Cooperative Gene Regulation by Microrna Pairs and Their
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Published online 29 May 2014 Nucleic Acids Research, 2014, Vol. 42, No. 12 7539–7552 doi: 10.1093/nar/gku465 Cooperative gene regulation by microRNA pairs and their identification using a computational workflow Ulf Schmitz1,*, Xin Lai2, Felix Winter1, Olaf Wolkenhauer1,3, Julio Vera2 and Shailendra K. Gupta1,4 Downloaded from 1Department of Systems Biology and Bioinformatics, University of Rostock, Rostock, Germany, 2Laboratory of Systems Tumor Immunology, Department of Dermatology, University Hospital Erlangen, Friedrich-Alexander-University Erlangen-Nuremberg, Germany, 3Stellenbosch Institute for Advanced Study (STIAS), Wallenberg Research Centre at Stellenbosch University, Stellenbosch, South Africa and 4Department of Bioinformatics, CSIR-Indian Institute of Toxicology Research, 226001 Lucknow, Uttar Pradesh, India http://nar.oxfordjournals.org/ Received December 22, 2013; Revised April 18, 2014; Accepted May 10, 2014 ABSTRACT fine-tuned through a cellular context-dependent regulation by multiple miRNAs, where miRNAs can either induce MicroRNAs (miRNAs) are an integral part of gene reg- translational repression or target mRNA degradation (4). ulation at the post-transcriptional level. Recently, it Thereby, the miRNA-target regulation machinery can real- at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 3, 2016 has been shown that pairs of miRNAs can repress the ize elaborate gene control functions, including noise buffer- translation of a target mRNA in a cooperative man- ing or homeostasis, and can ultimately mediate distinct tar- ner, which leads to an enhanced effectiveness and get expression patterns appropriate to the demand of differ- specificity in target repression. However, it remains ent biological processes (3,5,6). However, deregulated miR- unclear which miRNA pairs can synergize and which NAs have also been associated with the pathogenesis and genes are target of cooperative miRNA regulation. In the progression of many diseases, including cancer (7). An- this paper, we present a computational workflow for other remarkable aspect about miRNAs is that they provide the prediction and analysis of cooperating miRNAs a valuable source for diagnostic and prognostic markers for a growing number of human pathologies; especially those and their mutual target genes, which we refer to as miRNAs found in body fluids (8,9). Besides, miRNAs be- RNA triplexes. The workflow integrates methods of came a popular subject for the design of novel therapeutic miRNA target prediction; triplex structure analysis; interventions. For details see the reviews by Seto (10)and molecular dynamics simulations and mathematical Kasinski and Slack (11). modeling for a reliable prediction of functional RNA The phenomenon of cooperating miRNAs has, so far, not triplexes and target repression efficiency. In a case received extensive attention. It has been shown that pairs of study we analyzed the human genome and identi- miRNAs can synergistically regulate mutual targets to facil- fied several thousand targets of cooperative gene itate a more effective target repression (3,12–13), while the regulation. Our results suggest that miRNA coop- distance between the seed binding sites of a miRNA duet erativity is a frequent mechanism for an enhanced may affect the strength of target down-regulation (14). In target repression by pairs of miRNAs facilitating dis- this context, Sætrom et al. were able to determine an opti- mal seed site distance (13–35 nt) for miRNA cooperation tinctive and fine-tuned target gene expression pat- (13). The concept of miRNA cooperativity implies a possi- terns. Human RNA triplexes predicted and charac- ble sophisticated mechanism of regulation of miRNA tar- terized in this study are organized in a web resource gets (Figure 1). For example, we have shown previously that at www.sbi.uni-rostock.de/triplexrna/. higher quantities of miRNAs, as well as the phenomenon of synergistic target regulation, can work as an efficient noise INTRODUCTION buffer for target expression triggered by external stimuli MicroRNAs (miRNAs) are a well conserved and abundant (3). Furthermore, selective expression of cooperating miR- class of ∼22 nt long functional RNA molecules that regu- NAs could be adopted by cells to facilitate distinctive and late the expression of most protein coding genes at the post- fine-tune gene expression patterns to meet the requirements transcriptional level (1). Multiple predictions and growing in different biological scenarios (3,15). So far, only a few experimental evidence suggest