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Hypermorphic expression of centromeric retroelement-encoded small RNAs impairs CENP-A loading

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Abstract

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.

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Abbreviations

CENP:

Centromere protein

siRNA:

Small interfering RNA

RNAi:

RNA interference

dsRNA:

Double-stranded RNA

KERV:

Kangaroo endogenous retrovirus

kLTR:

KERV long terminal repeat

crasiRNA:

Centromere repeat-associated short interacting RNA

H3:

Histone 3

miRNA:

MicroRNA

piRNA:

Piwi interacting RNA

IVT:

In vitro transcription

IC:

Immunochemistry

FITC:

Fluorescein isothiocyanate

N.A.:

Numerical aperture

FACS:

Fluorescence activated cell sorting

RISC:

RNA-induced silencing complex

LNA:

Locked nucleic acid

miSAT:

Minor satellite

LINE:

Long interspersed nuclear element

HAC:

Human artificial chromosome

HJURP:

Holliday junction recognition protein

ACA:

Anti-centromere antibody

PKR:

Protein kinase R

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Acknowledgments

We thank the Center for Applied Genetics and Technology for instrumentation, S. Kasowitz for xlr3b dsRNA and A. Pask and M. Renfree for tammar material. A special thanks to A. Pask and B. Mellone for helpful discussion and comments on the manuscript and B. Mellone and I. Oderberg for SoftWoRx quantification protocols. This work was supported by grants from the UCRF and NSF to RJO.

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Authors and Affiliations

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

    Dawn M. Carone, Chu Zhang, Laura E. Hall, Craig Obergfell, Michael J. O’Neill & Rachel J. O’Neill

  2. Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA, 01655, USA

    Dawn M. Carone

  3. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, 01655, USA

    Benjamin R. Carone

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

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Carone, D.M., Zhang, C., Hall, L.E. et al. Hypermorphic expression of centromeric retroelement-encoded small RNAs impairs CENP-A loading. Chromosome Res 21, 49–62 (2013). https://doi.org/10.1007/s10577-013-9337-0

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