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. 2012 Sep;18(9):1612-23.
doi: 10.1261/rna.033142.112. Epub 2012 Jul 26.

Differential association of microRNAs with polysomes reflects distinct strengths of interactions with their mRNA targets

Natali Molotski  1 , Yoav Soen
Affiliations

Differential association of microRNAs with polysomes reflects distinct strengths of interactions with their mRNA targets

Natali Molotski et al. RNA. 2012 Sep.

Abstract

While microRNAs have been shown to copurify with polysomes, their relative fraction in the translation pool (polysome occupancy) has not yet been measured. Here, we introduce a high-throughput method for quantifying polysome occupancies of hundreds of microRNAs and use it to investigate factors affecting these occupancies. Analysis in human embryonic stem cells (hESCs) and foreskin fibroblasts (hFFs) revealed microRNA-specific preferences for low, medium, or high polysome occupancy. Bioinformatics and functional analysis based on overexpression of endogenous and chimeric microRNAs showed that the polysome occupancy of microRNAs is specified by its mature sequence and depends on the choice of seed. Nuclease treatment further suggested that the differential occupancy of the microRNAs reflects interactions with their mRNA targets. Indeed, analysis of microNRA•mRNA duplexes showed that pairs involving high occupancy microRNAs exhibit significantly higher binding energy compared to pairs with low occupancy microRNAs. Since mRNAs reside primarily in polysomes, strong interactions lead to high association of microRNAs with polysomes and vice versa for weak interactions. Comparison between hESCs and hFFs data revealed that hESCs tend to express lower occupancy microRNAs, suggesting that cell type-dependent translational features may be affected by expression of a particular set of microRNAs.

