Proteomics: the Nucleolus Weighs in Dispatch

Proteomics: The Nucleolus Weighs In Dispatch

Jason K. Ospina and A. Gregory Matera likely occurs near the border of the fibrillar centre and dense fibrillar component; processing of nascent pre- rRNAs takes place in the dense fibrillar component, Numerous studies have indicated that the nucleolus followed by assembly of ribosomal subunits in the is involved in a variety of cellular processes besides granular component [7,8]. its well-known function in ribosome biosynthesis. A Aside from these well-established functions, new recent study describing the nucleolar proteome roles for the nucleolus are surfacing. The occasional opens the way to new avenues of research on this identification of previously unknown nucleolar proteins important nuclear suborganelle. has provided the raw materials for studies concerning novel nucleolar functions [9–11]. Approaches that exploit protein–protein interactions, such as the yeast Recent advances in mass spectrometry, in combina- two-hybrid system and co-immunoprecipitation analy- tion with database analysis [1], have allowed investi- sis, have provided a means for identification of a gators to identify the protein components of extremely modest number of nucleolar proteins. However, iden- complex mixtures, including spliceosomes, interchro- tifying the balance of the nucleolar proteome should matin granule clusters, centrosomes and spindle pole provide a basis for future research into these ancillary bodies, nuclear pore complexes and even the Golgi functions. apparatus. As reported in this issue of Current Biology, As nucleoli are not bounded by membranes, and the Lamond and colleagues [2] have now used this intact structure is not differentially extractable by approach in a proteomic analysis of the human nucle- salts, the usual biochemical fractionation methods do olus. They identified several hundred proteins, many of not pertain. Instead, nucleoli must be detached from which are encoded by novel or uncharacterized genes. the nucleoplasmic milieu by extensive sonication, Furthermore, a subset of these proteins were fused followed by sucrose centrifugation. The purity and with yellow fluorescent protein (YFP) and localised by integrity of the fractions can then be confirmed by fluorescence microscopy. One such protein, described light and electron microscopy at each step of the pro- in a companion paper [3], was found to localise to a cedure. Once a suitably pure fraction has been novel nuclear domain, and to undergo a dramatic reor- obtained, identification of the proteins can proceed. ganization to the nucleolar periphery when transcription is inhibited with actinomycin D. Nucleolar Proteomic Analysis The foundation for proteomics was established some The Nucleolus: a Tough Nut to Crack thirty years ago, with the building of protein databases The nucleolus, first described over a century and a and the practice of two-dimensional gel electrophore- half ago, functions in ribosomal RNA (rRNA) synthesis, sis [1]. The advent of tandem and laser desorption mass processing and ribosome assembly ([4] and refer- spectrometry techniques, combined with the sequenc- ences therein). Nucleoli are thought to play a role in ing of a handful of genomes to create maps of pre- various other activities, such as the maturation of dicted peptide masses, has greatly enhanced the power certain mRNAs, the sequestration of regulatory mol- and applicability of high-throughput biochemistry. ecules and the maturation of the telomerase and Lamond and colleagues [2] tackled the composition signal recognition particle ribonucleoproteins (reviewed of the human nucleolus using the procedure described in [4,5]). The identification of proteins that localise to above. Their analysis of HeLa cell nucleoli resulted in the nucleolus will provide a crucial first step to many the identification of 271 separate proteins, 31% of studies focused on understanding nucleolar function, which are encoded by novel or as yet uncharacterized dynamics and interaction with other subnuclear genes. Existing evidence supports the nucleolar asso- structures. ciation of greater than half of the known proteins iden- The mammalian nucleolus is a large (5–10 µm) tified from the proteomic study. This group of proteins structure that disassembles in late prophase and is enriched in RNA-modifying enzymes, ribosomal pro- reforms in telophase [6]. Nucleoli form in response to teins and factors involved in the transcription of rRNA transcription of ribosomal DNA (rDNA) repeats that are genes. Numerous other nucleic acid and nucleotide- often tandemly arrayed in so-called nucleolar organizer binding proteins, DEAD box proteins, kinases, chaper- regions (NORs). There are three distinct subdomains ones and translation factors were also identified. With within nucleoli, called the dense fibrillar component, such a diverse assortment of associated proteins, it granular component and fibrillar centre. Transcription seems likely that we have only scratched the surface of nucleolar functionality. Department of Genetics, Center for Human Genetics, Center for RNA Molecular Biology and Program in Cell Biology, Case Paraspeckles Western Reserve University and University Hospitals of Once you have done all this work, why not do it again Cleveland, 10900 Euclid Avenue Cleveland, Ohio 44106-4955, under different conditions? Further investigation USA. E-mail: [email protected] revealed that actinomycin D treatment resulted in Dispatch R30

