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, 36 (7), 2353-65

Identification of Functional microRNAs Released Through Asymmetrical Processing of HIV-1 TAR Element

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Identification of Functional microRNAs Released Through Asymmetrical Processing of HIV-1 TAR Element

Dominique L Ouellet et al. Nucleic Acids Res.

Abstract

The interaction between human immunodeficiency virus type 1 (HIV-1) and RNA silencing pathways is complex and multifaceted. Essential for efficient viral transcription and supporting Tat-mediated transactivation of viral gene expression, the trans-activation responsive (TAR) element is a structured RNA located at the 5' end of all transcripts derived from HIV-1. Here, we report that this element is a source of microRNAs (miRNAs) in cultured HIV-1-infected cell lines and in HIV-1-infected human CD4+ T lymphocytes. Using primer extension and ribonuclease (RNase) protection assays, we delineated both strands of the TAR miRNA duplex deriving from a model HIV-1 transcript, namely miR-TAR-5p and miR-TAR-3p. In vitro RNase assays indicate that the lack of a free 3' extremity at the base of TAR may contribute to its low processing reactivity in vivo. Both miR-TAR-5p and miR-TAR-3p down-regulated TAR miRNA sensor activity in a process that required an integral miRNA-guided RNA silencing machinery. miR-TAR-3p exerted superior gene downregulatory effects, probably due to its preferential release from HIV-1 TAR RNA by the RNase III Dicer. Our study suggests that the TAR element of HIV-1 transcripts releases functionally competent miRNAs upon asymmetrical processing by Dicer, thereby providing novel insights into viral miRNA biogenesis.

