Developmental control of microRNA arm choice

MicroRNAs (miRNAs) are fundamental modulators of gene function. These approximately 22 nucleotide single stranded RNA molecules direct the activity of proteins that repress translation and degrade mRNAs. More than 10,000 miRNAs have been identified since their discovery a decade ago. Bioinformatic studies suggest that a third to a half of animal genes are regulated by miRNAs, signifying their huge impact in virtually every biological process. A miRNA can make functional mature products from either arm of the hairpin precursor but are commonly found to produce a single dominant product. Since sequences from opposite arms of the hairpin are not similar, each miRNA arm will regulate distinct sets of genes. The fidelity of the arm choice is critical for correct regulation of target mRNAs. For many miRNAs, choice of miRNA arm is highly reproducible. However, high-throughput sequencing of small RNAs in diverse animals and tissues has found that some miRNAs have different dominant arms depending on where and when they are expressed. The genes affecting arm choice are unknown. This has motivated our proposed study to understand genes involved in mediating this choice. This project we propose will take advantage of our discovery of arm switching in an ancient, highly conserved family of miRNAs present in all animals. We will utilize non-vertebrate models (the fruit fly and flour beetle) to understand the residues in the miRNA precursor that contribute to arm choice and identify genes that mediate choice of dominant miRNA arm direct miRNA function. The regulation of arm usage has broad significance, contributing to the fields of developmental biology, tissue repair and regeneration, and pathological conditions such as cancer.

Development of multi-cellular organisms is accomplished through the regulated action of gene function. The recent discovery of microRNAs (miRNAs) has exposed a hidden layer of gene regulation. miRNAs are ancient modulators of RNA transcript stability and translation. A key step in miRNA processing is the cleavage of the precursor stem-loop by the Dicer enzyme to produce a ~22 bp RNA duplex. One RNA molecule from the duplex (the mature miRNA) is preferentially incorporated into the RNA-induced silencing complex (RISC). Recent deep sequencing experiments have found that the mature miRNA may switch between precursor arms and that choice of arm may be modulated in different tissues and times during development. We have identified evolutionary events that have switched the dominant mature product in members of the ancient mir-100/10 family. The sequences from the mature 5' and 3' miRNA arms are not similar; therefore homologous miRNAs producing dominant sequences from opposite arms will regulate distinct target mRNAs. Developmental control of arm choice provides a completely unexplored mechanism to modulate the function of a single miRNA gene locus. We propose to utilize our discovery of arm switching in miR-10 to identify sequences and genes that regulate miRNA arm choice. We will construct chimeric miR-10 primary transcripts to identify the RNA sequence motifs underlying arm choice. A Drosophila cell culture based genome-wide siRNA screen will be used to identify genes that mediate miRNA arm choice. Candidate regulators of miRNA arm usage will be further characterized to identify genes that are evolutionary conserved and tissue-specifically expressed. We propose that this will define the set of candidates that reveal how control of miRNA arm choice directs different developmental outcomes. This work will describe the protein components and miRNA sequences that constitute a novel mechanism for controlling and modulating gene function in development and evolution.

The most identifiable impact of this work will be on the biological research community. The beneficiaries of this work will be primarily academic researchers that study RNA biology, particularly those with interest in miRNA biology. Advances in understanding the regulation of miRNA function have broad relevance for virtually every field of biology as the regulation of gene function plays a role in most biological process. Therefore this proposal has a potentially large impact on the greater biological community. A second group of beneficiaries will be non-academic individuals in the commercial and private sector that exploit the miRNA pathway to develop tools for genetic research and those that design assays for profiling of development and disease. The data resulting from this proposal has potential to result in improved design of a number of existing commercial products. Specifically, the outcomes of aim 1 in this proposal will provide a description of the sequences that control tissue specific control of miRNA arm choice. The use of artificially constructed miRNA like hairpins has become a common tool to modulate gene function that many companies exploit. The design of assays and constructs using these hairpins often suffer from problems such as 'off target effects' where the functions of unanticipated genes are disrupted along with the intended target. Some of these effects may be due to the production of functional RNAs from alternate hairpin arms. The ability to predict and control when and where this occurs would provide a significant improvement in these tools. A second pool of beneficiaries will be companies that develop assays directed at profiling miRNAs expression. Understanding the mechanisms of arm choice will allow the identification of these alternate miRNA arms to be included in assays and to provide a better interpretation for assay results. The two primary methods for ensuring impact will be communication of results through publication in international journals and presentations at national and international meetings. Dr. Ronshaugen will perform these activities and training will be provided to develop the skills of the PDRA to assist in publicizing results. Novel methods for integrating larger data sets with existing heavily accessed web resources will also be explored. miRBase, the predominant miRNA repository is currently maintained in the Faculty of Life Sciences at UoM and a collaboration to facilitate this is in place. The timescale for realizing the impact on academic research will be short, with publications, presentations, and web based resources for data access providing rapid availability for individual use. The development of miRNA assays incorporating the descriptive results of regulated miRNA arm use described in this work may also be relatively rapid in development (~3-5 years). Incorporation of anticipated results into novel therapeutic RNA molecules is however likely to be more long range (~8-10 years).