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, 43 (6), 982-92

A Primate Herpesvirus Uses the Integrator Complex to Generate Viral microRNAs

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A Primate Herpesvirus Uses the Integrator Complex to Generate Viral microRNAs

Demián Cazalla et al. Mol Cell.

Abstract

Herpesvirus saimiri (HVS) is a γ-herpesvirus that expresses Sm class U RNAs (HSURs) in latently infected marmoset T cells. By deep sequencing, we identified six HVS microRNAs (miRNAs) that are derived from three hairpin structures located immediately downstream of the 3' end processing signals of three of the HSURs. The viral miRNAs associate with Ago proteins and are biologically active. We confirmed that the expression of the two classes of viral noncoding RNAs is linked by identifying chimeric HSUR-pre-miRNA transcripts. We show that HVS miRNA biogenesis relies on cis-acting elements specifically required for synthesis and processing of Sm class RNAs. Knockdown of protein components in vivo and processing assays in vitro demonstrated that HVS does not utilize the Microprocessor complex that generates most host miRNAs. Instead, the Integrator complex cleaves to generate the 3' end of the HSUR and the pre-miRNA hairpin. Exportin-5 and Dicer are then required to generate mature viral miRNAs.

Figures

Figure 1
Figure 1. HVS miRNAs are located directly downstream of HSUR genes
(A) Genomic locations of HSURs, protein-coding genes, and miRNAs. The first 7.4 kbp of the HVS A11 genome are shown (Albrecht et al., 1992). (B) Predicted secondary structures of primary transcripts for HSURs and miRNAs. The 3′ ends of mature HSURs are marked by black arrowheads. Mature miRNAs are highlighted in gray. Pre-miRNAs hairpin structures are boxed with dashed lines. (C) RT-PCR identification of transcripts containing both HSURs and miRNAs in marmoset T cells latently infected with HVS. Arrows show the primers used to amplify sequences of mature HSURs (P1+P2) or longer primary transcripts (P1+P3). The mature HSUR is in black, the pre-miRNA hairpin in dark gray, and the intervening sequence (containing the 3′ box, not depicted) in light gray. The lower panel shows fractionation of products obtained in the presence (+) or absence (−) of reverse transcriptase (RT) with size markers.
Figure 2
Figure 2. HVS miRNAs are part of active RISC complexes
(A) Northern blot showing co-immunoprecipitation of HVS miRNAs or host-encoded miR-16 from extracts of virally transformed marmoset T cells with control (C) anti-HA (lane 3) or anti-Ago (αAgo, lane 5) antibody. I: Input (5%); S: Supernatant (5%); P: Pellet (100%). Host U6 serves as a loading control. (B) Luciferase assays confirm the biological activity of HVS miRNAs. 293T cells were co-transfected with Firefly-based reporters containing artificial 3′ UTRs with four perfect target sites for each of the six HVS miRNA, together with vectors expressing HSUR2, 4, or 5 either alone (pBS-HSUR2, pBS-HSUR4, or pBS-HSUR5) or together with their corresponding miRNAs (pBS-H2ΔmiR, pBS-H4ΔmiR, or pBS-H5ΔmiR). Averages of three independent experiments with SD are shown.
Figure 3
Figure 3. Mutational analysis of cis elements required for expression of HSUR4-linked miRNAs
The HSUR4 proximal sequence element (PSE) and the primary transcript containing HSUR4 and its linked miRNAs (highlighted in gray) are shown. The HSUR4 transcription initiation site (+1), nucleotides deleted (ΔPSE), and mutations disrupting the Sm-binding site or the 3′ box are indicated. Lower panels show Northern blots probed for HSURs 2 and 4, and for hvsA-miR-HSUR2-5p and hvsA-miR-HSUR4-5p. Total RNA was isolated from 293T cells transfected with either empty vector (lane 1) or a plasmid containing the first 7.4 kbp of HVS A11 genome carrying wild-type HSUR4 (lane 2), a promoter deletion (ΔPSE, lane 3), a mutated Sm-binding site (mtSm, lane 4), or a mutated 3′ box (mt3′box, lane 5).