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Figures

FIGURE 1.
FIGURE 1.
High-throughput method for measuring polysome occupancy of microRNAs. (A) Schematics of the approach. MicroRNA occupancies were computed based on the levels measured in the “polysome-bound” and “ribosome-free” fractions as indicated at the bottom. (B) Occupancy values for a few microRNAs, including miR-923 (right bar), which was shown to be a fragment of the 28S rRNA (Griffiths-Jones 2006, 2007; Griffiths-Jones et al. 2008). (C) Unbound, 80S, and polysomal fractions of high (let-7b), medium (miR-31), and low (miR-598) occupancy microRNAs indicates primary association with either the unbound or polysome fractions. (D,E) Validation of the high-throughput measurement by higher resolution profiles of representative microRNAs in hFFs (D) and using individual, microRNA-specific assays (E). Shown in D are levels of three microRNAs in seven equal volume (1-mL) fractions within the polysome-bound pool. Fractions are indicated along the OD profile in A. Displayed levels are relative to the levels detected in the ribosome-free, unbound pool (Unbound). Shown in E are occupancy (left y-axis, dark gray) and relative expression (right y-axis, light gray) for miR-21, miR-31, and let-7b, each measured using individual, microRNA-specific Taqman assays. (F) Occupancy of two representative microRNAs, miR-31 and let-7b, following treatment with 100 nM of rapamycin or DMSO (control). Occupancies were computed based on the polysome-bound and ribosome-free fractions as illustrated in A. Results in B, C, E, and F are displayed as mean +/− standard error (SE).
FIGURE 2.
FIGURE 2.
Dependence of microRNA occupancy on cellular context. (A) Distributions of microRNA occupancies evaluated in undifferentiated human embryonic stem cells (hESCs) and human foreskin fibroblasts (hFFs). (B) Color-coded heat map displaying side-by-side measurements of microRNA occupancies in hESCs and hFFs. (C,D) Correlation between hESC and hFF microRNAs with respect to occupancy (C) and abundance (D). Note the higher correlation with respect to occupancy (C vs. D, P-value = 0.009 based on Fisher's r-to-z transformation).
FIGURE 3.
FIGURE 3.
MicroRNAs that are highly expressed in hESCs have a stronger bias toward low occupancy compared to hFF microRNAs. (A) Left: Occupancy values for hESC microRNAs that are ranked according to their expression level. Right: Box plot distributions of occupancies of hESC microRNAs divided into groups of high, medium, and low expression. (B) Same as A for hFFs. Note the significantly larger inverse correlation between occupancy and expression in hESCs compared to hFFs (−0.51 vs. −0.23, P-value = 0.0035 based on Fisher's r-to-z transformation).
FIGURE 4.
FIGURE 4.
The mature form of the microRNA, but not its abundance, specifies its preference for low or high occupancy. Polysome occupancy and expression of let-7b (A) and miR-598 (B) measured in (1) hESCs transfected with the mature form of the microRNAs (overexpression in hESC), (2) hESCs transfected with negative control pre-microRNA (hESC), and (3) in hFFs. The tendencies for high and low occupancy of let-7b and miR-598 in hESCs were unaffected by the overexpression and were similar to the occupancy in hFFs. Values displayed as mean +/− SE.
FIGURE 5.
FIGURE 5.
MicroRNAs harboring identical seeds exhibit similar polysome occupancies. (A) Comparison of polysome occupancy and expression values for microRNAs harboring the same seed vs. microRNAs with nonidentical seeds. P-values are based on the two-sample Kolmogorov-Smirnov test. (B) Normalized histograms of occupancy differences for pairs of microRNAs sharing the same seed (Identical seed), and pairs of microRNAs with different seeds (Nonidentical seed). (C) Same as B for differences in expression as opposed to occupancy.
FIGURE 6.
FIGURE 6.
MicroRNA occupancy is functionally dependent on the choice of seed. (A) Schematics of chimeric microRNAs designed for testing relative contributions of the microRNA seed and body to the degree of its association with polysomes. (B) Polysome occupancies of the endogenous (left) and chimeric microRNAs (right) that were transfected into hESCs. Note the reversal in the difference between the occupancies of let-7b and miR-598 upon exchange of seeds. Values displayed as mean +/− SE.
FIGURE 7.
FIGURE 7.
High occupancy of microRNAs reflects their binding to the mRNA targets and not to the 80S ribosomal subunits. (A) Polysome occupancy of miR-21, miR-31, and let-7b with and without micrococcal nuclease treatment of hESCs (nuclease and control, respectively). Note the decrease in the occupancy of all tested microRNAs following nuclease treatment. (B–D) Relative abundance of miR-21 (B), miR-31 (C), and let-7b (D) in unbound, 80S, and polysome fractions with and without micrococcal nuclease treatment. Levels are relative to the abundance in the unbound fraction of untreated hESC (control). Note the negligible levels in the 80S fraction following nuclease treatment. Values displayed as mean +/− SE.
FIGURE 8.
FIGURE 8.
Polysome occupancy of microRNAs depends on the strength of interactions with their targets. (A) Normalized histograms of ΔΔG scores for four very high and very low occupancy microRNAs (>70% and <13%, respectively, in both hESCs and hFFs). Scores were computed by the PITA algorithm for each microRNA and its predicted mRNA targets. (B) Polysome occupancy (averaged across hESCs and hFFs) and average ΔΔG of the microRNAs in A. Inset displays the average occupancy vs. average ΔΔG for all the microRNAs that were detected in both hESCs and hFFs. Significance of the difference in average ΔΔG between the high and low occupancy microRNAs was computed based on permutation test, P-value < 1.5 × 10−6. (C) Same as A for empirical mRNA targets of let-7b and miR-598, representing high and low occupancy microRNA, respectively. (D) Functional analysis of the effect of seed swapping on ΔΔG. Replacement of let-7b seed with the seed of miR-598 (light blue) shifted the average ΔΔG of let-7b toward less negative values (blue arrow) and reduced the occupancy (Fig. 6B). Conversely, replacement of miR-598 seed with the seed of let-7b shifted the average ΔΔG toward more negative values (pink arrow) and increased the occupancy (Fig. 6B). (E) Box plot distributions of occupancy values of the top 100 mRNA targets predicted by the PITA algorithm for let-7b and miR-598. (F) Box plot distributions of occupancy values of empirical mRNA targets of let-7b and miR-598, respectively. Displayed occupancies in E and F were measured in hESCs.
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