Article |
Address correspondence to Thoru Pederson, University of Massachusetts Medical School, 377 Plantation St., Worcester, MA 01605. Tel.: (508) 856-8667. Fax: (508) 856-8668. E-mail: thoru.pederson{at}umassmed.edu
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: nucleolus; signal recognition particle; SRP RNA; ribosome synthesis; peptide nucleic acids
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The significance of SRP components in the nucleolus is not presently understood. A plausible explanation is that the SRP is at least partially assembled in the nucleolus. In addition, because mature SRP interacts with cytoplasmic ribosomes, SRP assembly may be coordinated with ribosome assembly within the nucleolus, perhaps as a mutual quality control mechanism assuring proper assembly of each particle (Pederson and Politz, 2000). Because the tripartite structural organization of the nucleolus has been so extensively studied and defined in relation to the steps of ribosome biosynthesis (Goessens, 1984; Hadjiolov, 1985; Hernandez-Verdun, 1991; Spector, 1993; Shaw and Jordan, 1995; Scheer and Hock, 1999; Huang, 2002), we reasoned that an important step toward defining the role of SRP RNA in the nucleolus, and any potential interaction with ribosomal components, would be to determine the precise sites within the nucleolus at which it is localized.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
UBF binds upstream of the rDNA promoters, and is thought to bind both actively transcribing genes as well as those open for transcription (Junera et al., 1997). As shown in Fig. 2 (AF), SRP RNA only minimally overlapped with sites in NRK nucleoli that were immunostained for UBF. Similarly low levels of colocalization were also observed in HeLa cells expressing GFP-UBF to mark the fibrillar centers (Fig. 2, GL). Next, three-dimensional optical stacks of immunostained, in situhybridized cells were processed using a constrained interactive deconvolution algorithm (Swedlow et al., 1997; Wallace et al., 2001) to obtain a higher resolution map of the intranucleolar space occupied by SRP RNA and UBF. Again, at this refined resolution, only a limited amount of overlap at the edges of a few fibrillar centers was observed (Fig. 3).
|
|
|
|
Using constrained iterative deconvolution to increase the resolution of subnucleolar regions, the intensity distribution of SRP RNA signal (Fig. 6, red) was found to clearly differ from that of B23 (Fig. 6 F, green). Specifically, the most concentrated regions of SRP RNA (Fig. 6, G and H, red peaks in linescans) often did not overlap with the most concentrated regions of B23 (Fig. 6, G and H, green peaks in linescans). Although the fraction of SRP RNA that colocalized with B23 varied among nucleoli in the same cell and between cells, all nucleoli contained some SRP RNA signal that was concentrated in intranucleolar regions where B23, fibrillarin, and UBF were least concentrated.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The major goal of the present study was to resolve the intranucleolar distribution of SRP RNA within the nucleolus, particularly in relation to the stations of ribosome synthesis as defined by the classical tripartite organization of the nucleolus. Because we have hypothesized that SRP RNA in the nucleolus is related to the regulated construction of the overall translation machinery (Pederson and Politz, 2000), we wished to determine whether SRP RNA might be uniquely present in one, and only one, of the three nucleolar compartments, as a clue to its possible role at a discrete step in ribosome biosynthesis. In the present study we have used the RNA polymerase Ispecific transcription factor UBF to demarcate the fibrillar centers, the protein fibrillarin as a fiduciary landmark of the DFC of the nucleolus, and the protein B23 to identify the rRNA containing regions of the granular component. In all three cases, we used both specific antibodies as well as fluorescent protein expression to identify these components of the nucleolus, combined with the highly sensitive detection of SRP RNA. Additionally, we performed dual in situ hybridization experiments to resolve the spatial relationship between 28S rRNA and SRP RNA. We found that very little SRP RNA was present in the fibrillar centers and the DFC. Rather, a portion of the SRP RNA was localized with B23 in the granular component and, surprisingly, the remainder was concentrated in rRNA deficient regions of the nucleolus.
