Cellular mRNA Activates Transcription Elongation by Displacing 7SK RNA

The positive transcription elongation factor P-TEFb is a pivotal regulator of gene expression in higher cells. Originally identified in Drosophila, attention was drawn to human P-TEFb by the discovery of its role as an essential cofactor for HIV-1 transcription. It is recruited to HIV transcription complexes by the viral transactivator Tat, and to cellular transcription complexes by a plethora of transcription factors. P-TEFb activity is negatively regulated by sequestration in a complex with the HEXIM proteins and 7SK RNA. The mechanism of P-TEFb release from the inhibitory complex is not known. We report that P-TEFb-dependent transcription from the HIV promoter can be stimulated by the mRNA encoding HIC, the human I-mfa domain-containing protein. The 3′-untranslated region of HIC mRNA is necessary and sufficient for this action. It forms complexes with P-TEFb and displaces 7SK RNA from the inhibitory complex in cells and cell extracts. A 314-nucleotide sequence near the 3′ end of HIC mRNA has full activity and contains a predicted structure resembling the 3′-terminal hairpin of 7SK that is critical for P-TEFb binding. This represents the first example of a cellular mRNA that can regulate transcription via P-TEFb. Our findings offer a rationale for 7SK being an RNA transcriptional regulator and suggest a practical means for enhancing gene expression

Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A

<p>Abstract</p> <p>Background</p> <p>Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture.</p> <p>Results</p> <p>Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression.</p> <p>Conclusion</p> <p>At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication <it>ex vivo</it>. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs.</p

The Human I-mfa Domain-Containing Protein, HIC, Interacts with Cyclin T1 and Modulates P-TEFb-Dependent Transcription

Positive transcription elongation factor b (P-TEFb) hyperphosphorylates the carboxy-terminal domain of RNA polymerase II, permitting productive transcriptional elongation. The cyclin T1 subunit of P-TEFb engages cellular transcription factors as well as the human immunodeficiency virus type 1 (HIV-1) transactivator Tat. To identify potential P-TEFb regulators, we conducted a yeast two-hybrid screen with cyclin T1 as bait. Among the proteins isolated was the human I-mfa domain-containing protein (HIC). HIC has been reported to modulate expression from both cellular and viral promoters via its C-terminal cysteine-rich domain, which is similar to the inhibitor of MyoD family a (I-mfa) protein. We show that HIC binds cyclin T1 in yeast and mammalian cells and that it interacts with intact P-TEFb in mammalian cell extracts. The interaction involves the I-mfa domain of HIC and the regulatory histidine-rich region of cyclin T1. HIC also binds Tat via its I-mfa domain, although the sequence requirements are different. HIC colocalizes with cyclin T1 in nuclear speckle regions and with Tat in the nucleolus. Expression of the HIC cDNA modulates Tat transactivation of the HIV-1 long terminal repeat (LTR) in a cell type-specific fashion. It is mildly inhibitory in CEM cells but stimulates gene expression in HeLa, COS, and NIH 3T3 cells. The isolated I-mfa domain acts as a dominant negative inhibitor. Activation of the HIV-1 LTR by HIC in NIH 3T3 cells occurs at the RNA level and is mediated by direct interactions with P-TEFb

Activation of HIV-1 promoter by HIC cDNA requires its 3'UTR.

<p>(A) Schematic representation of HIC RNAs. The boxed area represents the protein coding region, including the C-terminal I-mfa domain. Full-length HIC cDNA is referred to as HIC(+). HIC(−) lacks all but the first 109 nt of the 2,821 nt HIC 3'UTR. (B) NIH 3T3 cells were co-transfected with the pcDNA3.1-FLAG-HIC vectors specified (2 µg), the HIV-1 LTR-firefly luciferase (100 ng) and CMV-Renilla luciferase (20 ng) reporter plasmids, pcDNA3.1-HA-Tat (5 ng) and pcDNA3.1-HA-cyclin T1 (T1; 100 ng) as indicated, and pcDNA3.1 empty vector to maintain a constant amount of DNA in each sample. Transactivation was measured at 24 hours post-transfection and expressed as fold Tat transactivation, calculated as the relative firefly:Renilla luciferase activity normalized to the value obtained without Tat, cyclin T1 or HIC. Data represent the average of three experiments with standard errors. (C) Expression of HIC protein. Extracts of cells (10 µg protein) transfected with Tat, cyclin T1 and the indicated HIC expression vectors were analyzed by western blotting using anti-FLAG antibody. (D) Tat transactivation was measured in the presence of HIC cDNAs carrying or lacking the 3'UTR. Assays were conducted as in panel B. (E) Reduced amounts of HIC(−) vector were compared to 2 µg of HIC(+) and assayed as in panel B. Data represent the average of two experiments. Inset: HIC protein in cell extracts monitored as in panel C.</p

The HIC 3'UTR fragments are sufficient to increase gene expression.

<p>(A) NIH 3T3 cells were transfected with the pcDNA3.1-FLAG vectors indicated, together with other plasmids as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001010#pone-0001010-g001" target="_blank">Fig. 1B</a>. The pcDNA3.1-FLAG vectors expressed regions of the HIC protein and cDNA shown. Note that the HICΔ1(+) construct is identical to HIC(+) except for a point mutation that truncates the protein immediately before its I-mfa domain. Data represent the average of three experiments with standard errors and are presented as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001010#pone-0001010-g001" target="_blank">Fig. 1B</a>. (B) HIC protein expression was monitored as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001010#pone-0001010-g001" target="_blank">Fig. 1C</a>. (C) Deletions expressing the indicated regions of the 3'UTR were assayed as in panel A. Asterisks (*) indicate that the activities of the two shortest fragments were statistically different from the full-length 3'UTR (P = 0.0188 and 0.0181, respectively, in a Student's t-Test (two-tailed distribution, two-sample unequal variance)). (D) Total RNA was analyzed by relative quantitative RT-PCR using primers against the 3′ end of the HIC 3'UTR, and β-actin mRNA as an internal standard, producing fragments of 101 and 294 bp, respectively.</p

Similarity in RNA structure between the 3′ terminal region of HIC mRNA and regions in 7SK RNA.

