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, 10 (12), 4385-402

Assembly of the Nuclear Transcription and Processing Machinery: Cajal Bodies (Coiled Bodies) and Transcriptosomes

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Assembly of the Nuclear Transcription and Processing Machinery: Cajal Bodies (Coiled Bodies) and Transcriptosomes

J G Gall et al. Mol Biol Cell.

Abstract

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem-loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.

Figures

Figure 1
Figure 1
A model of Cajal body function. The essential feature of the model is the preassembly of RNA transcription and processing complexes in the Cajal body. It is suggested that complexes involving pol I, pol II, and pol III are assembled in the Cajal body before transport to the appropriate genes on the chromosomes. The pol II complexes are stored in the B-snurposomes, where they are visible in electron micrographs as 20- to 30-nm particles, which we call pol II transcriptosomes. It is postulated that similar pol I and pol III transcriptosomes are assembled in the matrix of the Cajal body.
Figure 2
Figure 2
(A) Phase-contrast (left) and fluorescent (right) images of a single lampbrush chromosome with scattered nucleoli, Cajal bodies, and B-snurposomes from a spread preparation of a Xenopus GV. The oocyte had been injected 4.5 h earlier with 20 mM BrUTP (253 ng in 23 nl). Strong BrU label in the lampbrush chromosome U loops is revealed by immunostaining with an anti-BrU antibody (right). Nucleoli are weakly stained, but Cajal bodies and B-snurposomes are completely negative. (B) Single loop from a lampbrush chromosome of the newt Notophthalmus. The oocyte had been injected 1 h earlier with 20 mM BrUTP (253 ng in 23 nl). The left panel shows a phase-contrast image of the loop. The following three panels show staining of the loop axis with an antibody against pol II (mAb H5, green), BrU incorporation (red), and a merge of the two images. Pol II and newly transcribed RNA are precisely colocalized. (C) Differential interference contrast (DIC) and immunofluorescence images of loops from a Notophthalmus lampbrush chromosome after double staining for pol II (mAb H14, green) and the Sm epitope (mAb Y12, red). Pol II occurs only along the axis, whereas splicing snRNPs are present throughout the RNP matrix of the loop. (D) Loop from a Notophthalmus lampbrush chromosome after double staining for pol II (mAb H14, green) and the 77-kDa subunitof the cleavage stimulation factor CstF (red). CstF77 occurs through the matrix of a single “thin-to-thick” transcription unit. (E) The left panel shows the DIC image of a single Cajal body with an attached B-snurposome and an internal B-like inclusion (arrowhead). Nearby are six free B-snurposomes. The following three panels show the same field after staining for the SR protein SC35 (red), the Cajal body marker coilin (green), and a merge of the two images. The B-snurposomes and the inclusion stain strongly for SC35; coilin is limited to the matrix of the Cajal body, which also stains weakly for SC35.
Figure 3
Figure 3
Fluorescent in situ hybridization of splicing snRNAs (U1, U2, U4, U5, and U6) in Cajal bodies, B-snurposomes, and nucleoli from spread GV preparations of Xenopus. Each pair of panels shows the fluorescent image on the left and the corresponding DIC image on the right. CB, Cajal body; N, nucleolus. (A) U1 is weakly detectable in B-snurposomes, but the Cajal body and nucleoli are barely above the background level of unhybridized controls. (B) Truncated U1 accumulates in Cajal bodies of oocytes treated with a deoxyoligonucleotide that removes the cap and first 20 nucleotides from the 5′ end. B-snurposomes and nucleoli are similar to those in untreated oocytes. (C) U2 is strong in B-snurposomes and easily detectable in the matrix of the Cajal body. (D) U4 is strong in B-snurposomes, but the Cajal body and nucleoli are at background level. (E) U5 is strong in B-snurposomes and detectably above background in the matrix of the Cajal bodies. (F) U6 is strong in B-snurposomes and gives the strongest reaction of all the splicing snRNAs in the matrix of the Cajal bodies.
Figure 4
Figure 4
Detection of RNA polymerases and TFIIIA by immunofluorescent staining. Each pair of panels shows the fluorescent image on the left and the corresponding DIC image on the right. CB, Cajal body; N, nucleolus. (A) Stain is brilliant in the matrix of Cajal bodies with a polyclonal antibody against the largest subunit of pol I, RPA194. For unknown reasons the nucleoli are very much weaker, although this antibody stains nucleoli well in HeLa cells. (B) Pol II stains strongly in B-snurposomes with mAb H5, which detects the CTD when serine 2 is phosphorylated (Patturajan et al., 1998), whereas the matrix of the Cajal bodies is only weakly stained. The arrowhead points to stained B-like inclusion in one Cajal body. (C) Pol II stains strongly in the matrix of Cajal bodies with mAb H14, which detects the CTD when serine 5 is phosphorylated (Patturajan et al., 1998), whereas the B-snurposomes are essentially unstained. (D) Pol II stains strongly in the matrix of Cajal bodies with mAb 8WG16, which recognizes the nonphosphorylated CTD (Patturajan et al., 1998). (E) Pol III stains strongly in the matrix of the Cajal bodies with a polyclonal antibody against the 53-kDa subunit, RPC53. (F) Pol III transcription factor TFIIIA is readily demonstrable in Cajal bodies with a polyclonal antibody.