Figures

Figure 1.
Figure 1.
HIV-1 TAR RNA is bound and cleaved by recombinant Dicer into ∼23–24-nt miRNA products in vitro. (A) Structural similarities between HIV-1 TAR RNA and the hsa-let-7a-3 pre-miRNA. The arrows indicate the Dicer cleavage sites. (B) Left, EMSA. 32P-labeled TAR RNA was incubated in the absence or presence of Dicer, and the formation of Dicer·TAR complexes was analyzed by nondenaturing PAGE and autoradiography. (B) Right, Dicer RNase assays. 32P-labeled TAR RNA was incubated in the absence or presence of Dicer with MgCl2. The samples were analyzed by denaturing PAGE and autoradiography. M, indicates a 10-nt RNA size marker.
Figure 2.
Figure 2.
Detection of miRNAs released from TAR in HIV-1-infected cells. (A) Northern blot analysis of small RNAs (<200 nt) isolated from primary human CD4+ T lymphocytes infected for 48 h with VSV-G-pseudotyped HIV-1 (NL4-3 clone) or mock. A 5′ end labeled probe-recognizing nt 40–59 of HIV-1 TAR (upper panel) was hybridized and detected by autoradiography. The membrane was subsequently probed for the presence of the 22-nt miR-16-1 (middle panel) and of 5S RNA (lower panel). (B) RPA. Small RNAs (<200 nt) isolated from H9 cells wild-type (−) or chronically infected with HTLV-IIIMN NIH 1984 (MN) or HTLV-IIIRF NIH 1983 (RF) HIV-1 strains were analyzed by RPA. Protected RNAs were separated by denaturing PAGE (15%) and visualized by autoradiography. Open arrowheads indicate RNA species expected to be protected by probe −5/32 (upper panel) and 33/64 (lower panel), respectively.
Figure 3.
Figure 3.
Delineation of the miRNA duplex derived from the HIV-1 TAR element. (A and B) Small RNAs (<200 nt) isolated from HEK293 cells, transfected with or without pXP2-LTR-Fluc or pXP2-LTRΔTAR-Fluc, were analyzed by primer extension and RPA. (A) miR-TAR-3p. Left, Primer extension analysis using a deoxyribonucleotide complementary to TAR nt 61–44, and run in parallel with DNA size markers. The extended products were analyzed by denaturing PAGE. Right, RPA using RNA probes directed against HIV-1 TAR nt 33/64 and nt 40/64, and run in parallel with RNA size markers. Protected small RNAs were analyzed by denaturing PAGE. Open arrowheads indicate RNA species not supported by concurrent RPA analysis, whereas filled arrowheads indicate the RNA bands considered to establish the consensus TAR miRNA duplex. (B) miR-TAR-5p. Left, Primer extension analysis using a deoxyribonucleotide complementary to nt 23–9. Right, RPA using RNA probes directed against HIV-1 TAR nt −5/32 and nt 3/32. Filled arrowheads indicate the RNA bands considered to establish the consensus TAR miRNA duplex. (C) The consensus miR-TAR-5p:miR-TAR-3p duplex derived from HIV-1 TAR. The arrows indicate the cleavages sites deduced from primer extension and RPA results.
Figure 4.
Figure 4.
Structural determinants of HIV-1 TAR RNA processing by Dicer. (A) Structure of the HIV-1 TAR RNA variants. The structure of wild-type TAR RNA is shown in Figure 1A. (B–E) 32P-labeled TAR RNAs with (wild-type, WT) or (B) without the loop (no loop), (C) with a G/U loop, (D) with the bulge transposed to the right arm (bulge other strand) or (E) with a 2-nt (2-nt 3′ overhang) or 10-nt (10-nt 3′ overhang) extension at the 3′ end were incubated in the absence or presence of recombinant human Dicer with MgCl2. The cleavage products were analyzed by denaturing PAGE and autoradiography.
Figure 5.
Figure 5.
The miRNAs released from the HIV-1 TAR element function in gene regulatory processes in vivo. (A) Experimental scheme of the TAR miRNA functional assays. (B) HEK293 cells were cotransfected with psiTAR and a TAR miRNA sensor construct in which the Rluc reporter gene is coupled with a BS perfectly complementary to the miR-TAR-5p or miR-TAR-3p sequence. Results are expressed as mean ± SEM. (n = 6 experiments, in duplicate). *P < 0.05 or **P < 0.01 versus 0.25 ng psiTAR (one-way ANOVA). (C) HEK293 cells were cotransfected with pXP2-LTR-Fluc, a TAR miRNA sensor construct and pLacZ. Results of Rluc activity were normalized with β-galactosidase activity, and expressed as a percentage of Rluc activity obtained with pXP2-LTRΔTAR-Fluc lacking the TAR element. Results are expressed as mean ± SEM (n = 6–9 experiments, in duplicate). *P < 0.05 versus 100 ng pXP2-LTR-Fluc (one-way ANOVA). (D) H9 cells infected with HIV-1 strain HTLV-IIIMN or HTLV-IIIRF were transfected with miR-TAR-5p or miR-TAR-3p sensor construct and harvested 48 h later. Results of Rluc activity were normalized with those obtained from the unspecific sensor plasmid. Mean ± SEM (n = 2 in duplicate). *P < 0.05 versus the unspecific sensor (Student's t-test). The levels of HIV-1 p24 protein released from either HTLV-IIIMN or HTLV-IIIRF cell lines into the medium were measured by ELISA.
Figure 6.
Figure 6.
Dicer is required for the gene regulatory effects of miR-TAR-3p. (A) Immunoblot (IB) analysis showing Dicer protein down-regulation induced upon Dox treatment (upper panels), in parallel with actin control (lower panels). (B and C) Stable 293T-REx + pcDNA6TR cells conditionally expressing a shRNA directed against Dicer mRNA (shDICER-6 and shDICER-27), or a deleted region in Rluc mRNA (shNeg-13), were induced with 1 µg/ml of Dox for 12 h (+Dox) before transfection with plasmid expressing the (B) TAR RNA/miR-TAR-3p sensor, or (C) shRluc/Rluc sensor combination. Mean ± SEM (n = 4 experiments, in duplicate). *P < 0.05, **P < 0.01 or ***P < 0.005 versus prior induction (−Dox) (Student's t-test).
Figure 7.
Figure 7.
FMRP is required for optimal miR-TAR-5p and miR-TAR-3p function in vivo. (A and B) Fmr1 KO (TSV-40) and wild-type (Naïves) cells were transfected to coexpress TAR RNA (psiTAR) and (A) miR-TAR-5p sensor, or (B) miR-TAR-3p sensor construct. Results are expressed as mean ± SEM (n = 6 experiments, in duplicate). *P < 0.05 or **P < 0.01 versus Naïves (Student's t-test).
Figure 8.
Figure 8.
Asymmetric release of miR-TAR-3p upon HIV-1 TAR processing by Dicer. (A) Structure of the TAR G/U loop RNA or modified G/U loop RNA mutants, in which the A-U and G·U base pairs of the TAR stem were preserved, but positioned to have all the 32P-labeled Us either on the left arm (TAR U5) or the right arm (TAR U3). (B) These TAR substrates were incubated in the absence or presence of recombinant human Dicer with MgCl2. The cleavage products were analyzed by denaturing PAGE and autoradiography. The RNA species detected upon in vitro processing by Dicer contain specific TAR RNA structures and are denoted as follows: a, TAR RNA substrate; b, left arm + loop; c, long left arm precursor of miR-TAR-5p; d, right arm; e, loop.

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References

    1. Ambros V. The functions of animal microRNAs. Nature. 2004;431:350–355. - PubMed
    1. He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat. Rev. Genet. 2004;5:522–531. - PubMed
    1. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–297. - PubMed
    1. Ouellet DL, Perron MP, Gobeil LA, Plante P, Provost P. MicroRNAs in gene regulation: when the smallest governs it all. J. Biomed. Biotechnol. 2006;2006:69616. - PMC - PubMed
    1. Provost P, Dishart D, Doucet J, Frendewey D, Samuelsson B, Radmark O. Ribonuclease activity and RNA binding of recombinant human Dicer. EMBO J. 2002;21:5864–5874. - PMC - PubMed

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