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 bodies. How these subnuclear domains are established and maintained is not well understood. 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 mRNAs. 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.

Figure 1. Immobilization of protein components fails to assemble paraspeckles

a, Tethering PSP1 protein to a specific locus (arrow) in the C2C12 nucleus recruited p54nrb, but not Men ε/β ncRNAs, PSP1, PSF, or PSP2 protein therefore failing to form paraspeckles. Scale bar, 5 μm. b, Quantification of protein tethering experiments shows that none of the immobilized paraspeckle proteins can initiate the formation of paraspeckles (n>50 cells in each condition). c, Co-IP experiments show that paraspeckle proteins can interact with each other in the absence of RNA.

Figure 2. Men ε/β transcription induces de novo formation of functional paraspeckles

a, Schematic representation of an inducible system to visualize expression of the Men ε/β reporter ncRNAs and recruitment of paraspeckle proteins. A LacO array, a tetracycline response element (TRE) array, and 24 MS2 stem loop repeats are placed upstream of the Men ε/β gene allowing visualization of the locus by expression of ECFP-LacI. Transcription is initiated by the addition of DOX and nascent transcripts are visualized through binding of EYFP-MS2 to the MS2 stem loops. Not drawn to scale. b, Live-cell imaging shows that transcriptional induction of Men ε/β ncRNAs initiated de novo formation of paraspeckles labeled by PSP1 (arrow). Scale bars, 5 μm. c, De novo formed paraspeckles (arrow) can retain inverted repeat-containing mRNAs, similar to the endogenous paraspeckles (arrowhead), confirming that they are bona fide functional paraspeckles. d, Quantification shows that Men ε/β transcription initiated the recruitment of different paraspeckle proteins, but not SC35 and SF2/ASF, nuclear speckle RNA binding proteins; and resulted in the retention of different inverted repeat containing mRNAs, but not mCat2 mRNA (n>50 cells in each condition).

Figure 3. Maintenance of paraspeckles depends on active Men ε/β transcription

a, Left, paraspeckles, labeled by PSP1 antibody, were often found localized adjacent to Men ε/β gene loci, but not to Ctn RNA gene loci, in interphase nuclei of C2C12 cells. Scale bars, 5 μm. Right, quantification of the distance between paraspeckles and Men ε/β gene and Ctn RNA gene loci (n=100 paraspeckles in 20 cells each, mean±s.e.m.). b, Left, paraspeckle protein PSP1 redistributed to perinucleolar caps (arrowhead) upon transcriptional inhibition by DRB treatment and paraspeckles reassembled at the same location (arrow) after DRB wash-out. Right, qRT-PCR analysis shows Men ε/β transcript level upon DRB treatment (n=4, mean±s.e.m.). Note that 60-min DRB treatment did not affect the Men ε/β transcript level, but paraspeckles already disassembled at this time point. c, Withdrawing DOX to switch off Men ε/β transcription disassembled de novo formed paraspeckle (arrow) but had no effect on endogenous paraspeckles (arrowhead) demonstrating that the maintenance of paraspeckles is coupled with Men ε/β transcription.

Figure 4. Dynamic behavior of paraspeckles by live-cell imaging

a, Dynamics of paraspeckles through the cell cycle: both endogenous (arrowhead) and de novo formed paraspeckles (arrow) disassembled during mitosis, and re-assembled at the Men ε/β loci in the daughter nuclei during next G1 phase. Scale bars, 5 μm. b, Budding and splitting of paraspeckles during interphase: a paraspeckle assembled upon DOX induction at the Men ε/β locus, increased in size and formed clusters of paraspeckles.

Figure 5. Differential kinetics of Men ε/β ncRNAs and paraspeckle proteins

a, FRAP analyses of paraspeckles. Paraspeckle proteins exhibit a rapid exchange rate, while Men ε/β ncRNAs exhibit a much slower exchange. Top, cells expressing mCherry-PSP1 and EYFP-MS2 labeling Men ε/β ncRNAs were imaged before and after bleaching of endogenous (arrowhead) or de novo formed (arrow) paraspeckles. Scale bar, 5 μm. Bottom, kinetics of recovery of paraspeckle proteins after bleaching in nucleoplasm (blue cross), endogenous (open red circle) or de novo formed paraspeckles (solid black circle), and of Men ε/β ncRNAs (square) (n=8 for PSP1 and p54nrb, n=5 for PSF, and n=12 for Men ε/β ncRNAs, mean±s.e.m.). Intensity was normalized as such that the first time point pre-bleach was set as 1 and the first time point post-bleach was set as 0. Note that the scale of the X-axis of Men ε/β ncRNAs recovery profile is different from those of paraspeckle proteins. b, Protein dynamics during paraspeckle formation. Top, the intensity of EYFP-MS2 and mCherry-fused paraspeckle proteins at the transcription site was quantified and normalized over time. Bottom, kinetics of first 40 minutes after DOX induction is shown. Within the temporal resolution (5 min), all paraspeckle proteins examined were found to be recruited to newly formed paraspeckles instantly upon the detection of Men ε/β transcription.