Computational analysis of miRNA-mediated repression of translation: implications for models of translation initiation inhibition

Abstract

The mechanism by which miRNAs inhibit translation has been under scrutiny both in vivo and in vitro. Divergent results have led to the suggestion that miRNAs repress translation by a variety of mechanisms including blocking the function of the cap in stimulating translation. However, these analyses largely only examine the final output of the multistep process of translation. This raises the possibility that when different steps in translation are rate limiting, miRNAs might show different effects on protein production. To examine this possibility, we modeled the process of translation initiation and examined how the effects of miRNAs under different conditions might be explained. Our results suggest that different effects of miRNAs on protein production in separate experiments could be due to differences in rate-limiting steps. This analysis does not rule out that miRNAs directly repress the function of the cap structure, but it demonstrates that the observations used to argue for this effect are open to alternative interpretations. Taking all the data together, our analysis is consistent with the model that miRNAs may primarily repress translation initiation at a late step.

FIGURE 1.

Details of the computational model. (A) Depiction of the relevant states in the translational model. Each rate and species that is included in the model is indicated as well as their primary function. (B) Protein production predicted from the model plotted as a function of k1 and k2, lines A–D correspond to specific rates described in the text.

FIGURE 2.

Effects of translational inhibition on individual steps of translation. These graphs depict the percentage of unrepressed protein production by altered rate constants and their effects for m7G-capped (m7G, green, k1 = 2), A-capped (A, blue, k1 = 0.09), and an intermediately translated mRNA (yellow, k1 = 0.5) for comparison. These modeled mRNA are identical except for the initiation complex formation rates (k1). Protein production refers to the percentage of unrepressed production for a respective k1 value. The region of rates inhibited by miRNA is from 0% to 100% repression of the respective values, where 100% repression results in no protein production. The circles indicate the observed in vitro miRNA-mediated inhibition in Mathonnet et al. (2007) with m7G-capped mRNA to allow comparisons to the amount of translation that will occur with mRNA having reduced k1 values. (A–B) Protein production from increasing repression of cap-dependent k1 shown as total and relative amounts, respectively. (C–D) Protein production from increasing repression of cap-independent k1 shown as total and relative amounts, respectively. (E–F) Protein production from increasing repression of k2 shown as total and relative amounts, respectively. (G–H) Protein production from increasing repression of k3 shown as total and relative amounts, respectively.

FIGURE 3.

Effect of miRNA inhibition on EMCV IRES-driven translation. (A) Depiction of the relative rates used in modeling cap-dependent and -independent (EMCV IRES) driven translation. The size of the arrow corresponds to the magnitude of the rate indicated. Panels B–I display the results of modeling potential sites of miRNA action on IRES and cap-dependent translation depicting protein production. The region of rates inhibited by miRNA is from 0% to 100% repression of the respective values, where 100% repression results in no protein production. The circles on m7G cap lines indicate the observed in vitro miRNA-mediated inhibition in Mathonnet et al. (2007). The circles on the IRES lines indicate the amount of translation if repressed equivalently to the m7G mRNA. (B–C) Protein production from increasing repression of cap-dependent k1 shown as total and relative amounts, respectively. (D–E) Protein production from increasing repression of cap-independent k1 shown as total and relative amounts, respectively. (F–G) Protein production from increasing repression of k2 shown as total and relative amounts, respectively. (H–I) Protein production from increasing repression of k3 shown as total and relative amounts, respectively.

FIGURE 4.

Resistance of eIF4F enhanced translation to miRNA-mediated translational repression. (A) Depiction of the rates and species used for modeling translation incorporating eIF4F and 60S subunit joining factors. For panels B–M, the results of modeling potential sites of miRNA action on mRNA with standard (1×) or elevated (10×) amounts of eIF4F depicting protein production, with the percentage representing that versus unrepressed translation for each condition. The region of rates inhibited by miRNA is from 0% to 100% repression of the respective values, where 100% repression results in no protein production. The amount of eIF4F modeled is either the standard extract concentration (green, 1×) or 10-fold increased eIF4F (blue, 10×), which is indicated in the respective graphs. The circles on 1× lines indicate the observed in vitro miRNA-mediated inhibition of m7G mRNA with no additional eIF4F in Mathonnet et al. (2007). The circles on the 10× lines indicate the amount of translation if repressed equivalently to the mRNA with no additional eIF4F. (B–C) Protein production from increasing repression of cap-dependent k1 shown as total and relative amounts, respectively. (D–E) Protein production from increasing repression of cap-independent k1 shown as total and relative amounts, respectively. (F–G) Protein production from increasing repression of k2 shown as total and relative amounts, respectively. (H–I) Protein production from increasing repression of k3 shown as total and relative amounts, respectively. (J–K) Protein production from increasing repression of eIF4F shown as total and relative amounts, respectively. (L–M) Protein production from increasing repression of 60S subunit joining factors shown as total and relative amounts, respectively.