Figure 5
Figure 5
Each pair of panels shows the fluorescent image on the left and the corresponding DIC image on the right. CB, Cajal body; N, nucleolus. (A) Five B-snurposomes and a nucleolus stained with an antipeptide antibody against TFIIF (RAP74). (B) A Cajal body from the same preparation as A, printed at lower contrast. The Cajal body stains much more intensely than its associated B-snurposomes. (C) A Cajal body, nucleoli, and B-snurposomes stained with an antipeptide antibody against the 100-kDa subunit of the cleavage/polyadenylation specificity factor CPSF. The Cajal body stains intensely. (D) A Cajal body, three B-snurposomes, and a nucleolus stained with an antipeptide antibody against the 77-kDa subunit of the cleavage stimulation factor CstF. The Cajal body is the most intensely stained structure.
Figure 6
Figure 6
Electron micrographs of thin sections through nuclear organelles, from a spread preparation of a Xenopus GV fixed in 4% glutaraldehyde and contrasted with OsO4, uranyl acetate, and lead citrate. (A) A nucleolus, a B-snurposome, and a Cajal body with two attached B-snurposomes. (B) A single B-snurposome composed of a relatively uniform aggregation of high-contrast 20- to 30-nm particles, which we call pol II transcriptosomes. (C) Cajal body from A at higher magnification showing that the matrix is heterogeneous in composition. The attached B-snurposomes are identical in appearance to free B-snurposomes. (D) At higher magnification the Cajal body matrix displays scattered 20- to 30-nm particles that look like pol II transcriptosomes, interspersed with numerous larger particles of lower contrast. (E) Boundary between the Cajal body matrix (below) and an attached B-snurposome (above) showing similar high-contrast particles in both compartments.
Figure 7
Figure 7
Spread GV contents from oocytes injected with capped, fluorescein-labeled RNAs. The fluorescent signal was enhanced with goat anti-fluorescein labeled with Alexa 488. Each pair of panels shows the fluorescent image on the left and the corresponding DIC image on the right. CB, Cajal body; N, nucleolus. (A) Two hours after cytoplasmic injection of U2 snRNA, only the matrix of the Cajal bodies is labeled. (B) Twenty-two hours after injection of U2 snRNA, the matrix of the Cajal bodies is still labeled, but now the B-snurposomes are also labeled. (C) Five hours after nuclear injection of U5 snRNA, only the matrix of the Cajal bodies is significantly labeled. (D) Fifteen minutes after nuclear injection of U3 snoRNA, the matrix of Cajal bodies and the dense fibrillar region of the nucleoli are labeled. Individual Cajal bodies and nucleoli vary greatly in intensity.
Figure 8
Figure 8
Distribution of HA-tagged CstF77 in a GV spread, detected with an antibody against the HA tag. The fluorescent image is on the left; the corresponding DIC image is on the right. CB, Cajal body; N, nucleolus; B, B-snurposomes. The oocyte was injected 24 h previously with a transcript synthesized in vitro from a full-length cDNA clone of CstF77. The Cajal body is the most intensely stained structure, but label is detectable in the chromosome and the B-snurposomes.
Figure 9
Figure 9
Nucleolar proteins in the Cajal body. Each pair of panels shows the fluorescent image on the left and the corresponding DIC image on the right. CB, Cajal body; N, nucleolus. (A) Fibrillarin detected with mAb 17C12. The matrices of the Cajal bodies and the nucleoli are equally stained. (B) Nopp140 detected with mAb No114. The matrix of the Cajal body stains more intensely than the dense fibrillar zone of the nucleoli. (C) NO38 (B23) detected with mAb No185. This GV was isolated and spread in the absence of Mg2+. Under these conditions the nucleoli lose much of their granular zone, only the matrix of the Cajal bodies remains intact, and the B-snurposomes disappear. Such partially solubilized Cajal bodies retain their NO38, as shown here, as well as their fibrillarin and Nopp140. (D) Nucleolin detected with mAb P71A4, after Mg2+-free spreading. Nucleolin is readily detectable in the nucleoli but is absent from Cajal bodies.
Figure 10
Figure 10
Diagram of an oocyte Cajal body and a list of its known molecular components. The Cajal body consists of a spherical matrix, a variable number of B-snurposomes attached to its surface, and B-like inclusions.

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