atlantic University, Boca Raton, FL 33431, USA.
Nucleic Acids Research (Impact Factor: 9.11). 12/2009; 38(4):1249-60. DOI: 10.1093/nar/gkp1105
Source: PubMed


HIV-1 gene expression requires both viral and cellular factors to control and coordinate transcription. While the viral factor
Tat is known for its transcriptional transactivator properties, we present evidence for an unexpected function of Tat in viral
splicing regulation. We used a series of HIV-1 reporter minigenes to demonstrate that Tat’s role in splicing is dependent
on the cellular co-transcriptional splicing activators Tat-SF1 and CA150. Surprisingly, we show that this Tat-mediated splicing
function is independent from transcriptional activation. In the context of the full-length viral genome, this mechanism promotes
an autoregulatory feedback that decreases expression of tat and favors expression of the env-specific mRNA. Our data demonstrate that Tat-mediated regulation of transcription and splicing can be uncoupled and suggest
a mechanism for the involvement of specific transcriptional activators in splicing.

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Available from: Massimo Caputi
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    • "For example, the regulatory Tat and Rev proteins are produced from fully spliced transcripts. Tat enhances transcription from the viral LTR promoter (Das et al., 2011 and references therein) and influences splice site selection (Jablonski et al., 2010). Rev stimulates nuclear export of the unspliced and singly spliced viral RNAs. "
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    ABSTRACT: The 5' leader region of the HIV-1 RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA hairpin influences splicing efficiency. In addition, splicing may be modulated by binding of splicing-regulatory proteins, in particular SR proteins SF2/ASF (SRSF1), SC35 (SRSF2), SRp40 (SRSF5) and SRp55 (SRSF6), to sequence elements in the SD region. The role of RNA structure and SR protein binding in splicing control was previously studied by functional analysis of mutant SD sequences. The interpretation of these studies was complicated by the fact that most mutations simultaneously affect both structure and sequence elements. We therefore tried to disentangle the contribution of these two variables by designing more precise SD region mutants with a single effect on either the sequence or the structure. The current analysis indicates that HIV-1 splicing at the major 5'ss is modulated by both the stability of the local RNA structure and the binding of splicing regulatory proteins.
    Full-text · Article · Mar 2015 · Journal of General Virology
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    • "The HIV Tat protein, a small basic intrinsically disordered protein, is well known to interact with the TAR element on the HIV mRNA and recruits transcription factors to promote HIV gene expression [26]. However it is clear that the Tat protein is multi-functional in nature and may also influence RNA interference [27], splicing [28], and notably mRNA capping. The Tat protein stimulates the capping of nascent HIV transcripts by either a direct interaction with the Mce1 component of the human capping enzyme [29,30] or through stimulating the phosphorylation of the CTD of the large subunit of RNA Pol II [31]. "
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    ABSTRACT: Like most cellular mRNAs, the 5' end of HIV mRNAs is capped and the 3' end matured by the process of polyadenylation. There are, however, several rather unique and interesting aspects of these post-transcriptional processes on HIV transcripts. Capping of the highly structured 5' end of HIV mRNAs is influenced by the viral TAT protein and a population of HIV mRNAs contains a trimethyl-G cap reminiscent of U snRNAs involved in splicing. HIV polyadenylation involves active repression of a promoter-proximal polyadenylation signal, auxiliary upstream regulatory elements and moonlighting polyadenylation factors that have additional impacts on HIV biology outside of the constraints of classical mRNA 3' end formation. This review describes these post-transcriptional novelties of HIV gene expression as well as their implications in viral biology and as possible targets for therapeutic intervention.
    Full-text · Article · Dec 2013 · AIDS Research and Therapy
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    • "In recent work, we show that depletion of Tat-SF1 by gene silencing did not affect basal or Tat-dependent transcription from an HIV-1 CAT reporter in HeLa and HEK293 cells [66]. These data contradict the results obtained by Caputi and coworkers, which have recently reported that down-regulation of Tat-SF1 by siRNAs induces a decrease in transcription and Tat-mediated activation of an HIV-1 reporter minigene in HEK293 cells [67]. In this later work, the effect of reduction of TCERG1 expression was also analyzed. "
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    ABSTRACT: Control of RNA polymerase II (RNAPII) release from pausing has been proposed as a checkpoint mechanism to ensure optimal RNAPII activity, especially in large, highly regulated genes. HIV-1 gene expression is highly regulated at the level of elongation, which includes transcriptional pausing that is mediated by both viral and cellular factors. Here, we present evidence for a specific role of the elongation-related factor TCERG1 in regulating the extent of HIV-1 elongation and viral replication in vivo. We show that TCERG1 depletion diminishes the basal and viral Tat-activated transcription from the HIV-1 LTR. In support of a role for an elongation mechanism in the transcriptional control of HIV-1, we found that TCERG1 modifies the levels of pre-mRNAs generated at distal regions of HIV-1. Most importantly, TCERG1 directly affects the elongation rate of RNAPII transcription in vivo. Furthermore, our data demonstrate that TCERG1 regulates HIV-1 transcription by increasing the rate of RNAPII elongation through the phosphorylation of serine 2 within the carboxyl-terminal domain (CTD) of RNAPII and suggest a mechanism for the involvement of TCERG1 in relieving pausing. Finally, we show that TCERG1 is required for HIV-1 replication. Our study reveals that TCERG1 regulates HIV-1 transcriptional elongation by increasing the elongation rate of RNAPII and phosphorylation of Ser 2 within the CTD. Based on our data, we propose a general mechanism for TCERG1 acting on genes that are regulated at the level of elongation by increasing the rate of RNAPII transcription through the phosphorylation of Ser2. In the case of HIV-1, our evidence provides the basis for further investigation of TCERG1 as a potential therapeutic target for the inhibition of HIV-1 replication.
    Full-text · Article · Oct 2013 · Retrovirology
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