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Direct visualization of the co-transcriptional assembly of a nuclear body by noncoding RNAs

Abstract

The cell nucleus is a highly compartmentalized organelle harbouring a variety of dynamic membraneless nuclear bodies1,2,3,4. How these subnuclear domains are established and maintained is not well understood5,6,7,8. Here, we investigate the molecular mechanism of how one nuclear body, the paraspeckle, is assembled and organized. Paraspeckles are discrete ribonucleoprotein bodies found in mammalian cells and implicated in nuclear retention of hyperedited mRNAs9,10,11. We developed a live-cell imaging system that allows for the inducible transcription of Men ɛ/β (also known as Neat1; ref. 12) noncoding RNAs (ncRNAs) and the direct visualization of the recruitment of paraspeckle proteins. Using this system, we demonstrate that Men ɛ/β ncRNAs are essential to initiate the de novo assembly of paraspeckles. These newly formed structures effectively harbour nuclear-retained mRNAs confirming that they are bona fide functional paraspeckles. By three independent approaches, we show that it is the act of Men ɛ/β transcription, but not ncRNAs alone, that regulates paraspeckle maintenance. Finally, fluorescence recovery after photobleaching (FRAP) analyses supported a critical structural role for Men ɛ/β ncRNAs in paraspeckle organization. This study establishes a model in which Men ɛ/β ncRNAs serve as a platform to recruit proteins to assemble paraspeckles.

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Figure 1: Immobilization of protein components fails to assemble paraspeckles.
Figure 2: Men ɛ/β transcription induces de novo formation of functional paraspeckles.
Figure 3: Maintenance of paraspeckles depends on active Men ɛ/β transcription.
Figure 4: Dynamic behaviour of paraspeckles by live-cell imaging.
Figure 5: Differential kinetics of Men ɛ/β ncRNAs and paraspeckle proteins.

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Acknowledgements

We thank J. Caceres, G. G. Carmichael, L.-L. Chen, Y. Kurihara, and A. I. Lamond for reagents, S. Hearn and Z. Lazar for assistance in microscopy, C. Berasain, M. Bodnar, M. Eckersley-Maslin, M. Huebner, I. R. Kumaran, J. Li, E. Reis, J. E. Wilusz, and R. Zhao of the Spector laboratory for discussions and comments. Y.S.M. is supported by a National Cancer Center Postdoctoral Fellowship. B.Z. is supported by a Department of Defense Prostate Cancer Research Program Postdoctoral Fellowship. This work was supported by grants to D.L.S. from NIH (NIGMS 42694 and 5PO1CA013106-38).

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Y.S.M., H.S., and D.L.S. designed the experiments. Y.S.M., H.S., and B.Z. performed experiments and analysed data. Y.S.M. and D.L.S. wrote the paper.

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Correspondence to David L. Spector.

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Mao, Y., Sunwoo, H., Zhang, B. et al. Direct visualization of the co-transcriptional assembly of a nuclear body by noncoding RNAs. Nat Cell Biol 13, 95–101 (2011). https://doi.org/10.1038/ncb2140

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