Tethered Dhh1 still functions under conditions in which translation initiation is limited.

<p>(A) Northern blot analysis of steady state <i>M/GFP</i> levels from both wild-type and <i>cdc33-1</i> (eIF4E mutant cells) cells co-expressing either MS2 alone or tethered Dhh1 grown at the restrictive temperature (37°C) for 1 h. Blots were first probed for the reporter, then were stripped and reprobed for endogenous <i>PGK1</i>. Relative quantitation of <i>M/GFP</i> signal is to the right of the gel. 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. (B) Northern blot analysis of steady state <i>M/GFP</i> levels in both wild-type and <i>prt1-1</i> (eIF3b mutant cells) cells co-expressing either MS2 alone or tethered Dhh1 grown at the restrictive temperature (37°C) for 1 h. Blots were probed and quantitated as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001342#pbio-1001342-g002" target="_blank">Figure 2A</a>. (C) Depiction of reporter mRNAs used in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001342#pbio-1001342-g002" target="_blank">Figure 2D and 2E</a>. Both reporters are derivatives of <i>PGK1pG</i> and as such are under control of the <i>GAL1</i> UAS; the pG tract has been replaced with two MS2 binding stem loops. Both reporters have also been engineered with an HA tag at the C-terminus of Pgk1 in order to distinguish the reporter from endogenous Pgk1 protein. The second reporter has a strong stem-loop engineered in the 5′ UTR. (D) Western blot analysis for Pgk1 and SL-Pgk1 proteins (with anti-HA) from wild-type cells co-expressing either MS2 alone or tethered Dhh1. Blots were stripped and reprobed with anti-Rpl5 antibody as a loading control. (E) Northern blot analysis for reporters in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001342#pbio-1001342-g002" target="_blank">Figure 2C</a> co-expressed with either MS2 alone or tethered Dhh1 in wild-type cells. Blots were stripped and reprobed for <i>SCR1</i> as a loading control.</p

A novel function of Dhh1 is to repress a late step in translation.

<p>We hypothesize that Dhh1 may function directly on 40S ribosomal subunits based on our earlier findings in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001342#pbio.1001342-Coller3" target="_blank">[12]</a> and the documented interaction of Dhh1 with ribosomes (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001342#pbio-1001342-g004" target="_blank">Figure 4</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001342#pbio.1001342-Drummond1" target="_blank">[38]</a>). If Dhh1 were to function on free 40S subunits, the consequence would be repression of translation at initiation, as was seen in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001342#pbio.1001342-Coller3" target="_blank">[12]</a> (depicted in the left side of the figure). Based on our findings in this article, action of Dhh1 on already assembled polyribosomes in vivo would lead to repression of translation at a late, post-initiation step (depicted in the right side of the figure). Repression of ribosome movement could either be direct repression of ribosomes or possibly further consolidation of already slowed ribosomes. Repressed polyribosomal mRNA can then either be decapped or stored depending on the biological context and activity of the decapping enzyme.</p

Dhh1 protein associates with slowly translocating polyribosomes.

<p>(A) Extracts from <i>dhh1Δ</i> cells expressing HBHT-tagged Dhh1 were separated by velocity sedimentation on 15%–45% sucrose gradients and protein was extracted from each fraction by TCA precipitation. SDS-PAGE was performed, protein was transferred to PVDF membrane, and Dhh1 was detected by Western blotting with anti-RGS-His antibody. −HCHO, without formaldehyde crosslinking; +HCHO, with formaldehyde crosslinking; +RNase A, with ribonuclease A. (B) Representative polyribosome traces from extracts of cells treated without (−NaCl) and with (+NaCl) 1 M NaCl. (C) Same analysis as in (A) for HBHT-Dhh1 association with polyribosomes from cells treated with or without 1 M NaCl. (D) Same analysis as in (C) of mutant Dhh1(D195A, E196A). (E) Ribosome affinity purification was performed on extracts from crosslinked cells resuspended in media without 1 M NaCl, expressing both <i>RPL16a-ZZ</i> and <i>DHH1-HA</i> or <i>DHH1-HA</i> alone (untagged). Shown is a Western blot probed for Dhh1 using anti-HA antibody. (In, one-tenth input; S, one-tenth supernatant; P, pellet). (F) same analysis as (E), but with cells treated with 1 M NaCl.</p

Tethering Dhh1 leads to accumulation of ribosomes on reporter mRNA.

<p>(A) Extracts from <i>dcp2Δ</i> cells expressing <i>M/GFP</i> and co-expressing either MS2 alone or tethered Dhh1 were separated by velocity sedimentation on sucrose gradients. RNA was extracted from each fraction and Northern blot was performed for <i>M/GFP.</i> The bottom panel is a representative ethidum bromide stained agarose gel showing the localization of 25S and 18S rRNA in sucrose gradients. (B) Quantification of signal from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001342#pbio-1001342-g003" target="_blank">Figure 3A</a>. Signal for each gradient was totaled and each fraction is represented as a percentage of the total. (C) Extracts from <i>dcp2Δ RPL16a-ZZ</i> cells co-expressing <i>M/GFP</i> and either MS2 alone or tethered Dhh1 were subjected to ribosome affinity purification followed by RNA isolation and agarose-formaldehyde gel electrophoresis. Ethidium bromide staining was used to visualize 18S rRNA (In, one-tenth input; P, pellet). (D) qRT-PCR for various RNAs from the ribosome affinity purification in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001342#pbio-1001342-g003" target="_blank">Figure 3C</a> to detect <i>U1</i>, <i>MFA2</i>, and <i>M/GFP</i>. ΔC_t between the pellet and the input were determined for each RNA, signal from <i>U1</i> in cells expressing MS2 alone was set to 1, and all other samples were expressed relative to <i>U1</i>. (E) Northern blot data from (A) were graphed as the ratio of <i>M/GFP</i> signal when Dhh1 was tethered to when MS2 was tethered for each fraction. (F) The same ribosome affinity purification was performed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001342#pbio-1001342-g003" target="_blank">Figure 3C and 3D</a>, except purified material was separated by velocity sedimentation on sucrose gradients. RNA was extracted from each fraction and <i>M/GFP</i> was detected by qRT-PCR. The ΔC_t was calculated for each fraction comparing the situation in which Dhh1 was tethered to the situation in which MS2 alone was tethered.</p

The DEAD-Box Protein Dhh1 Promotes Decapping by Slowing Ribosome Movement

<div><p>Translational control and messenger RNA (mRNA) decay represent important control points in the regulation of gene expression. In yeast, the major pathway for mRNA decay is initiated by deadenylation followed by decapping and 5′–3′ exonucleolytic digestion of the mRNA. Proteins that activate decapping, such as the DEAD-box RNA helicase Dhh1, have been postulated to function by limiting translation initiation, thereby promoting a ribosome-free mRNA that is targeted for decapping. In contrast to this model, we show here that Dhh1 represses translation in vivo at a step subsequent to initiation. First, we establish that Dhh1 represses translation independent of initiation factors eIF4E and eIF3b. Second, we show association of Dhh1 on an mRNA leads to the accumulation of ribosomes on the transcript. Third, we demonstrate that endogenous Dhh1 accompanies slowly translocating polyribosomes. Lastly, Dhh1 activates decapping in response to impaired ribosome elongation. Together, these findings suggest that changes in ribosome transit rate represent a key event in the decapping and turnover of mRNA.</p> </div

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.</p