Effects of the yeast RNA-binding protein Whi3 on the half-life and abundance of CLN3 mRNA and other targets - PubMed (original) (raw)
Effects of the yeast RNA-binding protein Whi3 on the half-life and abundance of CLN3 mRNA and other targets
Ying Cai et al. PLoS One. 2013.
Abstract
Whi3 is an RNA binding protein known to bind the mRNA of the yeast G1 cyclin gene CLN3. It inhibits CLN3 function, but the mechanism of this inhibition is unclear; in previous studies, Whi3 made no observable difference to CLN3 mRNA levels, translation, or protein abundance. Here, we re-approach this issue using microarrays, RNA-Seq, ribosome profiling, and other methods. By multiple methods, we find that the whi3 mutation causes a small but consistent increase in the abundance of hundreds of mRNAs, including the CLN3 mRNA. The effect on various mRNAs is roughly in proportion to the density of GCAU or UGCAU motifs carried by these mRNAs, which may be a binding site for Whi3. mRNA instability of Whi3 targets may in part depend on a 3' AU rich element (ARE), AUUUUA. In addition, the whi3 mutation causes a small increase in the translational efficiency of CLN3 mRNA. The increase in CLN3 mRNA half-life and abundance together with the increase in translational efficiency is fully sufficient to explain the small-cell phenotype of whi3 mutants. Under stress conditions, Whi3 becomes a component of P-bodies or stress granules, but Whi3 also acts under non-stress condition, when no P-bodies are visible. We suggest that Whi3 may be a very broadly-acting, but mild, modulator of mRNA stability. In CLN3, Whi3 may bind to the 3' GCAU motifs to attract the Ccr4-Not complex to promote RNA deadenylation and turnover, and Whi3 may bind to the 5' GCAU motifs to inhibit translation.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. mRNAs rich in GCAUs have increased abundance in whi3 mutants by microarray.
Genes were divided into quartiles according to their GCAU density (Materials and methods). For each quartile, change in mRNA abundance is shown in (A) whi3 mutants relative to wild-type; (B), WHI3 over-expressors relative to wild-type. As a control, genes were divided into quartiles according to their CGUA density (an irrelevant control motif). For each quartile, change in mRNA abundance is shown in (C) whi3 mutants relative to wild-type; and (D) WHI3 over-expressors relative to wild-type. Changes are shown as the log2 of the ratio of abundance in whi3 divided by abundance in WT (A, C) or the ratio of abundance in WHI3X4 divided by abundance in WT (B, D).
Figure 2. mRNAs rich in GCAUs have increased abundance in whi3 mutants by RNA-Seq.
Total RNA from whi3 or WT cells was extracted and sequenced using a HiSeq. As in Fig. 1, genes were divided into quartiles according to their GCAU density (Materials and methods). For each quartile, relative mRNA abundance is shown for whi3 mutants relative to wild-type as the log2 of the ratio of abundance.
Figure 3. CLN3 mRNA levels are increased in the whi3 mutant.
WT, whi3 and WHI3x4 cells were grown in YPD to log phase and total RNA was isolated and reverse transcribed into cDNA. RNA levels of CLN3 and other two Whi3 targets, MID2 and MTL1, were measure by Q-PCR and normalized using ACT1. The fold change in the abundance of CLN3, MID2 and MTL1 mRNAs in whi3 and WHI3x4 cells relative to WT cells is shown.
Figure 4. CLN3 mRNA half-life is increased in the whi3 mutant.
pGAL-CLN3 WHI3 (CYL104a), pGAL-CLN3 whi3 (CYL105a), and pGAL-CLN3 whi3 whi4 (CYL133a) cells were grown in YP medium with 2% raffinose and 2% galactose to log phase. 30 ml of culture was collected as t0. Glucose was added to a final concentration of 2% to stop CLN3 transcription. Cells were collected at 2, 5, 10 and 15 min. (A) CLN3 mRNA levels and (B) GAL1 mRNA levels were measured by Q-PCR and normalized using ACT1 for each sample. Lines show a first order rate fit of the decay data. Shaded regions indicate the 95% confident interval of the fit. (black, WT; red, whi3; blue, whi3 whi4) Decay of GAL1 is a control. (C)LM-PAT analysis of CLN3 poly(A) tail length in xrn1 (lane 1), xrn1 whi3 (lane 2–4), WT (lane 5), whi3 (Lane 6) and ccr4 (lane 7).
