Tethered Dhh1 represses translation independent of decapping.

<p>(A) Diagram of reporter mRNAs Dhh1 was tethered to. Each reporter was expressed under control of the <i>GAL1</i> UAS, and each reporter has two MS2 binding stem-loops engineered in its 3′ UTR. First reporter, <i>MFA2</i>; second reporter, <i>M/GFP</i>; third reporter, <i>P/GFP</i>. Transcriptional shut-off analysis of <i>MFA2</i> in either wild-type cells (B) or <i>dcp2Δ</i> cells (C) expressing either MS2 alone or tethered Dhh1. RNA was isolated from cells collected at each time point and Northern blot for the reporter was performed. Blots were stripped and reprobed for <i>SCR1</i> as a loading control. Half-lives are reported in minutes to the right of the gels. (D) Western blot analysis of GFP from either <i>M/GFP</i> or <i>P/GFP</i> co-expressed with either MS2 alone or tethered Dhh1 in wild-type cells or <i>dcp2Δ</i> cells. Blots were stripped and reprobed for Pgk1 as a loading control. (E) Relative quantitation of GFP protein signal normalized to Pgk1 protein signal from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001342#pbio-1001342-g001" target="_blank">Figure 1D</a>. For a given experiment, signal with MS2 alone tethered was set to 100% and signal with Dhh1 tethered was expressed as a percentage of tethering MS2 alone.