The Characterization of microRNA-Mediated Gene Regulation as Impacted by Both Target Site Location and Seed Match Type

<div><p>MicroRNAs (miRNAs) are small RNA molecules that play important roles in gene regulation and translational repression. The mechanisms that facilitate miRNA target binding and recognition have been extensively studied in recent years. However, it is still not well known how the miRNA regulation is affected by the location and the flanking sequences of miRNA target sites. In this study, we systematically quantify the contribution of a wide spectrum of target sites on miRNA-mediated gene expression regulation. Our study investigates target sites located in four different gene regions, including 3' untranslated regions, coding sequences, 5′ untranslated regions and promoter regions. We have also introduced four additional non-canonical types of seed matches beyond the canonical seed matches, and included them in our study. Computational analysis of quantitative proteomic data has demonstrated that target sites located in different regions impact the miRNA-mediated repression differently but synergistically. In addition, we have shown the synergistic effects among non-canonical seed matches and canonical ones that enhance the miRNA regulatory effects. Further systematic analysis on the site accessibility near the target regions and the secondary structure of the mRNA sequences have demonstrated substantial variations among target sites of different locations and of different types of seed matches, suggesting the mRNA secondary structure could explain some of the difference in the miRNA regulatory effects impacted by these different target sites. Our study implies miRNAs might regulate their targets under different mechanisms when target sites vary in both their locations and the types of seed matches they contain.</p></div

The mean of Z_ΔΔG of each sliding window near miRNA target sites.

<p>A) Target sites located in different gene regions. B) Target sites of different seed match types.</p

Signal-to-noise ratios of different types of seed matches in different gene regions.

<p>The signal-to-noise ratio is the ratio of the number of matches of each seed match type in the miRWalk dataset and the number of matches obtained from random shuffles of mRNA sequences.</p

The average log2 protein fold change after miRNA overexpression for each gene group containing different types of seed matches.

<p>Number, the total number of genes in each gene group.%(<−0.1), the percentage of genes in the group was down-regulated with a log2 protein fold change less than −0.1 and considered as true targets. P-value, the statistical significance of the percentage of true targets in a target group calculated by the Fisher's exact test and subsequently adjusted for multiple testing. Gene groups in italics indicate the percentage of true target genes in the group is not significant compared to the background model (“None” group). For groups with dual sites of different seed match types, only those with the percentage of true targets significantly higher than the background model (corrected p-value<0.05) are listed. The full list of combinations of two seed match types is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108260#pone.0108260.s006" target="_blank">Table S2</a>.</p><p>The average log2 protein fold change after miRNA overexpression for each gene group containing different types of seed matches.</p

A performance comparison of different computational miRNA target identification methods.

<p>The methods were evaluated by the independent benchmark dataset obtained by the pSILAC. Sensitivity, the proportion of true targets that are identified. Precision, the proportion of identified targets that are true targets.</p><p>A performance comparison of different computational miRNA target identification methods.</p

Genes with target sites significantly enriched in different gene regions have different degrees of secondary structure in the 5′UTRs and 3′UTRs.

<p>A) Genes with target sites enriched in 3′UTRs have a greater degree of secondary structure in the 5′UTRs than genes without target sites in any region (None). B) Genes with target sites enriched in 5′UTRs have a greater degree of secondary structure in the 3′UTRs than genes with target sites enriched in Promoters.</p

Synergism between target sites located in different gene regions.

<p>A) Cumulative distributions of protein log 2 fold changes of genes containing seed matches as indicated. Observed values for genes with one seed in 3′UTRs and one seed in CDSs are in red, simulated values for genes containing two independent seeds in 3′UTRs and CDSs were calculated as described in the Methods section. B) Similar as A) but with 3′UTR and 5′UTR seed combination. C) Cumulative distributions of protein log 2 fold changes of gene groups combining seed matches in multiple regions.</p

Synergism between different types of seed matches.

<p>A) Cumulative distributions of protein log 2 fold changes of genes containing only seed matches as indicated. Observed values for genes with one seed match of 2t8A1 and one seed match of 1t8Mi are in red, simulated values for genes containing two independent seed matches of types 2t8A1 and 1t8Mi were calculated as described in the Methods section. B) Similar as A) but with 2t7A1 and 1t8In seed combination. C) Cumulative distributions of protein log 2 fold changes of genes groups combining multiple types of seed matches.</p

Growth Kinetics and Morphological Evolution of ZnO Precipitated from Solution

This work characterizes the nucleation and growth kinetics of zinc oxide (ZnO) precipitated from aqueous hexamethylenetetramine (HMTA) zinc nitrate (Zn­(NO₃)₂) solutions observed by in situ and ex situ transmission electron microscopy. Quantitative comparisons between in situ beam-induced precipitation, in situ thermally activated precipitation, ex situ thermally activated precipitation, and ex situ electrochemistry provide insights into the rate limiting mechanism and the chemistry governing the reactions. All experiments indicate that isotropic ZnO precipitates directly from solution. These particles begin to aggregate and grow anisotropically shortly after nucleation. The conversion to anisotropic growth does not rely on coalescence despite the fact that the two are often observed to occur in concert. The results indicate that the reaction pathway for in situ beam-induced growth more closely mimics ex situ electrochemistry than ex situ chemistry. In situ and ex situ thermally activated growth processes proceed in a similar manner, although particle transport and aggregation are limited by the in situ geometry

Growth Kinetics and Morphological Evolution of ZnO Precipitated from Solution

This work characterizes the nucleation and growth kinetics of zinc oxide (ZnO) precipitated from aqueous hexamethylenetetramine (HMTA) zinc nitrate (Zn­(NO₃)₂) solutions observed by in situ and ex situ transmission electron microscopy. Quantitative comparisons between in situ beam-induced precipitation, in situ thermally activated precipitation, ex situ thermally activated precipitation, and ex situ electrochemistry provide insights into the rate limiting mechanism and the chemistry governing the reactions. All experiments indicate that isotropic ZnO precipitates directly from solution. These particles begin to aggregate and grow anisotropically shortly after nucleation. The conversion to anisotropic growth does not rely on coalescence despite the fact that the two are often observed to occur in concert. The results indicate that the reaction pathway for in situ beam-induced growth more closely mimics ex situ electrochemistry than ex situ chemistry. In situ and ex situ thermally activated growth processes proceed in a similar manner, although particle transport and aggregation are limited by the in situ geometry