that many genes are targets cases of synergistic target regulation by cooperating miR- of concerted miRNA regulation (1–3). Their expression is NAs have been identified and confirmed. Vella et al. were *To whom correspondence should be addressed. Tel: +49 381 498 7576; Fax: +49 381 498 7572; Email: [email protected] C The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 7540 Nucleic Acids Research, 2014, Vol. 42, No. 12 other prediction algorithms (17,18). However, only those target sites of conserved miRNAs and with good prediction scores (mirSVR score) were considered. Experimentally val- idated miRNA-target interactions were derived from the miRTarBase database (19). Secondary structure and minimum free energy prediction Figure 1. General principle of cooperative target regulations by pairs of Downloaded from miRNAs. The illustration on the left shows how a target mRNA can be 3 untranslated region (UTR) sequences of target genes repressed by either a single miRNA or by a pair of cooperating miRNAs. were extracted from the RefSeq gene track of the Uni- While in the first case miRNA and target form a duplex structure, the sec- versity of California Santa Cruz (UCSC) table browser ond case leads to the formation of a RNA triplex. On the right side, we il- (GRCh37/hg19; (20)). MiRNA sequences were extracted lustrated the repressive effect on the target that is induced either by a single miRNA (red and blue lines) or by two cooperating miRNAs (green dashed from the miRBase database (release 20; (21)). For struc- line). Even if the expression of the cooperating miRNAs is only mildly up- ture prediction of the RNA complexes, sequences of ma- http://nar.oxfordjournals.org/ regulated an enhanced repressive effect can be observed as compared to ture miRNAs and the target subsequence that encloses both the cases where single miRNAs are highly up-regulated. miRNA target sites were used. Secondary structure and triplex-free energy (TFE; a.k.a. Gibbs-free energy G)of the first to validate this phenomenon in Caenorhabditis ele- RNA triplexes were determined with the mfe tool from gans (12). Later, this was confirmed in HeLa cells by Sætrom the NUPACK software package (22). In NUPACK the full et al., who also determined the seed distance constraint re- partition function (except for pseudo-knots) of RNA com- quired for optimal target repression (13). In our own previ- plexes is computed in dilute solution. RNA triplexes as de- picted in Supplementary Figure S1 were visualized using the ous work we have analyzed this phenomenon and validated at Universitaet Erlangen-Nuernberg, Wirtschafts- und Sozialwissenschaftliche Z on August 3, 2016 it in human SK-Mel-147 melanoma cells for the case of p21 command line version of the RNA structure drawing tool which is regulated by miRNA-93 and miR-572 (3). VARNA (version 3.9; (23)). In the present work, we propose a workflow for the iden- tification and analysis of novel animal RNA triplexes com- Statistical analysis posed of two cooperating miRNAs and a mutual target All statistical analyses were performed using the R statisti- mRNA. We have implemented the workflow and analyzed cal computing software (version 3.0.1). the human genome for cases of putatively cooperating miR- NAs and their respective common targets. More specifically, we first made a whole human genome analysis in orderto 3D structure modeling and MDS identify putative RNA triplexes based on predicted target 3D model design. For the detailed 3D model of two sites and their respective seed site distance. Second, we ana- miRNA-Argonaute hybrids (miR-20a/hAgo2) attached to lyzed the local secondary structure of the identified triplexes one stretch of target 3 UTR (NEURL1B mRNA) we re- and computed the equilibrium probabilities of the inherent trieved the crystal structure of miR-20a/hAgo2 determined complexes (based on a partition function). Then, we deter- by Elkayam et al. (24) through X-ray crystallography from mined their thermodynamic profiles by performing molec- the Protein Data Bank (PDB id: 4F3T). All nucleotides ular dynamics simulations (MDS). Finally, we constructed and amino acid residues missing in the complex were mod- a kinetic model of synergistic target regulation by cooperat- eled using AccelrysR discovery studio package. The ac- ing miRNAs and simulated the target repression efficiency. curacy of the modeled miR-20a was ascertained by cal- In summary, our results indicate that the phenomenon of culating the backbone root-mean-square deviation of its cooperative miRNA-target repression is a prevalent mech- selected bases from the template which was less than 4 anism of post-transcriptional