The intranucleolar distribution pattern of SRP RNA observed in the present investigation renders unlikely a model where the SRP RNA is intimately associated with precursor ribosomes throughout their synthesis and assembly. SRP RNA is not concentrated in either fibrillar centers or the DFC and therefore it probably does not play a role during rRNA transcription or early processing in any direct, interactive fashion, although it remains possible that very low concentrations of SRP RNA might carry out functional interactions with formative ribosomes at these sites.
Rather, our results indicate that SRP RNA might interact with nucleolar ribosomes much later during assembly, perhaps within the B23-rich portion of the granular component. However, the SRP RNA that was localized within the granular component displayed a heterogeneous distribution and was not closely correlated with the abundance of B23 protein. The most concentrated regions of SRP RNA were often not coincident with concentrated regions of B23 protein, indicating that portions of SRP RNA and B23 protein have different locations within the granular component.
Studies at the electron microscopic level have generally conveyed the granular component as having a fairly compact and generally homogenous particulate appearance (for review see Hadjiolov, 1985). A protein termed No55 identified by Ochs et al. (1996) using a rare human patient autoimmune serum, and not presently known to be involved in ribosome biosynthesis, was found to be distributed fairly homogeneously throughout the granular component. However, B23, which is involved in a rRNA processing step (Savkur and Olson, 1998; Okuwaki et al., 2002), exhibits a somewhat uneven distribution within the granular component (Ochs et al., 1996; unpublished data). Our finding that a substantial portion of the SRP RNA is present in regions at which B23 is not concentrated (and yet are not DFC) may indicate that within the granular component itself, particular activities are spatially organized into different functional domains. Such activities may require different levels of SRP RNA as compared with B23. Indeed, it is not known whether all of the "granules" of the granular component that are visualized by electron microscopy actually represent nascent ribosomal subunits, or whether there are other particles present also. Our results raise the possibility that a portion of the particulate texture of the granular component might in part represent nascent SRPs, and that regions containing these particles are identifiable at the fluorescent light microscopy level.
Consistent with our results showing that SRP RNA is concentrated in regions other than fibrillar centers, the DFC or B23-rich regions of the nucleolus, we also found that SRP RNA was most concentrated in regions of the nucleolus that were deficient in 28S rRNA. This suggests that this portion of SRP RNA may not directly interact with ribosomes during assembly, but instead is sequestered from those areas. To the best of our knowledge, this intranucleolar distribution pattern has not been previously observed, and it raises a number of intriguing issues. Do these SRP RNArich areas have different structural characteristics that can be detected using electron microscopy, now that we are alerted to their existence? What else might colocalize within this domain of the nucleolus? More and more factors apparently unrelated to ribosome biogenesis are being found in the nucleolus, including several growth factors, components of gene silencing machinery, anaphase exit equipment and telomerase (Pederson, 1998a, 1998b; Mitchell et al., 1999; Carmo-Fonseca et al., 2000; Olson et al., 2000; Visintin and Amon, 2000; Wong et al., 2002). These various factors would not necessarily be expected to associate with the nucleolar regions dedicated to ribosome biosynthesis (and indeed might well disrupt ribosome synthesis if they did). Thus, it is conceivable that the SRP RNA-rich areas of the nucleolus discovered in the present study may define territories in which activities other than ribosome biosynthesis predominate. An intriguing possibility, among others, is that the functions taking place in this compartment may nonetheless be coordinated with the ribosome synthesis pathway, perhaps in a cell cycle regulated way.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PNA probes and in situ hybridization
PNA probes complementary to nucleotides 88102, 208221, and 231245 of rat (conserved in human) SRP RNA were synthesized by PE Biosystems. Each PNA was labeled with a rhodamine group at its 5' end. A PNA probe complementary to nucleotides 231245 of Schizosaccharomyces pombe SRP RNA was also synthesized for use as a control (Results). Cy3-labeled PO backbone oligodeoxynucleotides complementary to SRP RNA were as described earlier (oligo 1 is complementary to nucleotides 208240, oligo 2 to 118151, oligo 3 to 249284; Politz et al., 2000).
