Deep annotation of Drosophila melanogaster microRNAs yields insights into their processing, modification, and emergence

Abstract

Since the initial annotation of miRNAs from cloned short RNAs by the Ambros, Tuschl, and Bartel groups in 2001, more than a hundred studies have sought to identify additional miRNAs in various species. We report here a meta-analysis of short RNA data from Drosophila melanogaster, aggregating published libraries with 76 data sets that we generated for the modENCODE project. In total, we began with more than 1 billion raw reads from 187 libraries comprising diverse developmental stages, specific tissue- and cell-types, mutant conditions, and/or Argonaute immunoprecipitations. We elucidated several features of known miRNA loci, including multiple phased byproducts of cropping and dicing, abundant alternative 5′ termini of certain miRNAs, frequent 3′ untemplated additions, and potential editing events. We also identified 49 novel genomic locations of miRNA production, and 61 additional candidate loci with limited evidence for miRNA biogenesis. Although these loci broaden the Drosophila miRNA catalog, this work supports the notion that a restricted set of cellular transcripts is competent to be specifically processed by the Drosha/Dicer-1 pathway. Unexpectedly, we detected miRNA production from coding and untranslated regions of mRNAs and found the phenomenon of miRNA production from the antisense strand of known loci to be common. Altogether, this study lays a comprehensive foundation for the study of miRNA diversity and evolution in a complex animal model.

Adaptive evolution of testis-specific, recently evolved, clustered miRNAs in Drosophila

“…For example, many testis-restricted miRNAs are members of genomic clusters. In and of itself, this is not particularly notable since 54% of D. melanogaster miRNAs are clustered (Ruby et al 2007;Berezikov et al 2011;Mohammed et al 2013), and we detected the specific expression of clustered miRNAs in other tissues. However, the representation of clustered miRNAs among testis-restricted loci (84%) was notably much higher compared with the number of clustered miRNAs among head-restricted loci (21%) or with clustered Drosophila miRNAs in general (Fig.…”

“…Although we and others have generated hundreds of Drosophila small RNA libraries, the small size of this animal has precluded broad assessment of tissue-specific libraries, to the extent that has been done with mammals. Only a few fly tissues have been subjected to small RNA sequencing, particularly the adult head, ovary, and testis (Berezikov et al 2011). To identify spatially restricted miRNAs, we compared small RNA libraries from these tissues with other whole-animal data (Supplemental Table 1), including various stages of embryonic development.…”

Testing hypotheses on the rate of molecular evolution in relation to gene expression using microRNAs

“…Errors in processing either reduced the number of functioning molecules or created toxic effects on their own. Although errors in protein processing are difficult to study directly (34), errors in miR processing are measurable by deep sequencing, because mature miRs with a terminus off by even 1 bp can be observed (32,(35)(36)(37)(38).…”

“…Knowing that the error rate is conserved, influenced by slight changes in the backbone sequence and that the error products are likely functional, we can test the toxic-error hypothesis directly. Under this hypothesis, miRNA processing error is constrained by selection and the selective pressure is stronger for more highly expressed genes (32). Fig.…”

The use of high-throughput sequencing methods for plant microRNA research

“…Supportive results were shown in the previous study on the processing of the miRNA precursors of Drosophila melanogaster. 50 In that study, 50 observed that a portion of sRNA-seq reads could be mapped onto the "loop" regions of certain miRNA precursors.…”