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A new class of retroviral and satellite encoded small RNAs emanates from mammalian centromeres

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Abstract

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.

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Acknowledgements

We thank Judy Brown and Andrew Pask for critical evaluation of this manuscript and Marilyn Renfree and Andrew Pask for tammar wallaby material. This work was supported by a grant from the UCONN Research Foundation and an National Science Foundation award to RO.

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Authors and Affiliations

  1. Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, 06269, USA

    Dawn M. Carone, Mark S. Longo, Gianni C. Ferreri, Laura Hall, Melissa Harris, Nicole Shook, Kira V. Bulazel, Benjamin R. Carone, Craig Obergfell & Michael J. O’Neill

  2. Center for Applied Genetics and Technology, Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT, 06269, USA

    Rachel J. O’Neill

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  1. Dawn M. Carone
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Correspondence to Rachel J. O’Neill.

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Communicated by: R. Allshire

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Fig. S1

Calibration for fiber FISH. a M. musculus chromosome 11 BAC (RP23-234K24, accession number AL596181) mapped to fibers (blue gray) of M. musculus; b and c M. eugenii chromosome 6 BAC (ME_Kba-583J6, accession number AC166217) mapped to fibers (blue gray) of M. eugenii. Both probes were detected with avidin–FITC (green) (GIF 98 KB)

High resolution image file (EPS 1.55 MB)

Fig. S2

CREST and CENP-B recognize centromeres of M. eugenii. A partial cell, containing both compact centromeric chromatin and extended centromere chromatin fibers (panel 1, DAPI-stained, blue gray) detected with CREST anticentromere antibodies (panel 2, antihuman Texas Red) and anti-CENP-B (panel 3, antimouse FITC, green). While both antibodies detected compact chromatin (bottom left of panels 2–4), only CREST recognized extended chromatin fibers (indicated with an arrow). The merge of all three wavelengths is shown in panel 4 (GIF 38.3 KB)

High resolution image file (EPS 2.66 MB)

Fig. S3

The CENP-B binding domain of the unbound ChIP product is highly mutated. The unbound ChIP sat23 product obtained from chromatin immunoprecipitation with an antibody to CENP-B contains three 1-bp mismatches (highlighted in bold) compared to the tammar wallaby CENP-B box consensus (top) and sat23 sequence obtained from the bound fraction (CenpB bound sat23; bottom). The bases critical to binding of CENP-B are highlighted in red (Bulazel et al. 2006). The presence of mismatches within this conserved binding domain indicates that the sequence obtained in the unbound fraction has lost its ability to bind CENP-B (Bulazel et al. 2006) and is most likely located more distal to the centromere core (GIF 16.9 KB)

High resolution image file (EPS 687 KB)

Fig. S4

Small RNA are homologous to KERV-1 LTR and KERV-1 gag and sat23. Size-fractionated small RNA pool was dephosphorylated, end-labeled with γ32P, and hybridized to Southern blots containing PCR products corresponding to KERV-1 LTR (lane 1), sat23 (lane 2), and KERV-1 gag (lane 3) indicating that elements residing at the centromere produce small RNA. A noncentromeric sequence (B15) for the tammar wallaby (Bulazel et al. 2007) was included as a negative control (lane 4) (GIF 23.8 KB)

High resolution image file (EPS 2.51 MB)

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Carone, D.M., Longo, M.S., Ferreri, G.C. et al. A new class of retroviral and satellite encoded small RNAs emanates from mammalian centromeres. Chromosoma 118, 113–125 (2009). https://doi.org/10.1007/s00412-008-0181-5

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