The transcriptional framework of the eukaryotic centromere core has been described in budding yeast and rice, but for most eukaryotes and all vertebrates it remains largely unknown. The lack of large pericentric repeats in the tammar wallaby has made it possible to map and identify the transcriptional units at the centromere in a mammalian species for the first time. We show that these transcriptional units, comprised of satellites and a retrovirus, are bound by centromere proteins and that they are the source of a novel class of small RNA. The endogenous retrovirus from which these small RNAs are derived is now known to be in the centromere domain of several vertebrate classes. The discovery of this new RNA form brings together several independent lines of evidence that point to a conserved retroviral-encoded processed RNA entity within eukaryotic centromeres.
The pericentromere and centromere regions of the genome have previously been considered tightly compacted and transcriptionally inert. However, there is mounting evidence that these regions not only actively produce transcripts but that these pericentric and centromeric transcripts are also vital to maintaining genome stability and proper cell division. In this review, we define the pericentromere and centromere of eukaryotic chromosomes in terms of their histone modifications and their nascent transcripts. In addition, we present the currently known roles these transcripts play in heterochromatin formation, development, and differentiation, as well as their interaction with centromeric proteins, and ultimately centromere function. Recent work has added considerable complexity to the theoretical framework defining the innate requirement for pericentric and centromeric transcription. It is clear that maintaining a fine balance of transcriptional output is critical, as deviations from this balance result in centromere disfunction and genomic instability.
The proper functioning of centromeres requires a complex cascade of epigenetic events involving chromatin and kinetochore assembly; however, the precise mechanism by which this cascade proceeds is unknown. The pivotal event during kinetochore formation is the "loading," or deposition, of CENP-A. This histone H3 variant is specific to centromeres and replaces conventional H3 in centromeric chromatin. Failure to load CENP-A into mammalian centromeres in late telophase/early G1 of the cell cycle leads to malsegregation and cell division defects in subsequent cell cycles. Mounting evidence supports the hypothesis that an RNA component is involved, although how RNAs participate in centromere formation in mammals has remained unknown. Using the marsupial model, the tammar wallaby, we show that centromeric retroelements produce small RNAs and that hypermorphic expression of these centromeric small RNAs results in disruption of CENP-A localization. We propose that tight regulation of the processing of this new class of small RNAs, crasiRNAs, is an integral component of the epigenetic framework necessary for centromere establishment.
BackgroundSmall 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).ResultsUsing 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.ConclusionsThis 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.
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