General decapping activators target different subsets of inefficiently translated mRNAs
- University of Massachusetts Medical School, United States
Figures
Figure 1 with 4 supplements
Identification of transcripts differentially expressed in dcp2-N245, dcp2-E153Q-N245, and dcp2-E198Q-N245 cells.
(A) Violin and box plots displaying the average and median read count distributions of the RNA-Seq libraries from WT, dcp2-N245, dcp2-E153Q-N245, and dcp2-E198Q-N245 strains in three independent …
Figure 1—figure supplement 1
RNA-Seq libraries generated from WT, dcp2-N245, dcp2-E153Q-N245, and dcp2-E198Q-N245 strains exhibit good correlation between three different biological replicates.
Matrices showing the Pearson correlation coefficients among three independent experiments for RNA-Seq libraries generated from WT, dcp2-N245, dcp2-E153Q-N245, and dcp2-E198Q-N245 cells.
Figure 1—figure supplement 2
Yeast transcripts stabilized by inactivating the catalytic function of Dcp2 are mostly decapping substrates.
(A-D) Venn diagrams showing the extent of overlap between transcripts up-regulated in dcp2-E153Q-N245 and dcp2-E198Q-N245 cells and those up-regulated in dcp1∆ (A), dcp2∆ (B), xrn1∆ (C), or upf1/2/3∆…
Figure 1—figure supplement 3
Yeast transcripts destabilized by deletion of the large Dcp2 C-terminal domain are not normally typical decapping substrates.
(A-E) Venn diagrams depicting the extent of overlaps between the 264-transcript subset down-regulated only in dcp2-N245 cells (from Figure 1C) and those up-regulated in dcp1∆ (A), dcp2∆ (B), xrn1∆ (C…
Figure 1—figure supplement 4
Western blotting analysis of protein levels in cells with different dcp2 alleles.
N-terminal triple-HA tagged dcp2-N245, dcp2-E153Q-N245, and dcp2-E198Q-N245 were constructed and cloned into the yeast single copy expression vector pRS315. The resulting plasmids were transformed …
Figure 2
Elimination of the Dcp2 C-terminal domain deregulates mRNA decapping in vivo.
(A) Yeast cells harboring a deletion of the large Dcp2 C-terminal domain exhibit a significantly different genome-wide expression pattern from cells severely comprised in decapping activity or …
Figure 3 with 2 supplements
Identification of transcripts controlled by Pat1, Lsm1, and Dhh1.
(A) Violin and box plots displaying the average and median read count distributions of the RNA-Seq libraries from the WT, pat1Δ, lsm1Δ, and dhh1Δ strains in three independent experiments. (B) Venn …
Figure 3—figure supplement 1
RNA-Seq libraries generated from WT, pat1∆, lsm1∆, and dhh1∆ strains exhibit good correlation between three different biological replicates.
Matrices showing the Pearson correlation coefficients among three independent experiments for RNA-Seq libraries generated from WT, pat1∆, lsm1∆, and dhh1∆ cells. Libraries from strains shown here …
Figure 3—figure supplement 2
Western blotting analysis of Pat1, Lsm1, and Dhh1 levels in different mutant strains.
Deletions of PAT1, LSM1, or DHH1 were constructed in yeast strains harboring TAP-tagged PAT1, LSM1, or DHH1 alleles at the respective genomic loci. Cell extracts were prepared from yeast strains of …
Figure 4 with 1 supplement
Transcripts from different subgroups of mRNAs regulated by Pat1, Lsm1, or Dhh1 have distinct expression patterns in cells deficient in decapping or 5’ to 3’ exoribonuclease activities and also exhibit distinct extents of co-translational mRNA decay.
Transcripts up-regulated in pat1Δ, lsm1Δ, or dhh1Δ strains were divided into three non-overlapping Up-o-d, Up-o-pl, and Up-a-pld subgroups, representing transcripts up-regulated only in dhh1Δ cells, …
Figure 4—figure supplement 1
Inhibition of the 3’ to 5’ mRNA decay pathway partially restores the levels of transcripts down-regulated in pat1∆ and lsm1∆ cells, but not in dhh1∆ cells.
Yeast strains harboring double deletions of PAT1, LSM1, or DHH1 and SKI2 were constructed. Total RNA was isolated from these cells as well their isogenic wild-type and single deletion cells. The …
Figure 5
Validation of representative transcripts regulated by Pat1, Lsm1, or Dhh1.
