Yeast eIF4A enhances recruitment of mRNAs regardless of their structural complexity
- Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, United States
Figures
Figure 1 with 1 supplement
ATP hydrolysis by eIF4A stimulates recruitment of natural mRNA RPL41A as well as a synthetic 50-mer made up largely of CAA-repeats, presumed to be unstructured, with an AUG start codon 23 nucleotides from the 5'-end (CAA 50-mer).
The concentration of ATP and analogs was 2 mM. (A) Percentage of RPL41A recruited to the PIC versus time. (B) Percentage of CAA 50-mer recruited to the PIC versus time. The larger plot shows the …
-
Figure 1—source data 1
Individual measurements of the percent of RPL41A and CAA 50-mer recruited to the PIC over time.
- https://doi.org/10.7554/eLife.31476.004
Figure 1—figure supplement 1
RPL41A can form structural elements throughout its length whereas the CAA 50-mer has a low propensity to form structures.
RNA folds, predicted by mfold (Zuker, 2003), that are representative of the population of conformations adopted by (A–B) CAA 50-mer, ∆G > −1 kcal/mol; (C) RPL41A, ∆G = −46 kcal/mol; (D) chimeric RNA …
Figure 2 with 1 supplement
eIF4A and eIF4A•4G•4E ATPase activities are stimulated by the PIC.
ATPase activity of 5 µM eIF4A in the presence of saturating (5 mM) ATP•Mg2+ as a function of the concentration of capped RPL41A mRNA for (A) eIF4A (no PIC): kcat = 0.48 ± 0.04 min−1, = 80 nM; (B)…
-
Figure 2—source data 1
Individual kcat measurements of ATP hydrolysis.
- https://doi.org/10.7554/eLife.31476.007
-
Figure 2—source data 2
Related to Figure 2—figure supplement 1.
Control ATPase reactions and individual measurements of ATPase activity with uncapped RPL41A.
- https://doi.org/10.7554/eLife.31476.008
Figure 2—figure supplement 1
Control and data tables for the ATPase experiments.
(A) Controls for NADH enzyme-coupled microplate ATPase assay. The decrease in absorbance of 340 nm light was dependent on the presence of ATPase activity (5 µM eIF4A, 0.5 µM eIF4G•4E, together …
Figure 3 with 3 supplements
eIF4A stimulates recruitment of mRNAs regardless of their degree of structure.
(A) Schematic of mRNAs used in the study. mRNAs were capped unless otherwise noted but do not contain a poly(A) tail. Numbers on the mRNA indicate the total number of nucleotides in the …
-
Figure 3—source data 1
Individual measurements of endpoints and rates of mRNA recruitmentment for mRNAs 1–10.
- https://doi.org/10.7554/eLife.31476.013
-
Figure 3—source data 2
Related to Figure 3—figure supplement 1.
Observed rates (kobs) of mRNA recruitment for mRNAs 1–10 at varying concentrations of eIF4A.
- https://doi.org/10.7554/eLife.31476.014
Figure 3—figure supplement 1
eIF4A promotes recruitment of both structured and CAA-repeats mRNAs.
(A) RNAs used in the study (same as (A) in Figure 3, shown again here for convenience). (B–C) Observed rates (kobs) min−1 of mRNA recruitment as a function of the concentration of eIF4A. Data were …
Figure 3—figure supplement 2
Evidence that the designed hairpins in the 5'-UTRs of mRNAs 5 and 6 are formed and that mRNA 4 lacks secondary structure.
(A) RNAs 4–6 used in the study (same as in Figure 3A; shown again here for convenience). The fragments expected to be protected from 3'−5' RNase Exonuclease T digest are indicated in red. (B) RNAs …
Figure 3—figure supplement 3
A change in the rate-limiting step for mRNA recruitment may be responsible for the effect of mRNA structure on the values.
(A) An mRNA harboring a high degree of structure has a large barrier to mRNA recruitment posed by the need to resolve those structures in order to load the mRNA onto the PIC (left-hand barrier). As …
Figure 4 with 1 supplement
The 5'−7-methylguanosine cap inhibits mRNA recruitment in the absence of eIF4A.
Observed rates of mRNA recruitment (min−1) when eIF4A was not included in the reaction, in the presence (black bars) or absence (red bars) of the 5'-cap. (See Figure 3A Key for explanation of mRNA …
-
Figure 4—source data 1
Rates of mRNA recruitment measured with capped and uncapped mRNAs in the presence of saturating eIF4A (kmax) or in the absence of eIF4A ().
- https://doi.org/10.7554/eLife.31476.017
-
Figure 4—source data 2
Related to Figure 4—figure supplement 1.
Individual measurements of the observed rate of mRNA recruitment (kobs) with uncapped mRNAs at various concentrations of eIF4A.
- https://doi.org/10.7554/eLife.31476.018
Figure 4—figure supplement 1
The 5'−7-methylguanosine cap inhibits mRNA recruitment in the absence of eIF4A.
mRNA recruitment of (A) RNA 1 (CAA 50-mer), (B) RNA 4, and (C) RNA 7 in the presence or absence of a 5'-cap, as indicated to the right of each curve. Numbers to the right of the plots correspond to …
Figure 5
Possible models for the mechanism of action of eIF4A in promoting mRNA recruitment to the PIC.
(A) eIF4A modulates the conformation of the PIC to promote mRNA recruitment. In this model, binding of eIF4A•4G•4E to the PIC induces an opening of the mRNA entry channel of the 40S subunit …
Additional files
-
Supplementary file 1
RNAs used in the study.
- https://doi.org/10.7554/eLife.31476.020
-
Transparent reporting form
- https://doi.org/10.7554/eLife.31476.021