PO oligos complementary to rat 28S rRNA were as follows (see De Rijk et al., 1999 for database and nomenclature information): Oligo 1 in loop E11_1 (D7b), G*TACCGGCAC*GGACGCC*CGCGGCGCCCA*C; Oligo 2 in loop E9_1 (D-7a), C*GAGGGCAACGGAGGCCA*CGCCCG*CCCT*C; Oligo 3 in loop B13_1 (D1), G*ACGCCACAT*TCCCGCGCC*CGGCGCGCG*C; Oligo 4 in loop C1_1 (D2), C*CGCGCCGCCGGG*TCAATCC*CCGGGCGG*C; and Oligo 5 in loops H1_2, H1_3 (D12), A*GGCTC*CCGCACCGGACCCCGG*CCCGAC*C. *Indicates positions of aminohexyl-modified thymidine residues added during synthesis (Integrated DNA Technologies) and subsequently labeled with fluorescein (Politz and Singer, 1999). These five oligos were used together to detect 28S rRNA.
The methods used for cell fixation, permeabilization and in situ hybridization were as previously described (Politz et al., 2000), except that 3.8 ng of each of the three SRP PNAs (or 11.5 ng of yeast control PNA) in 25 µl total hybridization mixture was used per coverslip. Coverslips were then mounted in Prolong Antifade mounting medium (Molecular Probes) and dried overnight at room temperature before viewing.
Antibodies and immunofluorescence
UBF, a RNA polymerase Ispecific transcription factor specifically localized in fibrillar centers of the nucleolus, was detected with an antibody (Chan et al., 1991; Roussel et al., 1993; Dousset et al., 2000) provided by Daniele Hernandez-Verdun (Institut Jacques Monod, Paris, France). Fibrillarin, a protein specific to the DFC of the nucleolus, was detected with a monoclonal antibody we have previously used (Jacobson et al., 1995), provided by Edward Chan and Eng Tan (Scripps Research Institute, La Jolla, CA). B23, which identifies the granular component, was detected with a monoclonal antibody (Ochs et al., 1983) provided by Pui-Kwong Chan (Baylor College of Medicine, Houston, TX). Immunostaining and sequential in situ hybridization was performed using a minor modification of a protocol provided by Sui Huang (Northwestern University School of Medicine, Chicago, IL). Cells were grown on coverslips and fixed for 12 min in 4% (vol/vol) formaldehyde in PBS, containing 5 mM MgCl2. Fixation and all subsequent steps were performed at room temperature unless otherwise noted. Coverslips were washed three times in PBS containing 1% bovine serum albumin (PBSB) for 510 min each, incubated for 5 min in 0.5% Triton X-100 in PBSB, and then again washed three times in PBSB for 510 min each time. Coverslips were then incubated with the desired primary antibody (1:2,000 for anti-B23, 1:75 for anti-fibrillarin and 1:100 for anti-UBF) in PBSB for 1 h in a humidified chamber, washed three times in PBSB (10 min each), incubated with the appropriate secondary antibody (1:200 antimouse IgG for B23, 1:80 antimouse IgG for fibrillarin and 1:750 antihuman IgG for UBF; all secondary antibodies were from Sigma-Aldrich) in PBSB in humidified chambers for one hour and then washed three times in PBSB (10 min each). Cells were then refixed by immersing the coverslips in 2% (vol/vol) formaldehyde in PBS containing 5 mM MgCl2, for 5 min, followed by three rinses in 70% (vol/vol) ethanol. Coverslips were stored in absolute ethanol at 4°C for 1824 h before performing in situ hybridization as described above.
