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. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Virology. 2014 Dec 13;476:43–53. doi: 10.1016/j.virol.2014.11.021

Fig. 3. The PtvPyV2a, GggPyV1 and RacPyV miRNAs can negatively regulate early transcripts.

Fig. 3

(A) The biogenesis of PtvPyV2a, GggPyV1 and RacPyV miRNAs are Drosha dependent. 293T cells were transfected with an siRNA against Drosha or an irrelevant siRNA followed by co-transfection of the miRNA expression vectors with the siRNAs. Northern blot analysis was performed on the total RNA at 48 hours post transfection. The SV40 miRNA expression vector and the MHV68 miR-M1-7 expression vector were transfected as positive and negative control, respectively. The bands corresponding to the pre-miRNAs (white arrowheads) are indicated. Ethidium bromide-stained low-molecular-weight RNA served as a loading control. Irrelevant siRNA transfections are indicated by the “−” lanes and the Drosha siRNA transfections are indicated by the “+” lanes. (B) The biogenesis of PtvPyV2a, GggPyV1 and RacPyV miRNAs are Dicer dependent. Wild type or Dicer-deficient 293T cells are transfected with the corresponding miRNA expression vector. Northern blot analysis was performed on the total RNA at 48 hours post transfection. The SV40 miRNA and MHV68 miR-M1-7 expression vectors were transfected as positive controls. The bands corresponding to the pre-miRNAs (white arrowheads) and the mature miRNAs (black arrowheads) are indicated. As a loading control, ethidium bromide_stained low-molecular-weight RNA is shown. (C) The PtvPyV2a, GggPyV1 and RacPyV miRNAs can autoregulate early mRNA expression. The antisense Renilla luciferase construct for each of the three PyV was co-transfected with the firefly luciferase and various miRNA expression vectors into 293T cells. Normalized R. Luc for the different reporter constructs is shown. As a negative control, the empty pcDNA3.1neo vector was utilized. To show specificity of the reporter assay, an SV40 miRNA expression vector was co-transfected with a reporter construct containing the SV40 miRNA binding sites in the 3’ UTR.