Article

Rapid, Diffusional Shuttling of Poly(A) RNA between Nuclear Speckles and the Nucleoplasm

Department of Biochemistry, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Molecular Biology of the Cell (Impact Factor: 4.47). 04/2006; 17(3):1239-49. DOI: 10.1091/mbc.E05-10-0952
Source: PubMed

ABSTRACT

Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22 degrees C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy.

Download full-text

Full-text

Available from: Kevin Fogarty
  • Source
    • "Directly studying the dynamics of nuclear components, such as mRNAs in the nucleus of a living cell, will help to define the rules that govern the kinetics, locations, and interactions of proteins and nucleic acids relative to nuclear structure. Advanced microscopy techniques have improved image resolution or enabled fast tracking of individual molecules in living cells, allowing the nuclear mobility of different proteins, RNAs, and other molecules to be probed (Görisch et al., 2004; Shav-Tal et al., 2004; Politz et al., 2006; Grünwald et al., 2008). Currently available single-molecule imaging methods share the limitation that they can only image fast enough to accurately track single molecules in one optical plane (2D) or their 3D capability only allows visualization of small numbers of molecules within a limited field of view (Ragan et al., 2006; Huang et al., 2008; Backlund et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Imaging single proteins or RNAs allows direct visualization of the inner workings of the cell. Typically, three-dimensional (3D) images are acquired by sequentially capturing a series of 2D sections. The time required to step through the sample often impedes imaging of large numbers of rapidly moving molecules. Here we applied multifocus microscopy (MFM) to instantaneously capture 3D single-molecule real-time images in live cells, visualizing cell nuclei at 10 volumes per second. We developed image analysis techniques to analyze messenger RNA (mRNA) diffusion in the entire volume of the nucleus. Combining MFM with precise registration between fluorescently labeled mRNA, nuclear pore complexes, and chromatin, we obtained globally optimal image alignment within 80-nm precision using transformation models. We show that β-actin mRNAs freely access the entire nucleus and fewer than 60% of mRNAs are more than 0.5 µm away from a nuclear pore, and we do so for the first time accounting for spatial inhomogeneity of nuclear organization. © 2015 Smith et al.
    Full-text · Article · May 2015 · The Journal of Cell Biology
  • Source
    • "Thus, human Hub1 is able to associate with splicing speckles also independently of Snu66. This finding supports the above observation (Figure 3E) that binding of Hub1 to Snu66 is not essential for Hub1 function, and suggests that other surfaces of Hub1 may contribute to splicing factor association. Splicing speckles are typically highly dynamic as some of their protein and RNA content cycle continuously between speckles, sites of transcription and other nuclear locations (Misteli et al., 1997; Politz et al., 2006; Spector and Lamond, 2011). The splicing Figure 1 Conserved and divergent properties of Hub1. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Different from canonical ubiquitin-like proteins, Hub1 does not form covalent conjugates with substrates but binds proteins non-covalently. In Saccharomyces cerevisiae, Hub1 associates with spliceosomes and mediates alternative splicing of SRC1, without affecting pre-mRNA splicing generally. Human Hub1 is highly similar to its yeast homolog, but its cellular function remains largely unexplored. Here, we show that human Hub1 binds to the spliceosomal protein Snu66 as in yeast; however, unlike its S. cerevisiae homolog, human Hub1 is essential for viability. Prolonged in vivo depletion of human Hub1 leads to various cellular defects, including splicing speckle abnormalities, partial nuclear retention of mRNAs, mitotic catastrophe, and consequently cell death by apoptosis. Early consequences of Hub1 depletion are severe splicing defects, however, only for specific splice sites leading to exon skipping and intron retention. Thus, the ubiquitin-like protein Hub1 is not a canonical spliceosomal factor needed generally for splicing, but rather a modulator of spliceosome performance and facilitator of alternative splicing.
    Full-text · Article · May 2014 · Journal of Molecular Cell Biology
  • Source
    • "Indeed, photobleaching studies in living cells have shown that nuclear speckles concentrate a population of mobile poly(A) RNAs in continuous flux with the nucleoplasm (Molenaar et al., 2004; Politz et al., 2006), suggesting that a fraction of these polyadenylated RNAs are involved in post-transcriptional processing of pre-mRNAs before their nuclear export (reviewed in Hall et al., 2006; Melcák et al., 2000, 2001). In this context, we propose that the dynamic aggregation of poly(A) RNA/PABPN1 complexes into growing INIs gradually depletes the nuclear speckles of these molecular components, thereby interfering with the normal trafficking and post-transcriptional processing of polyadenylated mRNAs in speckles. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nuclear speckles are essential nuclear compartments involved in the assembly, delivery and recycling of pre-mRNA processing factors, and in the post-transcriptional processing of pre-mRNAs. Oculopharyngeal muscular dystrophy (OPMD) is caused by a small expansion of the polyalanine tract in the poly(A)-binding protein nuclear 1 (PABPN1). Aggregation of expanded PABPN1 into intranuclear inclusions (INIs) in skeletal muscle fibers is the pathological hallmark of OPMD. In this study what we have analyzed in muscle fibers of OPMD patients and in primary cultures of human myoblasts are the relationships between nuclear speckles and INIs, and the contribution of the former to the biogenesis of the latter. While nuclear speckles concentrate snRNP splicing factors and PABPN1 in control muscle fibers, they are depleted of PABPN1 and appear closely associated with INIs in muscle fibers of OPMD patients. The induction of INI formation in human myoblasts expressing either wild type GFP-PABPN1 or expanded GFP-PABPN1-17ala demonstrates that the initial aggregation of PABPN1 proteins and their subsequent growth in INIs occurs at the edges of the nuclear speckles. Moreover, the growing of INIs gradually depletes PABPN1 proteins and poly(A) RNA from nuclear speckles, although the existence of these nuclear compartments is preserved. Time-lapse experiments in cultured myoblasts confirm nuclear speckles as biogenesis sites of PABPN1 inclusions. Given the functional importance of nuclear speckles in the post-transcriptional processing of pre-mRNAs, the INI-dependent molecular reorganization of these nuclear compartments in muscle fibers may cause a severe dysfunction in nuclear trafficking and processing of polyadenylated mRNAs, thereby contributing to the molecular pathophysiology of OPMD. Our results emphasize the potential importance of nuclear speckles as nuclear targets of neuromuscular disorders.
    Full-text · Article · Jan 2012 · Neurobiology of Disease
Show more