Figure 5. Ribosome profile of CLN3 mRNA.
Ribosome densities in the CLN3 region are shown for WT (upper) and whi3 (middle) cells. The x-axis shows the footprint position relative to the sense strand of CLN3. The y-axis shows the read count for CLN3 normalized by total read count for all mRNAs (rpM is “reads per million reads”). The greater number of ribosome footprints over CLN3 in the whi3 strain (2.3 fold) is partly due to a greater abundance of CLN3 mRNA (1.5 fold) (as determined in the RNA-Seq part of the experiment), and partly due to an increased translational efficiency (1.5 fold). The bottom panel shows genomic features of CLN3. The 5′UTR and 3′UTR are shaded in gray. The arrows show the position of the upstream AUG (uAUG) and the start AUG of the ORF. The ticks show the position of GCAU sites.
Figure 6. Distribution of relative translational efficiencies in whi3 vs WT.
Translational efficiency (the number of ribosome footprints per mRNA, showing ribosome occupancy, divided by the number of RNA-Seq reads, showing mRNA abundance) was calculated for each gene. For each gene, the ratio of translational efficiency in whi3 cells to translational efficiency in WT cells was calculated. The log2 of this ratio is displayed. A value of 0 indicates that translational efficiency is the same in whi3 and WT cells. The position of CLN3 is indicated by the arrow; CLN3 has a relative translational efficiency of 1.5. This is statistically different from 1 (p<0.002).
Figure 7. Whi3 co-localizes with P-bodies/stress granules.
(A) Upper: Co-localization of chromosomal GFP tagged Whi3 (green) with P-body markers: Dcp2-RFP (pRP1155, magenta) and Edc3-mCherry (pRP1574, magenta) during log phase growth (+Glu) or after 30 min of glucose starvation (-Glu). Lower: Co-localization of chromosomal GFP tagged Whi3 (green) with stress granule marker Pub1-mCherry (pRP1661, magenta) after 10 min shift to 46°C. (B) Co-localization of Edc3-mCherry (magenta) and Whi3-GFP (green) after 30 min of glucose starvation. A partial co-localization is shown by the arrow. (C) Co-localization (or lack thereof) of GFP-tagged whi3 rrm or Q-domain mutants with P-body markers under stress. Upper: 30 min of glucose starvations. P-body markers are Dcp2-RFP (pRP1155, magenta) and Edc3-mCherry (pRP1574, magenta). Lower. 10 min of heat shock at 46°C. Stress granule marker is Pub1-mCherry (pRP1661, magenta). A scale bar of 2 µm is shown in each panel.
Figure 8. Under stress, CLN3 mRNA co-localizes with P-bodies/stress granules.
(A) Cells with MS2 loop tagged CLN3 in WHI3, whi3 or whi3 whi4 were cotransformed with Dcp2-RFP (pRP1155-LYS2) and MS2-GFP (YCplacIII-MS2-GFP) for co-localization of P-bodies and CLN3 mRNA. Cells were starved of glucose for 30 min. Dcp2 is shown in magenta and MS2-GFP is shown in green. Excess MS2-GFP in the nucleus appears as large green lumps. Small green foci indicate CLN3 mRNA. Micrographs were taken as Z-series and the images are shown as a projection of z-stacks. At higher contrast and brightness, additional CLN3 foci can be seen in some cells. (B) Upper. Cells with MS2 loop tagged PGK1 (PGK1-MS2L) were cotransformed with Edc3-mCherry (pRP1574) and MS2-GFP (YCplacIII-MS2-GFP). Cells were starved of glucose for 30 min. Edc3-mCherry is shown in magenta and MS2-GFP is shown in green. Lower. Control cells with no MS2 loops (BY4742) were transformed with Edc3-mCherry (pRP1574) and MS2-GFP (YCplacIII-MS2-GFP). No cytoplasmic GFP foci are seen. A scale bar of 2 µm is shown in panel A.
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