Walters, R.W., Bradrick, S.S. and Gromeier, M.
Notes: The authors investigated the mechanism of microRNA (miRNA)-mediated regulation of both endogenous mRNAs and artificial reporter constructs. To determine whether an m7G cap and poly(A) tail are required for repression, the authors created plasmids containing eight synthetic, tandem miR-30 recognition sequences in the 3´ untranslated region (UTR) of a Renilla luciferase gene under the control of a 5´UTR that confers either cap-dependent or cap-independent translation. They used these plasmids as a template for in vitro transcription, then transfected in vitro transcripts with and without an m7G cap and poly(A) tail into 293T cells, along with miR-30 miRNA (and miR-21 as a negative control). Similar experiments were performed by transfecting 293T cell with the Renilla luciferase constructs and miR-30 and miR-21 expression vectors. Finally, the authors exchanged the artificial 3´ UTR of the Renilla luciferase construct with the 3´ UTR of BACH1, a transcription factor that is regulated by miR-155, and cotransfected 293T cells with the BACH1 3´ UTR construct and an miR-115 expression vector to examine regulation via an endogenous 3´ UTR. The Promega Primer Extension System was used to compare miR-21, miR-30 and miR-155 RNA levels in transfected and untransfected 293T cells to determine endogenous levels of these miRNAs. Renilla luciferase activity was determined using the Renilla Luciferase Assay System. The authors used Northern blot analysis and quantitative RT-PCR to determine Renilla luciferase RNA levels (and GAPDH levels for normalization purposes). The Plexor® One-Step qRT-PCR System was used for qRT-PCR. (4048)