Volume 37, Issue 1, Pages 135-142 (January 2010) A Mammalian Herpesvirus Uses Noncanonical Expression and Processing Mechanisms to Generate Viral MicroRNAs  Hal P. Bogerd, Heather W. Karnowski, Xuezhong Cai, Jinwook Shin, Michael Pohlers, Bryan R. Cullen  Molecular Cell  Volume 37, Issue 1, Pages 135-142 (January 2010) DOI: 10.1016/j.molcel.2009.12.016 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Transcription and Function of Wild-Type MHV68 MicroRNAs (A) Proposed structure of pri-miR-M1-7. Proposed cleavage sites defining the base of the pre-miR-M1-7 intermediate are indicated by arrows and Dicer cleavage sites by closed triangles. The previously cloned (Pfeffer et al., 2005) miR-M1-7-5p and miR-M1-7-3p miRNAs are highlighted. This structured RNA can be subdivided into a 5′ “tRNA” domain, a central stem loop I or “SLI” domain, and a 3′ “SLII” domain, as indicated. (B) Northern analysis of RNA samples derived from MHV68-infected S11 cells or 293T cells transfected with pmiR-M1-1, pmiR-M1-5/6, or pmiR-M1-7X. The end-labeled probes used are complementary to the mature miRNA and are indicated above each panel. The mobility of RNA size markers is indicated. (C) The biological activity of MHV68 miRNAs was confirmed by cotransfecting 293T cells with an Rluc-based reporter containing artificial 3′UTR target sites for each indicated MHV68 miRNA, together with a vector encoding the MHV68 miRNAs indicated at the base of the figure. Observed Rluc activities are given relative to a culture cotransfected with a control expression vector. Average of three separate experiments with SD is indicated. (D) 293T cells were mock transfected or transfected with pmiR-M1-1, pmiR-M1-5/6, pmiR-M1-7X, or pCMV-miR-30a and, where necessary, treated with the Pol II inhibitor α-amanitin. Total RNA was harvested at 48 hr posttransfection and mature miRNA expression detected by northern analysis. Molecular Cell 2010 37, 135-142DOI: (10.1016/j.molcel.2009.12.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Functional Organization of MHV68 pri-miR-M1-7 (A) This functional analysis of the indicated pri-miR-M1-7 mutants was performed in 293T cells, as described in Figure 1C, except that a similar miR-30a-3p indicator construct was included as a control. Average of three experiments with SD indicated. (B) Northern analysis of mature miRNA and miRNA precursor expression in 293T cells using the same plasmids analyzed in (A). The 32P-labeled oligonucleotide probes used were complementary to miR-M1-7-3p (lanes 1–6) or miR-30a (lanes 7–9). RNA size markers are indicated. U6 was used as a loading control. (C) Primer extension analysis to map the 5′ end of miR-M1-7-5p expressed in 293T cells transfected with the indicated expression vectors. The end-labeled primer is 16 nt in length, while mature miR-M1-7-5p is 22 nt (Figure 1A). Mock-transfected 293T cells were used as a control. Molecular Cell 2010 37, 135-142DOI: (10.1016/j.molcel.2009.12.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 RNAi Analysis of the Cofactor Requirements for miR-M1-7 Processing (A) Northern analysis of the expression of mature miR-30a-3p in HeLa cells treated with a control siRNA or two distinct siRNAs specific for tRNase Z (Z1 and Z2) or Drosha (D1 and D2) and then transfected with pCMV-miR-30a. U6 RNA was used as a loading control. (B) Similar to (A), except that these blots analyze HeLa cells cotransfected with the indicated siRNAs and either pmiR-M1-7X (lanes 1–4 and 14–16), pmiR-M1-7ΔSLII (lanes 5–7), pH1/M1-7 (lanes 8–10), or pH1/M1-7ΔSLII (lanes 11–13). Size markers are indicated, and U6 was used as a loading control. (C) Quantitation of the level of expression of full-length wild-type pri-miR-M1-7 (tRNA-SLI+SLII), the miR-M1-7 tRNA-SLI intermediate, the SLI pre-miR-M1-7 intermediate, and mature miR-M1-7-3p in HeLa cells transfected with pmiR-M1-7 and depleted for either RNase Z or Drosha by RNAi. Human miR-30a was used as a control. Average of four experiments with SD indicated. Data are presented relative to the level of expression of each RNA species in cells treated with a control siRNA, which was set at 1. Molecular Cell 2010 37, 135-142DOI: (10.1016/j.molcel.2009.12.016) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 tRNase Z Cleaves pri-miR-M1-7 to Generate the Predicted pre-miRNA (A) (Upper panel) In vitro processing of a 132 nt transcript, consisting of the tRNA and SLI domains of MHV68 pri-miR-M1-7, and of the 260 nt pri-miR-K5 transcript. 32P-labeled transcripts were incubated in the presence of recombinant FLAG-tRNase Z or FLAG-Drosha isolated from overexpressing 293T cells and any resultant cleavage products separated by denaturing gel electrophoresis and visualized by autoradiography. Size markers are indicated at left. (Lower panel) The protein preparations used in this experiment were analyzed by western blot using a FLAG-specific antiserum. (B) Similar to (A), except that a 200 nt 32P-labeled transcript, consisting of the predicted full-length pri-miR-M1-7 (Figure 1A), was analyzed. Molecular Cell 2010 37, 135-142DOI: (10.1016/j.molcel.2009.12.016) Copyright © 2010 Elsevier Inc. Terms and Conditions