Unique small RNA signatures uncovered in the tammar wallaby genome

James Lindsay, Dawn M Carone, Judy Brown, Laura Hall, Sohaib Qureshi, Sarah E Mitchell, Nicholas Jannetty, Greg Hannon, Marilyn Renfree, Andrew Pask, Michael O’Neill, Rachel O’Neill

Small RNAs have proven to be essential regulatory molecules encoded within eukaryotic genomes. These short RNAs participate in a diverse array of cellular processes including gene regulation, chromatin dynamics and genome defense. The tammar wallaby, a marsupial mammal, is a powerful comparative model for studying the evolution of regulatory networks. As part of the genome sequencing initiative for the tammar, we have explored the evolution of each of the major classes of mammalian small RNAs in an Australian marsupial for the first time, including the first genome-scale analysis of the newest class of small RNAs, centromere repeat associated short interacting RNAs (crasiRNAs). Using next generation sequencing, we have characterized the major classes of small RNAs, micro (mi) RNAs, piwi interacting (pi) RNAs, and the centromere repeat associated short interacting (crasi) RNAs in the tammar. We examined each of these small RNA classes with respect to the newly assembled tammar wallaby genome for gene and repeat features, salient features that define their canonical sequences, and the constitution of both highly conserved and species-specific members. Using a combination of miRNA hairpin predictions and co-mapping with miRBase entries, we identified a highly conserved cluster of miRNA genes on the X chromosome in the tammar and a total of 94 other predicted miRNA producing genes. Mapping all miRNAs to the tammar genome and comparing target genes among tammar, mouse and human, we identified 163 conserved target genes. An additional nine genes were identified in tammar that do not have an orthologous miRNA target in human and likely represent novel miRNA-regulated genes in the tammar. A survey of the tammar gonadal piRNAs shows that these small RNAs are enriched in retroelements and carry members from both marsupial and tammar-specific repeat classes. Lastly, this study includes the first in-depth analyses of the newly discovered crasiRNAs. These small RNAs are derived largely from centromere-enriched retroelements, including a novel SINE. This study encompasses the first analyses of the major classes of small RNAs for the newly completed tammar genome, validates preliminary annotations using deep sequencing and computational approaches, and provides a foundation for future work on tammar-specific as well as conserved, but previously unknown small RNA progenitors and targets identified herein. The characterization of new miRNA target genes and a unique profile for crasiRNAs has allowed for insight into multiple RNA mediated processes in the tammar, including gene regulation, species incompatibilities, centromere and chromosome function.