Multi-protein bridging factor 1(Mbf1), Rps3 and Asc1 prevent stalled ribosomes from frameshifting
- University of Rochester, New York
Figures
Figure 1 with 3 supplements
MBF1 (Multi-protein Bridging Factor 1) prevents frameshifting at CGA codon repeats.
(A) Schematic of selection for mutants that frameshift at CGA codon repeats. The indicated CGA codon repeats plus one extra nucleotide were inserted upstream of the URA3 and GFP coding region (with …
Figure 1—figure supplement 1
Classification of dominant and recessive mutations and complementation of a recessive mutation.
(A) Analysis of complementation and dominant/recessive nature of mutations. Twelve MATa mutants were crossed with 20 MATα mutants, as well as with the wild type strains. An Ura+ phenotype of …
Figure 1—figure supplement 2
Confirmation that mutations in MBF1 are responsible for frameshifting.
(A) Plasmid-borne MBF1 gene suppressed the Ura+ phenotype of mutants P25 and P38 at 30°C. (B) Deletion of the MBF1 coding sequence in the parent GFP- strain resulted in GFP+ phenotype.
Figure 1—figure supplement 3
Mbf1 is conserved and frameshifting mutations do not exhibit sensitivity to 3-AT.
(A) Amino acid sequence alignment of Mbf1 protein from 11 eukaryotic species using MultAlin (http://multalin.toulouse.inra.fr/multalin/) (Corpet, 1988). The color of text corresponds to the …
Figure 2 with 1 supplement
Ribosomal protein Rps3 mediates reading frame maintenance with Mbf1 at CGA codon repeats.
(A) Left: Yeast ribosome from PDB: 3J78 (Svidritskiy et al., 2014) (light blue: small subunit; yellow: large subunit) showing Asc1/RACK1 (dark blue) and Rps3 (pink). Right: Residues of Rps3 in which …
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Figure 2—source data 1
(Source data file for Figure 2B–D).
- https://doi.org/10.7554/eLife.39637.009
Figure 2—figure supplement 1
RPS3 and mbf1Δ mutants do not affect mRNA levels of CGA-containing reporters.
(A) Scatter plot of RFP mRNA versus GFP mRNA of Rluc-GFP reporters containing in-frame or out-of-frame four Arg codons (AGA versus CGA) in wild-type (black), RPS3-K108E (yellow), mbf1Δ (green) and RP…
Figure 3 with 2 supplements
Mbf1 and Asc1 play distinct, but related, roles at CGA codon pairs.
(A) Analysis of effects of asc1Δ, mbf1Δ and asc1Δ mbf1Δ mutations on protein expression (fluorescence), mRNA levels and protein/mRNA of GLN4(1-99)-GFP reporters containing three CGA-CGA codon pairs …
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Figure 3—source data 1
Source data file for Figure 3A and C).
- https://doi.org/10.7554/eLife.39637.013
Figure 3—figure supplement 1
Analysis of effects of asc1Δ, mbf1Δ and asc1Δ mbf1Δ mutations on protein and mRNA expression of GFP reporters.
(A) Analysis of effects of asc1Δ, mbf1Δ and asc1Δ mbf1Δ mutations on protein expression of GLN4(1-99)-GFP reporters containing three Arg-Arg codon pairs (AGA-AGA versus CGA-CGA) in 0, +1, and −1 …
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Figure 3—figure supplement 1—source data 1
Source data file for Figure 3—figure supplement 1B.
- https://doi.org/10.7554/eLife.39637.014
Figure 3—figure supplement 2
Frameshifting is likely not due to reduction in Mbf1 protein in the asc1Δ mutant nor to limiting Asc1 protein in the mbf1Δ mutant.
(A) Western analysis of HA tagged Mbf1 in the asc1Δ mutant (three independent isolates shown) compared to the wild-type strain (four independent isolates shown) indicates that Mbf1 levels were …
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Figure 3—figure supplement 2—source data 2
Source data file for Figure 3—figure supplement 2B.
- https://doi.org/10.7554/eLife.39637.015
Figure 4 with 2 supplements
Mbf1 and Asc1 work at a common subset of inhibitory codon pairs and at a single inhibitory codon pair in a context-dependent manner.
(A) Frameshifted GFP/RFP fluorescence was detected at three inhibitory codon pairs (Gamble et al., 2016) in the mbf1Δ mutant, and at seven codon pairs in the asc1Δ mbf1Δ double mutant. Frameshifting …
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Figure 4—source data 1
Source data file for Figure 4A–E.
- https://doi.org/10.7554/eLife.39637.019
Figure 4—figure supplement 1
Analysis of effects of asc1Δ, mbf1Δ and asc1Δ mbf1Δ mutations on expression of different GFP reporters containing two sets of inhibitory codon pairs and programmed frameshifting site in TY1.
(A, B) Analysis of effects of the indicated mutations on expression of Gln4(1-99)-GFP reporters containing three copies of either (A) the Arg-Pro (AGA-CCA or CGA-CCG) codon pairs or (B) the Arg-Ile …
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Figure 4—figure supplement 1—source data 1
Source data file for Figure 4—figure supplement 1A-C.
- https://doi.org/10.7554/eLife.39637.020
Figure 4—figure supplement 2
Efficient frameshifting occurs at a single CGA-CGG pair in a particular context.