Representative transcripts from five of the subgroups (Up-o-d, Up-o-pl, Up-a-pld, Down-o-pl, and Down-a-pld) described in Figure 4 were selected and their steady-state levels in the indicated …
Figure 6
Decay rates of representative transcripts regulated by Pat1, Lsm1, or Dhh1.
Representative transcripts from the upregulated subgroups described in Figure 4 (Up-o-d, Up-o-pl, and Up-a-pld) were selected and their decay rates were determined by northern blot quantitation of …
Figure 7
Transcripts from different subgroups of mRNAs regulated by Pat1, Lsm1, and Dhh1 have distinct translational properties.
Boxplots were used to examine the distributions of average codon optimality scores, ribosome occupancies, and scaled protein abundances for transcripts from each of the six regulation subgroups …
Figure 8
Decapping activators have distinct targeting specificities and display dynamic regulation.
(A) Venn diagram depicting minimal significant overlaps between transcripts targeted by the Upf factors and those targeted by Dhh1 or Pat1 and Lsm1. (B) Two-dimensional clustering analysis of …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Chemical compound, drug | [α-32P]-dCTP | Perkin Elmer | Blu513Z | |
| Chemical compound, drug | Herring Sperm DNA | Promega | D1815 | |
| Peptide, recombinant protein | Taq DNA polymerase | Roche | 04-728-874-001 | |
| Peptide, recombinant protein | Baseline-ZERO DNase | Epicentre | DB0711K | |
| Peptide, recombinant protein | SuperScript II Reverse Transcriptase | Invitrogen | 18064–022 | |
| Strain, strain background (W303 or BY4741) | Yeast strains used in this study | This paper | Supplementary File 1 | Contains all yeast strains obtained or constructed in this study |
| Recombinant DNA reagent | Plasmids used in this study | This paper | Supplementary File 2 | Contain all plasmids constructed in this study |
| Sequence-based reagent | Oligonucleotides used in this study | This paper | Supplementary File 3 | Contains all oligonucleotides used in this study |
| Antibody | Mouse anti-HA monoclonal antibody | Sigma | H3663 | one to 4000 |
| Antibody | Rabbit anti-TAP Tag polyclonal antibody | Thermo Fisher | CAB1001 | one to 1000 |
| Antibody | Mouse anti-Pgk1 monoclonal antibody | Invitrogen | 459250 | one to 8000 |
| Commercial assay or kit | Ribo-Zero Gold rRNA Removal Kit (Yeast) | Illumina | MRZY1306 | |
| Commercial assay or kit | TruSeq Stranded mRNA LT Sample Prep Kit | Illumina | RS-122–2101 | |
| Commercial assay or kit | Agencourt RNA Clean XP Kit | Beckman-Coulter Genomics | A63987 | |
| Commercial assay or kit | Random Primed DNA labeling Kit | Roche | 11-004-760-001 | |
| Software, algorithm | RSEM | Li and Dewey, 2011 | http://deweylab.biostat.wisc.edu/rsem | |
| Software, algorithm | DESeq | Anders and Huber, 2010 | https://bioconductor.org/packages/release/bioc/html/DESeq2.html | |
| Other (Deposited Data) | R64-2-1 S288C sacCer3 genome assembly | Saccharomyces Genome Database (SGD) | https://downloads.yeastgenome.org/sequence/S288C_reference/genome_releases/ | |
| Other (Deposited Data) | Raw and analyzed data | This paper | GEO: GSE107841 | Contains raw and analyzed RNA-seq data |
| Other (Deposited Data) | Ribosomal profiling data | Young et al., 2015 | GEO: GSE69414 | |
| Other (Deposited Data) | Codon protection index data | Pelechano et al., 2015 | GEO: GSE63120 | |
| Other (Deposited Data) | Normalized yeast codon optimality scores | Pechmann and Frydman, 2013 | http://www.stanford.edu/group/frydman/codons | |
| Other (Deposited Data) | Scaled estimates of yeast proteomic data | Wang et al., 2012 | http://pax-db.org/ |
Additional files
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Supplementary File 1
Yeast strains used in this study.
- https://doi.org/10.7554/eLife.34409.017
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Supplementary File 2
Plasmids used in this study.
- https://doi.org/10.7554/eLife.34409.018
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Supplementary File 3
Oligonucleotides used in this study.
- https://doi.org/10.7554/eLife.34409.019
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Transparent reporting form
- https://doi.org/10.7554/eLife.34409.020
