The molecular basis of coupling between poly(A)-tail length and translational efficiency
- Howard Hughes Medical Institute, United States
- Whitehead Institute for Biomedical Research, United States
- Department of Biology, Massachusetts Institute of Technology, United States
Figures
Figure 1 with 2 supplements
PABPC overexpression uncouples poly(A)-tail length and TE in frog oocytes.
(A) Schematic of capped T7 transcripts with two different tail lengths, which were used as reporter mRNAs. Additional sequences beyond the HDV sequence are not depicted. (B) The effect of tail …
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Figure 1—source data 1
Source data for luciferase values shown in Figure 1 and Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/66493/elife-66493-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Supporting data for reporter experiments examining the effect of PAPBC levels on coupling between tail length and translation.
(A) The stability of reporters during in vitro translation. Shown are mRNA northern blots monitoring reporter mRNA levels over the course of the indicated in vitro translation reactions. (B) …
Figure 1—figure supplement 2
Additional reporter assays examining the effect of PAPBC levels on coupling between tail length and translation.
(A) The effect of tail length on reporter translation in vitro. This panel is as in Figure 1B, but uses Rluc reporters instead of Nluc reporters and shows the results of one replicate. (B) The …
Figure 2 with 1 supplement
Increased PABPC promotes translation of endogenous short-tailed mRNAs, thereby diminishing coupling between tail length and TE.
(A) The effect of PABPC on coupling between tail length and TE in frog oocytes. Shown is the relationship between TE and median poly(A)-tail length in oocytes injected with either water or mRNAs …
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Figure 2—source data 1
Source data for values shown in Figure 2D.
- https://cdn.elifesciences.org/articles/66493/elife-66493-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Increased PABPC increases translation of endogenous short-tailed mRNAs in frog oocytes.
(A) Increased relative translation of short-tailed mRNAs after overexpressing PABPC in oocytes. Shown for each gene with ≥100 poly(A) tags is the change in relative TE observed after overexpressing …
Figure 3 with 1 supplement
Limiting PABPC is required for intragenic coupling between poly(A)-tail length and TE.
(A) The PAL-TRAP method for measuring intragenic effects of tail length on TE. Ribosomes are sparsely tagged (red stars) so that highly translated mRNAs are more likely to contain tagged ribosomes …
Figure 3—figure supplement 1
Supporting data for PAL-TRAP analyses in frog oocytes.
(A) Incorporation of HA-tagged RPL3 into ribosomes. At the top is a sucrose-gradient polysome profile from frog oocytes injected with mRNAs encoding HA-tagged RPL3 and FLAG-tagged ePAB. At the …
Figure 4 with 3 supplements
PABPC depletion causes premature decay of shorter-tail cytoplasmic mRNAs in HeLa cells.
(A) The effect of PABPC knockdown on poly(A)-tail length. The plots compare median poly(A)-tail lengths in either PABPC1-knockdown cells (left) or PABPC1 and PABPC4 double-knockdown cells (right) to …
Figure 4—figure supplement 1
PABPC depletion is not sufficient to establish strong coupling between poly(A)-tail length and TE in HeLa cells.
(A) Western blot showing siRNA-mediated depletion of PABPC1 and PABPC4 in HeLa cells. (B) The effect of depleting PABPC on coupling between tail length and TE in HeLa cells. Shown is the …
Figure 4—figure supplement 2
Support and extension of experiments showing that PABPC depletion causes premature decay of short-tailed mRNAs in HeLa cells.
(A) The effect of PABPC knockdown on poly(A)-tail length in NIH3T3 cells. The plot compares median poly(A)-tail length in PABPC1-knockdown cells to that in control cells. Results are shown for mRNAs …
Figure 4—figure supplement 3
Measurements of mRNA half-lives in PABPC-depleted HeLa cells.
(A) Experimental scheme for measuring mRNA half-lives in HeLa cells. siRNA-transfected cells were incubated with 5-ethynyl uridine (5-EU), and cytoplasmic mRNA was harvested at the indicated time …
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Figure 4—figure supplement 3—source data 1
Source data for mRNA half-life values shown in Figure 4—figure supplement 3B–C.
- https://cdn.elifesciences.org/articles/66493/elife-66493-fig4-figsupp3-data1-v1.xlsx
Figure 5 with 1 supplement
Depletion of TUT4 and TUT7 attenuates premature decay of mRNA caused by PABPC depletion.
(A) Rescue of loss of shorter-tail mRNAs in PABPC-depleted HeLa cells by expressing either an siRNA-resistant human PABPC1, an siRNA-resistant PABPC1 coding for a mutant that does not bind eIF4G …
Figure 5—figure supplement 1
Minimal terminal uridylation activity in frog oocytes.
Plotted are the fractions of cytoplasmic mRNAs containing uridines near their termini, as detected in frog oocytes injected with mRNAs overexpressing the indicated proteins using tail-length …
Figure 6 with 2 supplements
Depletion of PABPC in mammalian cell lines has minimal effect on TE.
