The endonuclease Cue2 cleaves mRNAs at stalled ribosomes during No Go Decay
- Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, United States
- University of Toronto, Canada
Figures
Figure 1 with 1 supplement
Yeast overexpression screens identify a novel factor involved in NGD.
(A) Schematic of reporters used in genetic screens. Top is 'OPT'; middle is 'NGD-CGA'; bottom is 'NGD-AAA'. (B) Normalized reporter GFP levels. Violin plots show flow cytometry data from >4000 WT …
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Figure 1—source data 1
Overexpression screen results.
- https://doi.org/10.7554/eLife.49117.004
Figure 1—figure supplement 1
Validation of NGD reporters and screen results.
(A) Northern (left) and western blot (right) data for the indicated strains and reporters. (B) Venn diagram of top outliers from the overexpression screen. (C) Raw flow cytometry data from >4000 …
Figure 2 with 1 supplement
Cue2 domain structure and in vivo characterization of Cue2 in NGD.
(A) Domain organization of S. cerevisiae Cue2, with the ‘NoSMR’ mutant schematic below the WT Cue2. Putative ubiquitin binding domains indicated by asterisks. (B) Superimposition of homology model …
Figure 2—figure supplement 1
Characterizing Cue2 domains and confirming mutant in vivo expression levels.
(A) Sequence alignment of CUE domains of representative proteins. Conserved residues depicted in red on yellow background. Putative ubiquitin binding domains of Cue2 indicated by asterisks. (B) …
Figure 3
Canonical exonucleolytic decay by Xrn1 is the major contributor to NGD.
(A) Northern blot analysis of full length OPT and NGD-CGA reporter levels in the indicated strains. (B) Quantitated northern blot signals (in triplicate) from full length GFP/RFP mRNA levels for the …
Figure 4 with 2 supplements
Ribosome profiling analysis of NGD on reporter mRNAs in various genetic backgrounds.
(A–F) 16, 21 and 28 nt RPFs mapped to NGD-CGA reporter in ski2∆ (A), dom34∆ ski2∆ (B), cue2∆ dom34∆ ski2∆ (C), slh1∆ ski2∆ (D), slh1∆ dom34∆ ski2∆ (E), and cue2∆ slh1∆ dom34∆ ski2∆ (F) strains with …
Figure 4—figure supplement 1
Disome and monosome footprints are consistent in mapping the leading ribosome.
Comparison between monosome 21 nt RPFs and disome footprints. Disome footprints show predominant ribosome stall site at the 2nd to 5th CGA codons.
Figure 4—figure supplement 2
Hel2 is required for endonucleolytic cleavage at stall sites.
16, 21 and 28 nt RPFs on the NGD-CGA reporter from hel2∆ dom34∆ ski2∆ (A) and hel2∆ slh1∆ dom34∆ ski2∆ (B) strains.
Figure 5 with 1 supplement
In vitro cleavage of colliding ribosomes by purified Cue2-SMR.
(A) Absorbance at 260 nm (y-axis) for sucrose gradient of nuclease resistant trisomes treated with no enzyme (black), the SMR domain of Cue2 (pink), or the SMR-R402A mutant Cue2 (orange). (B) In …
Figure 5—figure supplement 1
Cycloheximide-induced cells give MNase-resistant trisome peak.
Sucrose gradients of undigested lysates from cells with low-dose cycloheximide treatment (blue), MNase digested lysates from cells with no drug treatment (gray), and MNase digested lysates from …
Figure 6 with 1 supplement
Cue2 targets prematurely polyadenylated mRNAs genome-wide for NGD.
(A) Ratio of 16 nt over 20–32 nt RPFs plotted in dom34∆ ski2∆ and cue2∆ dom34∆ ski2∆ strains. Genes in orange correspond to those with reproducibly decreased 16 nt RPFs upon CUE2 deletion. Pink dots …
Figure 6—figure supplement 1
Cue2-dependent 16 nt RPFs on prematurely polyadenylated mRNAs.
(A) Examples of reads with untemplated A’s on RNA14 (top) and YAP1 (bottom). (B) Ratio of 16 nt over 20–32 nt RPFs plotted from slh1∆ dom34∆ ski2∆ and cue2∆ slh1∆ dom34∆ ski2∆ strains. Genes in …
Figure 7
Multiple converging pathways for mRNA decay on NGD substrates.
Ribosome stalling triggers decay either by the exonuclease Xrn1 or by the endonuclease Cue2. The relative contributions of these pathways are modulated by the activity of Slh1 (member of the RQT …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | GFP primary antibody – mouse monoclonal | Takara | 632381 | (1:5000) |
| Antibody | FLAG primary antibody – mouse monoclonal | Sigma | F3165 | (1:5000) |
| Antibody | PGK1 primary antibody – mouse monoclonal | Invitrogen | 22C5D8 | (1:5000) |
| Antibody | HA primary antibody – rat monoclonal | Roche | 11867423001 | (1:5000) |
| Antibody | eEF2 primary antibody – rabbit polyclonal | Kerafast | ED7002 | (1:10000) |
| Software, algorithm | Rstudio | http://www.rstudio.com/ | ||
| Software, algorithm | Graphpad prism | www.graphpad.com | ||
| Software, algorithm | skewer | Jiang et al., 2014 | Version 0.2.2 | |
| Software, algorithm | seqtk | https://github.com/lh3/seqtk | ||
| Software, algorithm | STAR | Dobin et al., 2013 | STAR_2.5.3a_modified | |
| Software, algorithm | ImageQuant TL | GE Healthcare Life Sciences | Version 8.1 | |
| Software, algorithm | Spotfinder | Saeed et al., 2003 | ||
| Software, algorithm | SGAtools | http://sgatools.ccbr.utoronto.ca/ | ||
| Software, algorithm | Custom software | https://github.com/ greenlabjhmi/Cue2eLife | yeast_KD2.gff | Find information on use in Materials and methods section 'Analysis of ribosome profiling data' |
| Software, algorithm | Custom software | https://github.com/greenlabjhmi/Cue2eLife | StarAlignment_KD2.py | Find information on use in Materials and methods section 'Analysis of ribosome profiling data' |
| Software, algorithm | Custom software | https://github.com/greenlabjhmi/Cue2eLife | Screen_data_analysis.R | Find information on use in Materials and methods section 'Screen data analysis' |
| Strains | All strains | Supplementary file 1 | ||
| Recombinant DNA reagent | All plasmids | Supplementary file 1 | ||
| Sequence based reagent | All primers and oligos | Supplementary file 1 | ||
| Peptide, recombinant protein | Cue2-SMR, and Cue2-SMR-R402A | This study | Find information in Materials and methods section – 'Cue2 E. coli expression plasmids' and 'Cue2-SMR prep' |
Additional files
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Supplementary file 1
Methods supplementary table.
- https://doi.org/10.7554/eLife.49117.016
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Transparent reporting form
- https://doi.org/10.7554/eLife.49117.017
