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

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.     

Alzheimer-specific variants in the 3'UTR of Amyloid precursor protein affect microRNA function

Background

APP expression misregulation can cause genetic Alzheimer's disease (AD). Recent evidences support the hypothesis that polymorphisms located in microRNA (miRNA) target sites could influence the risk of developing neurodegenerative disorders such as Parkinson's disease (PD) and frontotemporal dementia. Recently, a number of single nucleotide polymorphisms (SNPs) located in the 3'UTR of APP have been found in AD patients with family history of dementia. Because miRNAs have previously been implicated in APP expression regulation, we set out to determine whether these polymorphisms could affect miRNA function and therefore APP levels.

Results

Bioinformatics analysis identified twelve putative miRNA bindings sites located in or near the APP 3'UTR variants T117C, A454G and A833C. Among those candidates, seven miRNAs, including miR-20a, miR-17, miR-147, miR-655, miR-323-3p, miR-644, and miR-153 could regulate APP expression in vitro and under physiological conditions in cells. Using luciferase-based assays, we could show that the T117C variant inhibited miR-147 binding, whereas the A454G variant increased miR-20a binding, consequently having opposite effects on APP expression.

Conclusions

Taken together, our results provide proof-of-principle that APP 3'UTR polymorphisms could affect AD risk through modulation of APP expression regulation, and set the stage for further association studies in genetic and sporadic AD.     

Uncoupling of lin-14 mRNA and protein repression by nutrient deprivation in Caenorhabditis elegans

In animals, microRNAs (miRNAs), typically, pair to sites of partial complementarity in the 3′-untranslated regions (3′UTRs) of target genes. Regulation by miRNAs often results in down-regulation of target mRNA and protein expression by mechanisms that are yet to be fully elucidated. Additionally, changes in environmental conditions have been shown to influence miRNA function in some cell culture systems. Here, we report the effect of nutrient deprivation on regulation of an endogenous miRNA target in developing worms. In Caenorhabditis elegans, the lin-4 miRNA recognizes multiple sites in the lin-14 3′UTR and directs mRNA degradation and translational repression, but it is unclear how these processes are coupled. In this study, we demonstrate that nutrient deprivation results in loss of lin-14 mRNA, but not protein, repression. In worms removed from feeding conditions, lin-14 mRNA reaccumulates despite the continued expression of lin-4 miRNA. The relative increase in lin-14 mRNA levels during nutrient deprivation is less pronounced in genetic mutants lacking lin-4 miRNA or the lin-14 3′UTR target sites. In conclusion, regulation of lin-14 at the mRNA and protein levels can be uncoupled by changes in culture conditions, indicating that miRNA function can be modulated by environment in multicellular organisms. The awareness that endogenous miRNA pathways can be sensitive to environment is an important consideration for elucidating the mechanism used by miRNAs to regulate target mRNA and protein expression.     

Inference of miRNA targets using evolutionary conservation and pathway analysis

Background

MicroRNAs have emerged as important regulatory genes in a variety of cellular processes and, in recent years, hundreds of such genes have been discovered in animals. In contrast, functional annotations are available only for a very small fraction of these miRNAs, and even in these cases only partially.

Results

We developed a general Bayesian method for the inference of miRNA target sites, in which, for each miRNA, we explicitly model the evolution of orthologous target sites in a set of related species. Using this method we predict target sites for all known miRNAs in flies, worms, fish, and mammals. By comparing our predictions in fly with a reference set of experimentally tested miRNA-mRNA interactions we show that our general method performs at least as well as the most accurate methods available to date, including ones specifically tailored for target prediction in fly. An important novel feature of our model is that it explicitly infers the phylogenetic distribution of functional target sites, independently for each miRNA. This allows us to infer species-specific and clade-specific miRNA targeting. We also show that, in long human 3' UTRs, miRNA target sites occur preferentially near the start and near the end of the 3' UTR. To characterize miRNA function beyond the predicted lists of targets we further present a method to infer significant associations between the sets of targets predicted for individual miRNAs and specific biochemical pathways, in particular those of the KEGG pathway database. We show that this approach retrieves several known functional miRNA-mRNA associations, and predicts novel functions for known miRNAs in cell growth and in development.

Conclusion

We have presented a Bayesian target prediction algorithm without any tunable parameters, that can be applied to sequences from any clade of species. The algorithm automatically infers the phylogenetic distribution of functional sites for each miRNA, and assigns a posterior probability to each putative target site. The results presented here indicate that our general method achieves very good performance in predicting miRNA target sites, providing at the same time insights into the evolution of target sites for individual miRNAs. Moreover, by combining our predictions with pathway analysis, we propose functions of specific miRNAs in nervous system development, inter-cellular communication and cell growth. The complete target site predictions as well as the miRNA/pathway associations are accessible on the ElMMo web server.     