Figure 4
Figure 4. HVS does not utilize the Microprocessor complex
(A) Northern blot analysis of the expression of hvsA-miR-HSUR4-5p, miR-142-3p, and EBER1 in 293T cells treated with a control siRNA (siCtrl) or a siRNA specific for DGCR8 (siDGCR8) for 48 hr, followed by co-transfection with siDGCR8 and three plasmids expressing HSUR4 and downstream miRNAs, miR-142, or EBER1. U6 provides a loading control. Western blots (lower right two panels) show the knockdown of DGCR8 (a cross-reacting band is marked by an asterisk) with GAPDH as a loading control. (B) 32P-labeled T7-transcribed RNA substrates corresponding to pri-miR-16-2 or to variants of the primary transcript for HSUR4 and its linked miRNAs were incubated with buffer alone (lanes 1-4) or immunoprecipitates containing FLAG-Drosha and FLAG-DGCR8 (lanes 5-8) expressed in 293T cells. Cleavage products were separated on a denaturing polyacrylamide gel. Pri-miRNA substrates are diagramed on the right, with the pre-miRNA hairpin in gray and the box representing the HSUR4-3′ box.
Figure 5
Figure 5. HVS miRNAs are exported by Exportin-5 and processed by Dicer
(A) Same as Figure 4A, but cells were treated with an Exportin-5 specific siRNA (siXPO5). Mature miRNAs and pre-miRNAs are shown. (B) Same as in (A), but cells were treated with a Dicer-specific siRNA (siDicer). (C) In vitro assays using purified Dicer (kind gift of J. Doudna). In vitro transcribed 32P-labeled RNAs corresponding to pre-miR-15b (lanes 1 and 2), pre-let-7 (lanes 3 and 4), or the viral pre-hvsA-miR-HSUR4 (lanes 5 and 6) were incubated in the presence of buffer alone (lanes 1, 3, and 5) or recombinant human Dicer (lanes 2, 4, and 6). Cleavage products were separated on a denaturing polyacrylamide gel, with size markers (M).
Figure 6
Figure 6. The Integrator complex is required for generation of HVS miRNAs
(A) Northern blot analysis of the expression of hvsA-miR-HSUR4-5p, miR-142-3p, and EBER1 in 293T cells pre-treated with a control siRNA (siCtrl) or a siRNA specific for SMN (siSMN) and then co-transfected with the same siRNAs and three plasmids expressing HSUR4 and its linked miRNAs, miR-142, or EBER1. The Western blot shows the SMN knockdown, with GAPDH as a loading control. (B) Same as (A), but with Int11-specific siRNA (siInt11). (C) Constructs expressed HSUR4 with its 3′ box and linked miRNAs from a U1 snRNA promoter (U1-HSUR4) or from a cytomegalovirus immediate-early gene (CMV) promoter (CMV-HSUR4), or only the 3′ box of HSUR4 (no snRNA sequence) with its linked miRNAs from the U1 (U1-H4ΔsnRNA) or CMV promoter (CMV-H4ΔsnRNA). The lower panel shows Northern blot analysis of the expression of hvsA-miR-HSUR4-5p and HSUR4 in 293T cells transfected with the above constructs. pBS-HSUR4 (see Figure S3) was a positive control for miRNA expression. U6 snRNA is a loading control.
Figure 7
Figure 7. Model of HVS miRNA biogenesis pathway
The Integrator complex recognizes the 3′ box and Int11 co-transcriptionally cleaves the HSUR-miRNA primary transcript. The released pre-snRNA molecule exits the nucleus for further processing, while the viral pre-miRNA undergoes export directed by Exportin-5 (XPO5) to the cytoplasm, where it is recognized and processed by Dicer to yield two mature Ago-associated HVS miRNAs.

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