Fluorescent fusion proteins
Plasmids encoding green fluorescent protein fusions to human UBF and human B23 were obtained from Sui Huang (Northwestern University School of Medicine; Chen and Huang, 2001). A plasmid encoding a yellow fluorescent protein fusion to human fibrillarin was obtained from Angus Lamond (University of Dundee, Dundee, Scotland). These fluorescent protein-encoding plasmids were transfected into NRK cells with Lipofectamine 2000 (Invitrogen Life Technologies) following manufacturer's instructions. In the case of YFP-fibrillarin, a stable cell line was constructed. HeLa cells were transfected as above and 18 h later the medium was replaced with fresh medium containing 800 µg/ml Geneticin (G-418; GIBCO BRL). The medium was changed twice weekly and the cells were cultured for 68 wk before subcloning and further selection.
Microscopy and image processing
Results were analyzed with a Leica DMIRB microscope equipped with a 100x objective (N.A. 1.4) and appropriate filter sets, and images were captured using a Quantix 57 CCD camera (Roper Scientific Photometrics). For high resolution spatial mapping, three-dimensional optical stacks (containing 21 consecutive 0.25 micron slices) were captured using a PIFOC microscope focusing drive (Polytec PI). Images were dark current subtracted, intensity scaled, and in some cases, subjected to two-dimensional nearest-neighbor deconvolution using Metamorph software. Alternatively, image stacks were processed by constrained iterative deconvolution (Applied Precision) using an empirical point-spread function (Swedlow et al., 1997; Wallace et al., 2001).
![]() |
Footnotes |
---|
![]() |
Acknowledgments |
---|
This work was supported by National Institutes of Health grant GM-21595, which requires us to state that the content of our paper is not the official position of the U.S. government.
Submitted: 6 August 2002
Revised: 25 September 2002
Accepted: 25 September 2002
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chan, E.K.L., H. Imai, J.C. Hamel, and E.M. Tan. 1991. Human autoantibody to RNA polymerase I transcription factor hUBF: molecular identity of nucleolus organizer region autoantigen NOR-90 and ribosomal RNA transcription upstream binding factor. J. Exp. Med. 174:12391244.[Abstract]
Chen, D., and S. Huang. 2001. Nucleolar components involved in ribosome biogenesis cycle between the nucleolus and nucleoplasm in interphase cells. J. Cell Biol. 153:169176.
De Rijk, P., E. Robbrecht, S. De Hoog, A. Caers, Y. Van de Peer, and R. De Wachter. 1999. Database on the structure of the large subunit ribosomal RNA. Nucleic Acid Res. 27:174178.
Dousset, T., C. Wang, C. Verheggen, D. Chen, D. Hernandez-Verdun, and S. Huang. 2000. Initiation of nucleolar assembly is independent of RNA polymerase I transcription. Mol. Biol. Cell. 11:27052717.
Grosshans, H., K. Deinert, E. Hurt, and G. Simos. 2001. Biogenesis of the signal recognition particle (SRP) involves import of SRP proteins into the nucleolus, assembly with the SRP-RNA, and Xpo1p-mediated export. J. Cell Biol. 153:745761.
Hadjiolov, A.A. 1985. Cell Biology Monographs. Vol. 12. Springer-Verlag, Vienna/Berlin. 263 pp.
Huang, S. 2002. Building an efficient factory: where is pre-rRNA synthesized in the nucleolus? J. Cell Biol. 157:739741.
Jacobson, M.R., L.-G. Cao, Y.-L. Wang, and T. Pederson. 1995. Dynamic localization of RNase MRP RNA in the nucleolus observed by fluorescent RNA cytochemistry in living cells. J. Cell Biol. 131:16491658.[Abstract]
Jacobson, M.R., and T. Pederson. 1998. Localization of signal recognition particle RNA in the nucleolus of mammalian cells. Proc. Natl. Acad. Sci. USA. 95:79817986.