Search for signatures in miRNAs associated with cancer

Since the first discovery in the early 1990''s, the predicted and validated population of microRNAs (miRNAs or miRs) has grown significantly. These small (~22 nucleotides long) regulators of gene expression have been implicated and associated with several genes in the cancer pathway as well. Globally, the identification and verification of microRNAs as biomarkers for cancer cell types has been the area of thrust for most miRNA biologists. However, there has been a noticeable vacuum when it comes to identifying a common signature or trademark that could be used to demarcate a miR to be associated with the development or suppression of cancer. To answer these queries, we report an in silico study involving the identification of global signatures in experimentally validated microRNAs which have been associated with cancer. This study has thrown light on the presence of significant common signatures, viz., - sequential and hybridization, which may distinguish a miR to be associated with cancer. Based on our analysis, we suggest the utility of such signatures in the design and development of algorithms for prediction of miRs involved in the cancer pathway.     

MiRComb: An R Package to Analyse miRNA-mRNA Interactions. Examples across Five Digestive Cancers

MicroRNAs (miRNAs) are small RNAs that regulate the expression of target mRNAs by specific binding on the mRNA 3''UTR and promoting mRNA degradation in the majority of cases. It is often of interest to know the specific targets of a miRNA in order to study them in a particular disease context. In that sense, some databases have been designed to predict potential miRNA-mRNA interactions based on hybridization sequences. However, one of the main limitations is that these databases have too many false positives and do not take into account disease-specific interactions. We have developed an R package (miRComb) able to combine miRNA and mRNA expression data with hybridization information, in order to find potential miRNA-mRNA targets that are more reliable to occur in a specific physiological or disease context. This article summarizes the pipeline and the main outputs of this package by using as example TCGA data from five gastrointestinal cancers (colon cancer, rectal cancer, liver cancer, stomach cancer and esophageal cancer). The obtained results can be used to develop a huge number of testable hypotheses by other authors. Globally, we show that the miRComb package is a useful tool to deal with miRNA and mRNA expression data, that helps to filter the high amount of miRNA-mRNA interactions obtained from the pre-existing miRNA target prediction databases and it presents the results in a standardised way (pdf report). Moreover, an integrative analysis of the miRComb miRNA-mRNA interactions from the five digestive cancers is presented. Therefore, miRComb is a very useful tool to start understanding miRNA gene regulation in a specific context. The package can be downloaded in http://mircomb.sourceforge.net.     

A non-canonical plant microRNA target site

Plant microRNAs (miRNAs) typically form near-perfect duplexes with their targets and mediate mRNA cleavage. Here, we describe an unconventional miRNA target of miR398 in Arabidopsis, an mRNA encoding the blue copper-binding protein (BCBP). BCBP mRNA carries an miR398 complementary site in its 5′-untranslated region (UTR) with a bulge of six nucleotides opposite to the 5′ region of the miRNA. Despite the disruption of a target site region thought to be especially critical for function, BCBP mRNAs are cleaved by ARGONAUTE1 between nucleotides 10th and 11th, opposite to the miRNA, like conventional plant target sites. Levels of BCBP mRNAs are inversely correlated to levels of miR398 in mutants lacking the miRNA, or transgenic plants overexpressing it. Introducing two mutations that disrupt the miRNA complementarity around the cleavage site renders the target cleavage-resistant. The BCBP site functions outside of the context of the BCBP mRNA and does not depend on 5′-UTR location. Reducing the bulge does not interfere with miR398-mediated regulation and completely removing it increases the efficiency of the slicing. Analysis of degradome data and target predictions revealed that the miR398-BCBP interaction seems to be rather unique. Nevertheless, our results imply that functional target sites with non-perfect pairings in the 5′ region of an ancient conserved miRNA exist in plants.     

