Structure, evolution and function of the bi-directionally transcribed iab-4/iab-8 microRNA locus in arthropods

Nucleic Acids Research, Mar 2013

Hui, Jerome H. L., Marco, Antonio, Hunt, Suzanne, Melling, Janet, Griffiths-Jones, Sam, Ronshaugen, Matthew

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Structure, evolution and function of the bi-directionally transcribed iab-4/iab-8 microRNA locus in arthropods

Nucleic Acids Research Structure, evolution and function of the bi-directionally transcribed iab-4/iab-8 microRNA locus in arthropods Jerome H. L. Hui 0 1 Antonio Marco 0 Suzanne Hunt 0 Janet Melling 0 Sam Griffiths-Jones 0 Matthew Ronshaugen 0 0 Faculty of Life Sciences, University of Manchester , Michael Smith Building, Oxford Road, Manchester M13 9PT , UK 1 School of Life Sciences, Chinese University of Hong Kong , Shatin , Hong Kong In Drosophila melanogaster, the iab-4/iab-8 locus encodes bi-directionally transcribed microRNAs that regulate the function of flanking Hox transcription factors. We show that bi-directional transcription, temporal and spatial expression patterns and Hox regulatory function of the iab-4/iab-8 locus are conserved between fly and the beetle Tribolium castaneum. Computational predictions suggest iab-4 and iab-8 microRNAs can target common sites, and cell-culture assays confirm that iab-4 and iab-8 function overlaps on Hox target sites in both fly and beetle. However, we observe key differences in the way Hox genes are targeted. For instance, abd-A transcripts are targeted only by iab-8 in Drosophila, whereas both iab-4 and iab-8 bind to Tribolium abd-A. Our evolutionary and functional characterization of a bi-directionally transcribed microRNA establishes the iab-4/iab-8 system as a model for understanding how multiple products from sense and antisense microRNAs target common sites. INTRODUCTION Specification of segmental identity on the head–tail axis of all animals is accomplished, in large part, by the highly coordinated expression of an ancient conserved complex of related transcription factors collectively known as Hox genes ( 1 ). Since the first molecular characterization of the Drosophila Hox complex, it has been recognized that numerous non-coding RNAs are interspersed between the protein coding genes ( 2,3 ). Subsequent work in other animals has found that non-coding RNAs are a prevalent feature of many animal Hox complexes (4). Insight into the function of a subset of these RNAs was facilitated by the discovery of microRNAs (miRNAs): 22-bp RNAs that direct translational repression. The Hox gene complex contains several miRNAs, which are themselves ancient genes, and their conserved positions within the complex suggest that this association arose near the time of the origin of the Hox complex itself ( 1 ). Only a small number of metazoan miRNAs conserved between distantly related metazoan animals date from this time ( 600 million years ago). Indeed, the oldest shared conserved animal miRNA is miR-100, which was duplicated in an ancestor of bilaterians to give rise to the Hox miR-10 miRNA ( 5 ). Studies of Hox complex miRNA function find that they seem to act in part by modulating the translation of Hox genes themselves. As translational repression occurs after processing in the cytoplasm, there is no obvious reason why a miRNA should target genes located nearby in the genome. Recent advances in sequencing technologies have enabled large-scale identification of miRNAs in animals ( 6,7 ). The available data suggest that only a tiny proportion of miRNA loci are transcribed in both sense and antisense directions. Indeed this bi-directional transcription has been shown for only a handful of loci in a variety of animals [e.g. Drosophila melanogaster (8) and human ( 9 )]. In these cases, the two distinct primary transcripts are independently processed by Drosha and Dicer to produce distinct mature miRNA sequences. The origin and conservation of bi-directional miRNA transcription and the functional relationships of products from sense and antisense transcription have not been extensively studied. The miRNAs produced from the D. melanogaster iab-4 locus in the Hox complex are currently the only functionally characterized sense/antisense miRNAs ( 10–13 ). The bi-directionally transcribed iab-4 locus produces two primary transcripts that fold into two distinct hairpin precursors, named dme-iab-4 and dme-iab-8 (the latter is also sometimes referred to as dme-iab-4-as). The spatial and temporal expression of the sense and antisense transcripts differ during embryogenesis ( 10,12,13 ). The mature miRNA products regulate translation of the Hox genes Ubx and abd-A in the fly ( 10–13 ). Interestingly, the vertebrate mir-196 and the insect iab-4 miRNAs are located in analogous regions in the Hox cluster and regulate homologous Hox genes, yet there is no evidence that the two miRNAs are themselves homologues ( 14 ). This has been suggested as an example of ‘genomic parallelism’, where selective pressure acts on miRNAs that have independently arisen in this locus, resulting in functional convergence to target the flanking Hox gene ( 15 ). Here, we investigate the evolution of bi-directional transcription at the iab-4/iab-8 miRNA locus. We use computational analysis and in vivo assays to identify and confirm iab-4 and iab-8 target sites in insect Hox UTRs (unt (...truncated)


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Hui, Jerome H. L., Marco, Antonio, Hunt, Suzanne, Melling, Janet, Griffiths-Jones, Sam, Ronshaugen, Matthew. Structure, evolution and function of the bi-directionally transcribed iab-4/iab-8 microRNA locus in arthropods, Nucleic Acids Research, 2013, pp. 3352-3361, Volume 41, Issue 5, DOI: 10.1093/nar/gks1445