Nuclear Speckles - Lamond Lab

1 Nuclear SpecklesDavid L. Spector1and Angus I. Lamond21 Cold Spring Harbor Laboratory, One Bungtown Road, Cold Spring Harbor, New York 117242 Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee,MSI/WTB/JBC Complex Dow Street, Dundee DD1 5EH, United Speckles , also known as interchromatin granule clusters, are Nuclear domainsenriched in pre-mRNA splicing factors, located in the interchromatin regions of the nucleo-plasm of mammalian cells. When observed by immunofluorescence microscopy, theyusually appear as 20 50 irregularly shaped structures that vary in size.

2 Speckles aredynamic structures, and theirconstituents can exchange continuously with the nucleoplasmand other Nuclear locations, including active transcription sites. Studies on the composition,structure, and dynamics of Speckles have provided an important paradigm for understandingthe functional organization of the nucleus and the dynamics of the gene mammalian cell nucleus is a highly com-partmentalized yet extremely dynamic org-anelle (reviewed in Misteli 2001a; Spector2006; Zhao et al. 2009). Many Nuclear factorsare localized in distinct structures, such asspeckles, paraspeckles, nucleoli, Cajal bodies,polycomb bodies, and promyelocytic leukemiabodies and show punctate staining patternswhen analyzed by indirect immunofluorescencemicroscopy (reviewed in Lamond et al.)

3 1998;Spector 2001; Spector 2006).In mammalian cells the pre-mRNA splicingmachinery, including small Nuclear ribonucleo-protein particles (snRNPs), spliceosome sub-units, and other non-snRNP protein splicingfactors, shows a punctate Nuclear localizationpattern that is usually termed a speckled pat-tern but has also been referred to as SC35 do-mains (Wansink et al. 1993) or splicing factorcompartments (Phair et al. 2000) (Figs. 1 and2). The first detailed description of the nucleardomains that we presently refer to as nuclearspeckles was reported by Santiago Ramo nyCajal in 1910 (Ramo n y Cajal 1910; reviewedin Lafarga et al.

4 2009). Ramo n y Cajal usedacid aniline stains to identify structures hereferred to as grumos hialinas (literally tran-slucent clumps ). The term Speckles was firstput forth in 1961 by J. Swanson Beck (Beck1961) upon examination of rat liver sectionsimmunolabeled with the serum of individualswith autoimmune disorders. Although the con-nection was not made at the time, these speckleshad been identified two years earlier by HewsonSwift (Swift 1959) at the electron microscopiclevel and called interchromatin particles. Swiftobserved that these particles were not randomlydistributed but that they occurred in localizedEditors: Tom Misteli and David L.

5 SpectorAdditional Perspectives on The Nucleus available at #2011 Cold Spring Harbor Laboratory Press; all rights reserved; doi: this article asCold Spring Harb Perspect Biol2011;3:a0006461 clouds, and cytochemical analysis indicatedthat they contained RNA (Swift 1959). However,the first link between pre-mRNA splicingand Nuclear Speckles or interchromatin gran-ule clusters came from an examination of thedistribution of snRNPs using anti-splicingfactor-specific antibodies, demonstrating aspeckled distribution pattern of snRNPs in cellnuclei (Lerner et al.)

6 1981; Perraud et al. 1979;Spector et al. 1983).It is now clear that much of the punctatelocalization of splicing factors observed by im-munofluorescence microscopy corresponds tothe presence of these factors in Nuclear specklesof variable size and irregular shape that arerevealed by electron microscopy as inter-chromatin granule clusters (IGCs) (Fig. 3). IGCsrange in size from one to several micrometersin diameter and are composed of 20 25 nmgranules that are connected in places by a thinfibril resulting in a beaded chain appearance(Thiry 1995b). These structures can be observedby electron microscopy without antibody la-beling (Thiry 1995b).

7 We will define Speckles here specifically as the IGC component of thesplicing factor labeling pattern, and distinguishthis from other Nuclear structures, includingperichromatin fibrils and Cajal bodies, whichalso contain splicing factors (reviewed in Fakan1994; Spector 1993).For some of the speckle components aspeckle targeting signal has been arginine/serine-rich domain (RS domain)of some SR pre-mRNA splicing factors has beenshown to be necessary and sufficient for the tar-geting of these factors to Nuclear Speckles (Caceres et al. 1997; Hedley et al.)

8 1995; Liet al. 1991). In addition, the threonine-prolinerepeats of SF3b1 (Eilbracht et al. 2001) andthe forkhead-associated domain in NIPP1(Jagiello et al. 2000) have also been implicatedin speckle-targeting. Most recently, Salichset al. (Salichs et al. 2009) performed a genome-wide analysis of homopolymeric histidine tractsand identified 86 human proteins that containstretches of five or more histidines. Of the 22 Figure form in the interchromatin cells showing splicing factors localized in aspeckled pattern as well as being diffusely distributedthroughout the nucleoplasm.

9 Bar illumination microscopy, usingthe OMX system (Applied Precision, Issaqua,Washington), of a HeLa cell expressing 100 nm resolution substructure can be observedwithin Speckles . In addition, the diffuse populationof SC35-EYFP is resolved as a granular of twelve optical sectionsthrough the center of a nucleus encompassing Image provided by Zsolt Lazar and R. IlengKumaran. Bar Spector and Lamond2 Cite this article asCold Spring Harb Perspect Biol2011;3:a000646that were Nuclear localized, 15 were shown to bepresent in Nuclear Speckles .

10 Based on these data,the polyHis-repeats were proposed to act as aspeckle-targeting signal that functions by actingas an interaction surface for resident nuclearspeckle constituents. Interestingly, these target-ing signals rely mostly on charge effects, beingbasic protein , structures similar to nuclearspeckles have been identified in the amphibianoocyte nucleus (Gall et al. 1999) and inDro-sophila melanogasterembryos when transcrip-tion increases upon cellularization duringcycle 14 (Segalat et al. 1992), but not in yeast(Potashkin et al. 1990). Importantly, not allnuclear proteins that show a speckle-like label-ing pattern by immunofluorescence microscopylocalize to IGCs.