<p>The 314 nt 3′ terminal region of the HIC 3′ UTR (nt 3,839–4,152) was aligned with the human 331 nt 7SK sequence using Foldalign. Two regions of HIC with significant score are shaded in gray. Region 1 was predicted to be structurally similar to stem 4 of 7SK (boxed) and region 2 to an alternative form of 7SK stem 2.</p

The 3'UTR of HIC is Necessary to Activate Tat Transactivation in HeLa and COS cells.

<p>(A) COS cells were co-transfected with the pcDNA3.1-FLAG-HIC vectors specified, the HIV-1 LTR-firefly luciferase (100 ng) and RSV-Renilla luciferase (100 ng) reporter plasmids, pcDNA3.1-HA-Tat (5 ng) and pcDNA3.1 empty vector to maintain a constant amount of DNA in each sample. Reduced amounts of HIC(−) vector were compared to 2 µg of HIC(+). Transactivation was measured at 24 hours post-transfection and expressed as fold Tat transactivation, calculated as the relative firefly:Renilla luciferase activity normalized to the value obtained without Tat and HIC. Data represent the average of two experiments. <i>Inset:</i> HIC protein in cell extracts monitored by western blotting using anti-FLAG antibody. (B) HeLa cells were co-transfected with pcDNA3.1-FLAG-HIC vectors specified, HIV-1 LTR-firefly luciferase (100 ng), CMV-Renilla luciferase (20 ng) reporter plasmids, pcDNA3.1-HA-Tat (5 ng) and pcDNA3.1 empty vector to maintain a constant amount of DNA in each sample. Reduced amounts of HIC(−) vector were compared to 2 µg of HIC(+). Transactivation was measured at 24 hours post-transfection and expressed as fold Tat transactivation, calculated as the relative firefly:Renilla luciferase activity normalized to the value obtained without Tat and HIC. Data represent the average of two experiments. <i>Inset:</i> HIC protein in cell extracts monitored by western blotting using anti-FLAG antibody.</p

The HIC 3'UTR acts at the transcriptional level.

<p>(A) NIH 3T3 cells were co-transfected with the two firefly luciferase reporter plasmids, and vectors expressing Tat, cyclin T1 and the indicated HIC vectors as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001010#pone-0001010-g001" target="_blank">Fig. 1B</a>. RNA was isolated from nuclear fractions 24 hours post-transfection, subjected to RNase protection assays <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001010#pone.0001010-Young1" target="_blank">[12]</a> and hybridized to probes for firefly luciferase RNA <i>(top),</i> or Renilla luciferase RNA to control for transfection efficiency <i>(bottom).</i> The control lacked cellular RNA but was digested with RNases A and T1. The probe lane contains 10% of the input probe RNA. Arrows indicate the position of protected luciferase probes. Bar graphs represent data averaged from two experiments calculated as relative firefly:Renilla RNA levels normalized to the sample with cyclin T1 but not Tat or HIC. Relative quantitative RT-PCR was performed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001010#pone-0001010-g003" target="_blank">Fig. 3D</a> to examine HIC 3'UTR and β-actin mRNA levels. (B) HIC Activates Tat-mediated CAT expression. NIH 3T3 cells were co-transfected with the reporter plasmids HIV-1-CAT (100 ng) and CMV-Renilla luciferase (20 ng), and the plasmids pcDNA3.1-HA-Tat (5 ng), pcDNA3.1-HA-cyclin T1 (T1) (100 ng), 2 µg pcDNA3.1-HIC(+) expression vector, or pcDNA3.1 empty vector as indicated. Transactivation was measured at 24 hours post-transfection and expressed as fold Tat transactivation, calculated as the relative CAT:Renilla luciferase activity normalized to the value obtained without Tat, cyclin T1 and HIC. Data represent the average of three experiments with standard errors. (C) The PCNA promoter is only slightly responsive to HIC(+). NIH 3T3 cells were co-transfected with100 ng HIV-1 LTR-firefly luciferase (light bars) or PCNA-firefly luciferase (dark bars) and other vectors as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001010#pone-0001010-g001" target="_blank">Fig. 1B</a>. (D) Tat is dispensable in the tethering system. NIH 3T3 cells were co-transfected with Gal4BD-HIV-1 LTR-firefly luciferase reporter plasmid and Gal4BD-T1 expression vector (100 ng each), 20 ng CMV-Renilla luciferase reporter plasmid, 5 ng pcDNA3.1-HA-Tat expression vector, and 2 µg pcDNA3.1 vectors expressing FLAG-HIC(+), FLAG-HIC(−) or the HIC 3'UTR alone as indicated. Data represent the average of three experiments with standard errors, presented as fold activation by Gal4BD-T1 at 24 hours calculated as the relative firefly:Renilla luciferase activity normalized to the value obtained without Gal4BD-T1, HIC or Tat. The schematic diagrams the HIV-1 LTR-firefly luciferase construct furnished with five upstream Gal4 sites, and the Gal4BD-cyclin T1 fusion protein (Gal4BD-T1).</p