paraspeckles function in transcription regulation, but should not bias the focus of future investigations. The fact that PSP1 was not detected in HeLa nucleoli under normal conditions by immunofluorescence or YFP overexpression was surprising, given that PSP1 was detectable by mass spectrometry in untreated nucleoli. This apparent paradox was resolved through Untreated Actinomycin D the use of ‘fluorescence loss in photobleaching’ (FLIP) Paraspeckles PSP1 experiments. By successive bleaching of the nucleo- CB lus, PSP1 was depleted from the nucleoplasmic Nucleolus paraspeckles. Thus, in transcriptionally active cells, PSP1 cycles between nucleoli and paraspeckles. Speckles Coilin Nucleolus Nucleoplasm Nucleolar Cap Formation Cytoplasm Interestingly, the nucleolar redistribution of paraspeckle proteins following transcriptional inhibition leads to their localization in perinucleolar caps (Figure 1). The PSP1-containing cap-like domains contain PSP2 and p54/nrb, and are distinct from the Current Biology caps that form under similar conditions and contain either TLS/FUS and associated proteins [15] or coilin Figure 1. Paraspeckles. and fibrillarin, the Cajal body signature protein and Paraspeckles represent novel nuclear domains that are often in box C/D snoRNP methylase, respectively [16]. At least close proximity to splicing factor compartments known as for coilin and fibrillarin, nucleolar cap accumulation is speckles. Paraspeckle domains are distinct from other nuclear detectable in wild-type mouse tissues [17]. Addition- suborganelles, such as Cajal bodies (CBs). In transcriptionally ally, the proteins found in nucleolar caps are simulta- active cells (left), PSP1 is enriched in paraspeckles and cycles between paraspeckles and nucleoli. Upon treatment with actin- neously found in both nucleoli and at least one other omycin D (right), PSP1 and coilin relocate to distinct perinucle- nuclear domain under normal conditions. These olar caps, whereas speckles remain in the nucleoplasm. observations provide evidence that accumulation in nucleolar caps is not, per se, an artifact of global tran- substantial nucleolar enrichment of eleven of the iden- scription inhibition and thus reflects a normal cellular tified proteins, further illustrating the dynamic nature process. of the nucleus [12]. Surprisingly, YFP-tagging of a The nucleolus is comprised of a surprisingly diverse subset of these eleven proteins failed to detect their array of proteins, whose investigation is sure to presence in the nucleolus. Immunofluorescence con- uncover novel functions. Nucleolar proteomic analy- firmed the YFP overexpression data for one of these ses such as those described here, will provide a foun- factors, called ‘paraspeckle protein 1’ (PSP1), and dation for many other investigations concerning revealed the existence of a novel nuclear subdomain, nucleolar functions and may even provide clues to the para-speckle. These domains are enriched in novel ribosome-associated factors. Additionally, the PSP1 and are distinct from nucleoli, Cajal bodies, identification of a novel nuclear domain, the gems, perinucleolar compartments, PML bodies, splic- paraspeckle, illustrates the power and applicability of ing factor domains (speckles) as well as the major proteomic analysis under different cellular conditions. sites of transcription [3]. As the name paraspeckle Elucidation of paraspeckle function and its relation- suggests, one of the interesting things about the local- ship to other nuclear subdomains should provide isation pattern of PSP1 is that paraspeckle domains additional insight into the functional organization of are often juxtaposed to splicing factor domains the nucleus. (Figure 1). Two other proteins that are enriched in nucleolar References fractions following actinomycin D treatment also 1. Pandey, A., and Mann, M. (2000). Proteomics to study localised to paraspeckles. One is a novel protein and genes and genomes. Nature 405, 837–846. 2. Andersen, J.S., Lyon, C.E., Fox, A., Leung, A., Lam, Y., was thus dubbed PSP2, while the other was previ- Steen, H., and Mann, M. (2002). Directed proteomic analy- ously identified as p54/nrb [4,13]. All three proteins sis of the human nucleolus. Curr. Biol., this issue. contain RNA-binding domains, but little else is known 3. Fox, A., Lam, Y., Leung, A., Lyon, C.E., Andersen, J.S., and about the function of PSP1. Subsequent to its identi- Mann, M. (2002). Paraspeckles: a novel nuclear domain. fication in the proteomic study, another group isolated Curr. Biol., this issue. PSP2 as a transcriptional activator that interacts with 4. Olson, M.O., Dundr, M., and Szebeni, A. (2000). The nucle- the thyroid hormone receptor binding protein [13]. olus: an old factory with unexpected capabilities. Trends Cell Biol. 10, 189–196. Furthermore, p54/nrb is proposed to be involved in 5. Pederson, T. (1998). The plurifunctional nucleolus. Nucleic transcriptional corepression by binding to the DNA- Acids Res. 26, 3871–3876. binding domains of steroid hormone receptors [14]. 6. Shaw, P.J., and Jordan, E.G. (1995). The nucleolus. Annu. These observations make it tempting to speculate that Rev. Cell Dev. Biol. 11, 93–121. Current Biology R31

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