Mutations in microRNA Binding Sites of CEP Genes Involved in Cancer

The CEP genes play a pivotal role in the replication of the cell. CEP family proteins form the major constituents of the centrosome and play a prominent role in centriole biogenesis and in cell replication. Alteration in CEP genes will result in disruption of cell cycle that may in turn cause cancer. In our study, we found that 16 of the CEP genes are a potential target to miRNA that binds to complementary sequences in 3′untranslated regions (UTR) of mRNA and stop them from translation. Single nucleotide polymorphisms (SNPs) occurring naturally in such miRNA binding site can alter the miRNA: mRNA interaction and can significantly alter gene expression. We developed a systematic computational pipeline that integrates data from well-established databases, followed stringent selection criteria and identified a panel of 44 high-confidence SNPs that may impair miRNA target sites in the 3′UTR of 16 genes. Further we performed expression analysis to shed light on the potential tissues that might be affected by mutation, enrichment analysis to find the metabolic functions of the gene, and network analysis to highlight the important interactions of CEP genes with other genes to provide insight that complex network will be disturbed upon mutation. In this study, we explored and prioritised the SNPs in CEP gene which could act as a potential target in centrosome-associated human disease. Our analysis would provide a thoughtful insight to wet lab researches to understand the expression pattern of CEP genes and binding phenomenon of mRNA and miRNA upon mutation, which is responsible for inhibition of translation process at genomic levels.     

Genetic Variations in Key MicroRNA Processing Genes and Risk of Head and Neck Cancer: A Case-Control Study in Chinese Population

MicroRNAs (miRNAs) have been reported to play a key role in oncogenesis. Genetic variations in miRNA processing genes and miRNA binding sites may affect the biogenesis of miRNA and the miRNA-mRNA interactions, hence promoting tumorigenesis. In the present study, we hypothesized that potentially functional polymorphisms in miRNA processing genes may contribute to head and neck cancer (HNC) susceptibility. To test this hypothesis, we genotyped three SNPs at miRNA binding sites of miRNA processing genes (rs1057035 in 3′UTR of DICER, rs3803012 in 3′UTR of RAN and rs10773771 in 3′UTR of HIWI) with a case-control study including 397 HNC cases and 900 controls matched by age and sex in Chinese. Although none of three SNPs was significantly associated with overall risk of HNC, rs1057035 in 3′UTR of DICER was associated with a significantly decreased risk of oral cancer (TC/CC vs. TT: adjusted OR  = 0.65, 95% CI  = 0.46–0.92). Furthermore, luciferase activity assay showed that rs1057035 variant C allele led to significantly lower expression levels as compared to the T allele, which may be due to the relatively high inhibition of hsa-miR-574-3p on DICER mRNA. These findings indicated that rs1057035 located at 3′UTR of DICER may contribute to the risk of oral cancer by affecting the binding of miRNAs to DICER. Large-scale and well-designed studies are warranted to validate our findings.     

Computational approaches for microRNA studies: a review

MicroRNAs (miRNAs) are one class of tiny, endogenous RNAs that can regulate messenger RNA (mRNA) expression by targeting homologous sequences in mRNAs. Their aberrant expressions have been observed in many cancers and several miRNAs have been convincingly shown to play important roles in carcinogenesis. Since the discovery of this small regulator, computational methods have been indispensable tools in miRNA gene finding and functional studies. In this review we first briefly outline the biological findings of miRNA genes, such as genomic feature, biogenesis, gene structure, and functional mechanism. We then discuss in detail the three main aspects of miRNA computational studies: miRNA gene finding, miRNA target prediction, and regulation of miRNA genes. Finally, we provide perspectives on some emerging issues, including combinatorial regulation by miRNAs and functional binding sites beyond the 3′-untranslated region (3′UTR) of target mRNAs. Available online resources for miRNA computational studies are also provided.     

3’UTR Shortening Potentiates MicroRNA-Based Repression of Pro-differentiation Genes in Proliferating Human Cells

Most mammalian genes often feature alternative polyadenylation (APA) sites and hence diverse 3’UTR lengths. Proliferating cells were reported to favor APA sites that result in shorter 3’UTRs. One consequence of such shortening is escape of mRNAs from targeting by microRNAs (miRNAs) whose binding sites are eliminated. Such a mechanism might provide proliferation-related genes with an expression gain during normal or cancerous proliferation. Notably, miRNA sites tend to be more active when located near both ends of the 3’UTR compared to those located more centrally. Accordingly, miRNA sites located near the center of the full 3’UTR might become more active upon 3''UTR shortening. To address this conjecture we performed 3'' sequencing to determine the 3'' ends of all human UTRs in several cell lines. Remarkably, we found that conserved miRNA binding sites are preferentially enriched immediately upstream to APA sites, and this enrichment is more prominent in pro-differentiation/anti-proliferative genes. Binding sites of the miR17-92 cluster, upregulated in rapidly proliferating cells, are particularly enriched just upstream to APA sites, presumably conferring stronger inhibitory activity upon shortening. Thus 3’UTR shortening appears not only to enable escape from inhibition of growth promoting genes but also to potentiate repression of anti-proliferative genes.