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Subnuclear targeting of the RNA-binding motif protein RBM6 to splicing speckles and nascent transcripts

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Abstract

RNA-binding motif (RBM) proteins comprise a large family of RNA-binding proteins whose functions are poorly understood. Since some RBM proteins are candidate alternative splicing factors we examined whether one such member of the family, RBM6, exhibited a pattern of nuclear distribution and targeting consistent with this role. Using antibodies raised against mouse RBM6 to immmunostain mammalian cell lines we found that the endogenous protein was both distributed diffusely in the nucleus and concentrated in a small number of nuclear foci that corresponded to splicing speckles/interchromatin granule clusters (IGCs). Tagged RBM6 was also targeted to IGCs, although it accumulated in large bodies confined to the IGC periphery. The basis of this distribution pattern was suggested by the targeting of tagged RBM6 in the giant nuclei (or germinal vesicles (GVs)) of Xenopus oocytes. In spread preparations of GV contents RBM6 was localized both to lampbrush chromosomes and to the surface of many oocyte IGCs, where it was confined to up to 50 discrete patches. Each patch of RBM6 labelling corresponded to a bead-like structure of 0.5–1 μm diameter that assembled de novo on the IGC surface. Assembly of these novel structures depended on the repetitive N-terminal region of RBM6, which acts as a multimerization domain. Without this domain, RBM6 was no longer excluded from the IGC interior but accumulated homogeneously within it. Assembly of IGC-surface structures in mammalian cell lines also depended on the oligomerization domain of RBM6. Oligomerization of RBM6 also had morphological effects on its other major target in GVs, namely the arrays of nascent transcripts visible in lampbrush chromosome transcription units. The presence of oligomerized RBM6 on many lampbrush loops caused them to appear as dense structures with a spiral morphology that appeared quite unlike normal, extended loops. This distribution pattern suggests a new role for RBM6 in the co-transcriptional packaging or processing of most nascent transcripts.

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Abbreviations

CB:

Cajal body

CELF1:

CUG-BP1 and ETR-3 Like Factor 1

DRB:

5,6-dichloro-1-β-d-ribofuranosylbenzimidazole

GV:

Germinal vesicle

IGC:

Interchromatin granule cluster

LBC:

Lampbrush chromosome

pol II:

RNA polymerase II

RBM6:

RNA-binding motif protein 6

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Acknowledgements

We are grateful to Rivka Dikstein for RBM6 antibodies and to Archa Fox and Angus Lamond for help with the analysis of paraspeckles. We also thank Tim Self for assistance with confocal microscopy and the MRC for a studentship to support EH.

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  1. Emma Heath

    Present address: Wellcome Trust Centre for Stem Cell Research, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QN, UK

Authors and Affiliations

  1. Centre for Genetics and Genomics, School of Biology, University of Nottingham, Queen’s Medical Centre, Nottingham, NG7 2UH, UK

    Emma Heath, Fred Sablitzky & Garry T. Morgan

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  1. Emma Heath
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Correspondence to Garry T. Morgan.

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Heath, E., Sablitzky, F. & Morgan, G.T. Subnuclear targeting of the RNA-binding motif protein RBM6 to splicing speckles and nascent transcripts. Chromosome Res 18, 851–872 (2010). https://doi.org/10.1007/s10577-010-9170-7

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