Junera, H.R., C. Masson, G. Geraud, J. Suja, and D. Hernandez-Verdun. 1997. Involvement of in situ conformation of ribosomal genes and selective distribution of upstream binding factor in rRNA transcription. Mol. Biol. Cell. 8:145156.[Abstract]
Mitchell, J.R., J. Cheng, and K. Collins. 1999. A box H/ACA small nucleolar RNA-like domain at the human telomerase RNA 3' end. Mol. Cell. Biol. 19:567576.
Ochs, R., M. Lischwe, P. O'Leary, and H. Busch. 1983. Localization of nucleolar phosphoproteins B23 and C23 during mitosis. Exp. Cell Res. 146:139149.[Medline]
Ochs, R.L., T.W. Stein, Jr., E.K. Chan, M. Ruutu, and E.M. Tan. 1996. cDNA cloning and characterization of a novel nucleolar protein. Mol. Biol. Cell. 7:10151024.[Abstract]
Okuwaki, M., M. Tsujimoto, and K. Nagata. 2002. The RNA binding activity of a ribosome biogenesis factor, nucleophosmim/B23, is modulated by phosphorylation with a cell cycle-dependent kinase and by association with its subtype. Mol. Biol. Cell. 13:20162030.
Pederson, T. 1998a. Growth factors in the nucleolus? J. Cell Biol. 143:279282.
Pederson, T. 1998b. The plurifunctional nucleolus. Nucleic Acids Res. 26:38713876.
Pederson, T., and J.C. Politz. 2000. The nucleolus and the four ribonucleoproteins of translation. J. Cell Biol. 148:10911095.
Politz, J.C., S. Yarovoi, S. Kilroy, K. Gowda, C. Zwieb, and T. Pederson. 2000. Signal recognition particle components in the nucleolus. Proc. Natl. Acad. Sci. USA. 97:5560.
Reddy, R., W.-Y. Li, D. Henning, Y.C. Choi, K. Nohga, and H. Busch. 1981. Characterization and subcellular localization of 7-8 S RNAs of Novikoff hepatoma. J. Biol. Chem. 25:84528457.
Roussel, P., C. Andre, C. Masson, G. Geraud, and D. Hernandez-Verdun. 1993. Localization of the RNA polymerase I transcription factor hUBF during the cell cycle. J. Cell Sci. 104:327337.
Savkur, R., and M.O.J. Olson. 1998. Preferential cleavage in pre-ribosomal RNA by protein B23 endoribonuclease. Nucleic Acids Res. 26:45084515.
Shaw, P.J., and E.G. Jordan. 1995. The nucleolus. Annu. Rev. Cell Dev. Biol. 11:93121.[CrossRef][Medline]
Spector, D.L. 1993. Macromolecular domains within the cell nucleus. Annu. Rev. Biochem. 9:265315.
Swedlow, J.R., J.W. Sedat, and D.A. Agard. 1997. Deconvolution in optical microscopy. In Deconvolution of Images and Spectra. P.A. Jansson, editor. Academic Press, New York. 284309.
Szebeni, A., and M.O.J. Olson. 1999. Nucleolar protein B23 has molecular chaperone activity. Protein Sci. 8:905912.[Abstract]
Vincent, W.S., and O.L. Miller, Jr. 1966. International Symposium on the Nucleolus. Its Structure and Function. Montevideo, Uruguay. J. Natl. Cancer Inst. Monograph. 23:1610.
Wallace, W., L.H. Schaefer, and J.R. Swedlow. 2001. A working person's guide to deconvolution in light microscopy. Biotechniques. 31:10761097.[Medline]
Wang, J., L.G. Cao, Y.L. Wang, and T. Pederson. 1991. Localization of pre-messenger RNA at discrete nuclear sites. Proc. Natl. Acad. Sci. USA. 88:73917395.[Abstract]