The DEAD-Box Protein Dhh1p Couples mRNA Decay and Translation by Monitoring Codon Optimality

Abstract

A major determinant of mRNA half-life is the codon-dependent rate of translational elongation. How the processes of translational elongation and mRNA decay communicate is unclear. Here, we establish that the DEAD-box protein Dhh1p is a sensor of codon optimality that targets an mRNA for decay. First, we find mRNAs whose translation elongation rate is slowed by inclusion of non-optimal codons are specifically degraded in a Dhh1p-dependent manner. Biochemical experiments show Dhh1p is preferentially associated with mRNAs with suboptimal codon choice. We find these effects on mRNA decay are sensitive to the number of slow-moving ribosomes on an mRNA. Moreover, we find Dhh1p overexpression leads to the accumulation of ribosomes specifically on mRNAs (and even codons) of low codon optimality. Lastly, Dhh1p physically interacts with ribosomes in vivo. Together, these data argue that Dhh1p is a sensor for ribosome speed, targeting an mRNA for repression and subsequent decay.

Figure 1. Codon optimality is a powerful determinant of mRNA stability

See also Figure S1 and Table S1. (A) Representation of the HIS3 mRNA reporter. Each reporter encodes the exact same polypeptide sequence, but is comprised of different codon composition of varying optimality. The average codon stabilization coefficient (CSC) and species-specific tRNA adaptation index (sTAI) for each construct is shown. (B) Northern blots of the HIS3 reporter series following transcriptional shut-off in a rpb1-1 strain (left panel). The right panel shows the same reporters recloned with the GAL1 inducible promoter. Shown are Northern blots following transcriptional inhibition with glucose. (C) Graphs the half-lives of the mRNA reporters in panel B.

Figure 2. Dhh1p selectively stimulates the decay of mRNAs with low codon optimality

See also Figure S2. (A) Representation of the synthetic mRNAs (SYN) and the encoded polypeptide sequence. Optimal (OPT) or non-optimal (NON-OPT) codons encoding the same peptide were used. The artificial peptide has no similarity to any known proteins. (B) The half-lives of SYN OPT and NON-OPT mRNAs in WT and different mutant strains were obtained from GAL1 shutoff experiments. Quantitations were normalized to the amount of SCR1 RNA. *Denotes average of 3 experiments. (C) Half-lives of HIS3 reporters from Figure 1B (GAL1 UAS constructs) in WT or dhh1Δ cells. Right panel indicates fold stabilization in a dhh1Δ cells vs. WT. (D) Quantification of steady state levels of mRNAs transcripts by RNA-Seq in dhh1Δ cells (RPKM) relative to WT cells (RPKM). mRNA transcripts are binned by sTAI, a numerical proxy for overall optimality. Shown are two biological replicates. A two-tailed Mann-Whitney test shows that low optimality mRNAs (sTAI = 0.25, Med. = 1.52) are enriched relative to high optimality mRNAs (sTAI = 0.55, Med. = 0.72) upon Dhh1p depletion, U = 1668, p < 2.2×10⁻¹⁶.

Figure 3. Dhh1p preferentially binds to mRNAs with low codon optimality

(A) Representation of the reporters and experimental design used for mRNA pulldown. A tag sequence was inserted in the 3'UTR of the SYN reporters for pulldown. (B) Northern blot for the SYN mRNAs pull-downs. PGK1 mRNA was probed as a control of specificity. o: optimal, n: non-optimal. (C) Western blot showing the amount of Dhh1p, Pab1p and GAPDH pulled down by the SYN mRNAs. Quantitations of Dhh1p were normalized to mRNA levels from eluates in b. (D) Reanalysis of previously performed CLIP-Seq on Dhh1p calculating enrichment of mRNA transcripts bound to Dhh1p relative to WT conditions, where transcripts are binned by sTAI. Shown are two biological replicates. A two-tailed Mann-Whitney test shows that low optimality mRNAs (sTAI = 0.25, Med. = 2.02) are preferentially bound to Dhh1p relative to high optimality mRNAs (sTAI = 0.55, Med. = 0.32), U = 304, p = 7.1×10⁻⁹.

Figure 4. Dhh1p senses the polarity of a stretch of non-optimal codons in an optimal mRNA

See also Figure S3. (A) Representation of PGK1 reporters with a stretch of 10 non-optimal codons at increasing distances from the initiating AUG. NC: Non-optimal Codons; NC0: no stretch, NC5, 25, 50, 63, 77: Non-optimal Codon stretch 5, 25, 50, 63, 77% away from the AUG. (B) Northern blots of the different PGK1 reporters after GAL-transcriptional shut-off, showing the remaining mRNA at the indicated time-points after shut-off. (C) Half-lives of the different PGK1 reporters calculated from the northern blots (quantitation was normalized to SCR1, loading controls not shown), in WT and dhh1Δ cells.

Figure 5. Dhh1p-mediated degradation is dependent on inefficient translation

See also Figure S3. (A) A stem loop (SL) was inserted in the 5'UTR of the previously described PGK1 reporters containing non-optimal codons at variable positions to inhibit translation. (B) Northern blot for steady-state abundance of the reporters with and without SL, and relative levels on the right. SCR1 was probed as a loading control. (C), A premature termination codon (PTC) was inserted immediately after the NC stretch of the reporters to prevent ribosome association downstream of the stretch. (D) Northern blot for steady-state abundance of the reporters with and without PTC, and relative levels below. SCR1 was probed as a loading control.

Figure 6. Dhh1p binds ribosomes and preferentially modulates ribosome occupancy on mRNAs with low codon optimality

See also Figure S4. (A) Dhh1p-TAP purification followed by mass spectrometry (left, Coomassie blue gel staining) or Northern blots and specific probing for different rRNAs or tRNA (right). (B) Plotting the ribosome occupancy (average number of ribosomes per mRNA transcript) for mRNA transcripts under constitutive Dhh1p OE relative to WT conditions, binning transcripts by sTAI. Shown are two biological replicates. A two-tailed Mann-Whitney test shows that low optimality mRNAs (sTAI = 0.25, Med. = 1.30) have increased ribosome occupancy relative to high optimality mRNAs (sTAI = 0.55, Med. = 0.72), U = 1364, p < 2.2×10⁻¹⁶ upon Dhh1p overexpression (C) Quantifying the ribosome footprint density in the A-site under Dhh1p OE or dhh1Δ relative to WT. The identity of the codon in the A-site was determined by using 28-nt fragments as outlined previously (Ingolia et al., 2009). (D) Schematic of the reporter used in polysome occupancy assays. (E) Northern blots were used to quantify the enrichment (relative fractional occupancy) of optimal and non-optimal HA-OST4 mRNA along a polysome gradient upon tethering catalytically active and inactive Dhh1p. Reported values are averaged across three samples and presented with standard error. Shown are representative northern blots for the non-optimal and optimal mRNAs upon tethering of catalytically active and inactive Dhh1p.

Figure 7. Model: Dhh1p is a general and essential sensor of ribosome speed during elongation

In this model, codon optimality influences the transit speed of ribosomes which in turns affects the association of the decay factor Dhh1p. Ribosomes are slowed down on non-optimal stretches, recruiting Dhh1p which may slow down ribosome movement further, and leads to mRNA decapping and degradation.