Sequence Relationships among C. elegans , D. melanogaster and Human microRNAs Highlight the Extensive Conservation of microRNAs in Biology Carolina Iba´ n˜ ez-Ventoso . , Mehul Vora., Monica Driscoll* Department of Molecular Biology and Biochemistry, Rutgers, The State University of New Jersey, Piscataway, New Jersey, United States of America Abstract microRNAs act in a prevalent and conserved post-transcriptional gene regulatory mechanism that impacts development, homeostasis and disease, yet biological functions for the vast majority of miRNAs remain unknown. Given the power of invertebrate genetics to promote rapid evaluation of miRNA function, recently expanded miRNA identifications (miRBase 10.1), and the importance of assessing potential functional redundancies within and between species, we evaluated miRNA sequence relationships by 59 end match and overall homology criteria to compile a snapshot overview of miRNA families within the C. elegans and D. melanogaster genomes that includes their identified human counterparts. This compilation expands literature documentation of both the number of families and the number of family members, within and between nematode and fly models, and highlights sequences conserved between species pairs or among nematodes, flies and humans. Themes that emerge include the substantial potential for functional redundancy of miRNA sequences within species (84/139 C. elegans miRNAs and 70/152 D. melanogaster miRNAs share significant homology with other miRNAs encoded by their respective genomes), and the striking extent to which miRNAs are conserved across species—over half (73/139) C. elegans miRNAs share sequence homology with miRNAs encoded also in both fly and human genomes. This summary analysis of mature miRNA sequence relationships provides a quickly accessible resource that should facilitate functional and evolutionary analyses of miRNAs and miRNA families. Citation: Iba´n˜ ez-Ventoso C, Vora M, Driscoll M (2008) Sequence Relationships among C. elegans, D. melanogaster and Human microRNAs Highlight the Extensive Conservation of microRNAs in Biology. PLoS ONE 3(7): e2818. doi:10.1371/journal.pone.0002818 Editor: Steven L. Salzberg, University of Maryland, United States of America Received January 28, 2008; Accepted June 19, 2008; Published July 30, 2008 Copyright: 2008 Iba´ n˜ez-Ventoso et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work has been supported by NIH grant R01 AG024882 and R21 AG029376. Competing Interests: The authors have declared that no competing interests exist. * E-mail: driscoll@biology.rutgers.edu . These authors contributed equally to this work. Introduction microRNAs (miRNAs) are small (16–29 nucleotide (nt)), non- coding RNAs that regulate gene expression at the post- transcriptional level [1–5]. Intensive research over the last several years has led to the appreciation that these tiny RNAs act via a highly prevalent, and generally conserved, gene expression regulatory mechanism that impacts development, homeostasis and disease. A major research challenge for the decade will be the elaboration of miRNA function in biology and the investigation of how microRNAs can be exploited for therapeutic application. To date, little is actually known about the biological functions of most miRNAs—the roles of only a small number have been experimentally elucidated [6,7]. Numerous studies have reported on miRNA expression profiles in cells, tissues, organisms, and disease states [8–31]. In addition, multiple bioinformatic efforts have predicted target mRNA transcripts to suggest candidate genes regulated by miRNA interactions e.g. [13,32,33–38]. The potential for complex cross-regulation that emerges from these general surveys is staggering, and appreciation for the complexity is further extended by observations that: 1) many mRNA transcripts include potential binding sites for multiple, distinct miRNAs, and 2) different miRNAs that share sequence similarity (especially in the 59 end seed region) can recognize the same binding sites on individual mRNA targets [39–42]. Against this backdrop, the need for understanding when and where miRNAs are expressed, what the relevant mRNA targets are, and what the complete miRNA family sequence relationships encoded by the genome are, is dramatically underscored. This work addresses the latter goal, with an emphasis on invertebrate genetic models that are likely to have a major impact on advancing understanding of miRNA function. Over the last few years, intensive discovery efforts have contributed to extensive additions and sequence changes to annotated miRBase miRNA compilations for C. elegans, D. melanogaster and humans as total numbers of mature miRNA sequences increased from 107 C. elegans, 79 D. melanogaster and 152 human (miRBase release 3.0, Jan. 2004) to 139 C. elegans, 152 D. melanogaster and 733 human (miRBase release 10.1, December 2007) [43–46]. Although it is anticipated that miRNAs will continue to be identified, (numbers of human miRNAs may be in the thousands (see Bentwich et al. [47]), it is likely that most of the abundant miRNAs have been identified in nematodes, flies and humans. Moreover, the majority of identified C. elegans miRNAs have been genetically deleted [48–50], an accomplishment that sets the stage for detailed evaluation of functions in this model. Initial studies support that evaluation of functional redundancies will be an important factor in this analysis [39,40,42,48] and that PLoS ONE | www.plosone.org 1 July 2008 | Volume 3 | Issue 7 | e2818