(A) Schematic of inserts in +1 frame in GLN4(1-99)-GFP reporters used to identify the contributions of individual CGA-CGG pairs to frameshifting. Sequences with all possible combinations of zero, …
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Figure 4—figure supplement 2—source data 2
Source data file for Figure 4—figure supplement 2B-E.
- https://doi.org/10.7554/eLife.39637.021
Figure 5
Frameshifting occurs in the +1 direction with the CGA codon in the P site and is modulated by tRNA competition at the A site.
(A) Schematic of the purification construct for the frameshifted peptide. An eight amino acid sequence with a single CGA-CGG pair from the RNA-ID reporter was inserted between a GST tag and an …
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Figure 5—source data 1
(Source data file for 5D).
- https://doi.org/10.7554/eLife.39637.023
Figure 6
Models for the interplay between the Mbf1/Rps3 and Asc1-mediated RQC pathways and for the role of Mbf1 in reading frame maintenance.
(A) Model of the two pathways that maintain reading frames of stalled ribosomes at CGA codons. Mbf1 and Rps3 act on stalled ribosomes to prevent frameshifting while Asc1 causes removal of many of …
Tables
Key resources table
| Reagent type | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Saccharomyces cerevisiae) | MBF1/ YOR298C-A | SGD:S000007253 | ||
| Gene (S. cerevisiae) | ASC1/ YMR116C | SGD:S000004722 | ||
| Gene (S. cerevisiae) | RPS3/ YNL178W | SGD:S000005122 | ||
| Strain, strain background (S. cerevisiae, MATa) | BY4741 | Open Biosystems | ||
| Strain, strain background (S. cerevisiae, MATα) | BY4742 | Open Biosystems | ||
| Genetic reagent (S. cerevisiae) | See Supplementary table 1 in Supplementary file 3 | this paper | ||
| Antibody | anti-HA High Affinity (Rat monoclonal) | Roche | 3F10, catalog# 11867423001; RRID:AB_10094468 | 1:3000 dilution |
| Antibody | Goat anti-Rat IgG-HRP conjugate | Jackson Immuno Research | catalog# 112-035-003; RRID:AB_2338128 | 1:5000 dilution |
| Antibody | anti-G-6-PDH (Rabbit monoclonal) | Sigma | catalog# A9521; RRID: AB_258454 | 1:5000 dilution |
| Antibody | Goat anti- Rabbit IgG- HRP conjugate | Biorad | catalog# 1706515; RRID:AB_11125142 | 1:10000 dilution |
| Recombinant DNA reagent | See Supplementary table 2 in Supplementary file 3 | this paper | ||
| Sequence- based reagent | See Supplementary table 3 in Supplementary file 3 | this paper | ||
| Sequence -based reagent | Random Primers | Invitrogen | catalog# 48190011 | |
| Commercial assay or kit | T4 DNA Polymerase, LIC-qualified | Novagen | catalog# 70099 | Used for ligation- independent cloning |
| Commercial assay or kit | Super Script II Reverse Transcriptase | Invitrogen | catalog# 18064014 | |
| Commercial assay or kit | RQ1 Rnase- Free Dnase | Promega | catalog# M6101 | |
| Commercial assay or kit | RiboMAX Large Scale RNA Production System-T7 | Promega | catalog# P1300 | |
| Commercial assay or kit | MicroSpin G-25 columns | GE | catalog# 27-5325-01 | |
| Commercial assay or kit | Fast SYBR Green Master Mix | Applied Biosystems | catalog# 4385612 | |
| Commercial assay or kit | Glutathione sepharose-4B | GE | catalog# 17-0756-01 | |
| Commercial assay or kit | MagStrep ‘type3’ XT beads | IBA | catalog# 2-4090-002 | |
| Chemical compound, drug | 5-FOA | USBiological | catalog# F5050 | |
| Chemical compound, drug | Complete mini EDTA-free protease inhibitor | Roche | catalog# 11836170001 | |
| Chemical compound, drug | leupeptin | Roche | catalog# 11017128001 | |
| Chemical compound, drug | pepstatin | Roche | catalog# 11359053001 | |
| Chemical compound, drug | Glutathione | Sigma | catalog# G4251 | |
| Chemical compound, drug | Biotin | IBA | catalog# 2-1016-002 | |
| Software, algorithm | MultAlin | (Corpet, 1988) | http://multalin.toulouse.inra.fr/multalin/ |
Additional files
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Supplementary file 1
Related to Figure 2.
Effects of RPS3-K108E, mbf1Δ and RPS3-K108E mbf1Δ mutants on GFP/RFP protein, mRNA and protein/mRNA from in-frame and frameshifted RLuc-Arg4-GFP/RFP.
- https://doi.org/10.7554/eLife.39637.025
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Supplementary file 2
Related to Figure 3.
Effects of asc1Δ, mbf1Δ and asc1Δ mbf1Δ mutations on GFP/RFP protein, mRNA and protein/mRNA from GLN4(1-99)-GFP reporters with three CGA-CGA codon pairs in the 0, +1, and −1 reading frames.
- https://doi.org/10.7554/eLife.39637.026
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Supplementary file 3
Related to Key Resources Table in Materials and methods Strains, plasmids and oligonucleotides used in these studies.
- https://doi.org/10.7554/eLife.39637.027
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Transparent reporting form
- https://doi.org/10.7554/eLife.39637.028