(A) Effect of PABPC knockdown on mRNA abundance (left), ribosome-footprint abundance (middle) and TE (right) in HeLa cells, comparing values in double-knockdown cells to those in control cells. For …
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Figure 6—source data 1
- https://cdn.elifesciences.org/articles/66493/elife-66493-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Depletion of PABPC in mammalian cell lines has minimal effect on TE.
(A) The effect of PABPC knockdown on protein synthesis in HeLa cells. Cells transfected with the indicated siRNAs were cultured for 48 hr, then treated with puromycin before harvesting for …
Figure 6—figure supplement 2
Rapid depletion of PABPC1 is not sufficient to establish detectable coupling between poly(A)-tail length and TE in HCT116 cells.
Shown is the relationship between TE and median poly(A)-tail length in cells that were either not treated with IAA, or treated with IAA and collected at the indicated time. Otherwise, this panel is …
Figure 7
Model for coupling between poly(A)-tail length and TE, and context-dependent roles of PABPC.
See text for details.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | HCT116 PABPC1-AID (sC152-C16) | This study | sC152-C16 | See Materials and methods for details |
| Cell line (human) | HCT116 PABPC1-AID (sC278-C2) | This study | sC278-C2 | See Materials and methods for details |
| Cell line (human) | HCT116 OsTIR | Natsume et al., 2016 | ||
| Cell line (human) | HeLa | Nam et al., 2014 | ||
| Cell line (human) | HeLa RPL3-3xHA (sC262-4) | This study | sC262-4 | See Materials and methods for details |
| Cell line (mouse) | NIH3T3 | Eisen et al., 2020 | ||
| Cell line (zebrafish) | ZF4 | ATCC (CRL-2050) | ||
| Antibody | Anti-β-actin (Rabbit monoclonal) | Cell Signalling Technology (D6A8) | 1:1000 dilution | |
| Antibody | Anti-AID (Mouse monoclonal) | MBL International (M214-3) | 1:1000 dilution | |
| Antibody | Anti-ePAB (Rabbit polyclonal) | Wilkie et al., 2005 | 1:2000 dilution | |
| Antibody | Anti-GFP (Mouse monoclonal) | Cell Signalling Technology (2955) | 1:1000 dilution | |
| Antibody | Anti-FLAG (Mouse monoclonal) | MilliporeSigma (F9291) | 1:1000 dilution | |
| Antibody | Anti-GAPDH (Mouse monoclonal) | Proteintech (60004) | 1:1000 dilution | |
| Antibody | Anti-HA (Mouse monoclonal) | Cell Signalling Technology (6E2) | 1:1000 dilution | |
| Antibody | Anti-PABPC1 (Rabbit polyclonal) | Cell Signalling Technology (4992S) | 1:1000 dilution | |
| Antibody | Anti-PABPC4 (Rabbit polyclonal) | Novus Biologicals (NB100-74594) | 1:1000 dilution | |
| Antibody | Anti-puromycin (Mouse monoclonal) | MilliporeSigma (MABE343) | 1:1000 dilution | |
| Antibody | Anti-RPL3 (Rabbit polyclonal) | Abcam (ab154882) | 1:1000 dilution | |
| Antibody | Anti-RPS15 (Rabbit polyclonal) | Proteintech (14957–1-AP) | 1:1000 dilution | |
| Antibody | Anti-RPS3 (Rabbit monoclonal) | Cell Signalling Technology (D50G7) | 1:1000 dilution | |
| Antibody | Anti-TUT4 (Rabbit polyclonal) | ABclonal (A5972) | 1:1000 dilution | |
| Antibody | Anti-TUT7 (Rabbit polyclonal) | Proteintech (25196–1-AP) | 1:1000 dilution | |
| Antibody | Anti-Vinculin (Mouse monoclonal) | Proteintech (66305) | 1:1000 dilution | |
| Antibody | IRDye 800CW Goat anti-Rabbit IgG (H + L) | LI-COR (926–32211) | 1:10,000 dilution | |
| Antibody | IRDye 680RD Goat anti-Mouse IgG | LI-COR (926–68070) | 1:10,000 dilution | |
| Sequence based reagent | All oligos | Supplementary file 1 | ||
| Recombinant DNA reagent | All plasmids | Supplementary file 2 | ||
| Other | Reference sequences | Supplementary file 3 | ||
| Other | Masked mitochondrial pseudogenes | Supplementary file 4 | ||
| Other | mRNA 3′-end annotations | Supplementary file 5 |
Additional files
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Supplementary file 1
Oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/66493/elife-66493-supp1-v1.xlsx
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Supplementary file 2
Plasmids used in this study.
- https://cdn.elifesciences.org/articles/66493/elife-66493-supp2-v1.xlsx
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Supplementary file 3
Reference sequences.
- https://cdn.elifesciences.org/articles/66493/elife-66493-supp3-v1.xlsx
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Supplementary file 4
Masked mitochondrial pseudogenes.
- https://cdn.elifesciences.org/articles/66493/elife-66493-supp4-v1.xlsx
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Supplementary file 5
mRNA 3′-end annotations.
- https://cdn.elifesciences.org/articles/66493/elife-66493-supp5-v1.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66493/elife-66493-transrepform-v1.docx
