Transcription Nucleoli

Microscopic examination of the mature neutrophils reveals two striking features a single multilobed nucleus and a dense, granular appearance of the cytoplasm (see Fig. 1.1a). The nucleus typically comprises two to four segments, and within this organelle the chromatin is coarsely clumped. Until recently, this abnormal chromatin structure was taken as evidence that the nucleus was transcriptionally inactive however, it is now appreciated that the mature neutrophil does perform active transcription ( 7.3), although rates of biosynthesis are somewhat lower than those observed in cells such as monocytes. There is no detectable nucleolus, so there can be only limited synthesis of ribosomal RNA in these cells. 

Bose S, Basu M, Banerjee AK (2004) Role of nucleolin in human parainfluenza virus type 3 infection of human lung epithelial cells. J Virol 78 8146-8158 Bouche G, Caizergues-Ferrer M, Bugler B, Amalric F (1984) Interrelations between the maturation of a 100 kDa nucleolar protein and pre rRNA synthesis in CHO cells. Nucleic Acids Res 12 3025-3035 Bouche G, Gas N, Prats H, Baldin V, Tauber JP, Teissie J, Amalric F (1987) Basic fibroblast growth factor enters the nucleolus and stimulates the transcription of ribosomal genes in ABAE cells undergoing GO-Gl transition. Proc Natl Acad Sci U S A 84(19) 6770-6774. 

Cellular RNAs vary widely in their size, structure, and lifespan. The great majority of them are ribosomal RNA (rRNA), which in several forms is a structural and functional component of ribosomes (see p.250). Ribosomal RNA is produced from DNA by transcription in the nucleolus, and it is processed there and assembled with proteins to form ribosome subunits (see pp.208, 242). The bacterial 16S-rRNA shown in Fig. A, with 1542 nucleotides (nt), is a component of the small ribosomae subunit, while the much smaller 5S-rRNA (118 nt) is located in the large subunit. 

Almost all of the RNA in the cell is synthesized in the nucleus, in this process, known as transcription, the information stored in DNA is transcribed into RNA (see p. 242). As mentioned above, ribosomal RNA (rRNA) is mainly produced in the nucleolus, while messenger and transfer RNA (mRNA and tRNA) are formed in the region of the euchromatin. Enzymatic duplication of DNA—replication—also only takes place in the nucleus (see p. 240). 

The nucleus is not capable of synthesizing proteins. All of the nuclear proteins therefore have to be imported—the histones with which DNA is associated in chromatin, and also the so-called non-histone proteins (DNA polymerases and RNA polymerases, auxiliary and structural proteins, transcription factors, and ribosomal proteins). Ribosomal RNA (rRNA) already associates with proteins in the nucleolus to form ribosome precursors. 

FIGURE 26-22 Processing of pre-rRNA transcripts in vertebrates. In step (T), the 45S precursor is methylated at more than 100 of its 14,000 nucleotides, mostly on the 2 -OH groups of ribose units retained in the final products. (5) A series of enzymatic cleavages produces the 18S, 5.8S, and 28S rRNAs. The cleavage reactions require RNAs found in the nucleolus, called small nucleolar RNAs (snoRNAs), within protein complexes reminiscent of spliceosomes. The 5S rRNA is produced separately. 

In eukaryotes, a 45S pre-rRNA transcript is processed in the nucleolus to form the 18S, 28S, and 5.8S rRNAs characteristic of eukaryotic ribosomes (Fig. 26-22). The 5S rRNA of most eukaryotes is made as a completely separate transcript by a different polymerase (Pol III instead of Pol I). 

Eukaryotic ribosomes contain four pieces of RNA (Tables 5-4 and 29-1), which are usually designated by their sedimentation coefficients. Tire 18S, 5.8S, and 28S RNAs are encoded as single transcriptional units with spacers separating the sequences that encode the mature RNAs. A typical animal cell contains several hundred copies of this transcriptional unit, all located in the DNA in the nucleolus (Fig. 28-15), and each having its own set of promoter sequences, enhancers, and transcription factors.47 520 522 The promoter sequences vary substantially among different species.523 The primary transcripts from these units are the sole product of RNA polymerase I. 

As is indicated in Fig. 28-15, transcription is thought to occur from the loops of DNA that form the nucleolar organizing region. The 100-kDa nucleolin, the major protein of the nucleolus, binds to the non-transcribed spacer sequences in the DNA.529-530 It also binds to the newly formed transcripts, as do various proteins that enter the nucleus from the cytoplasm.524531 More than 270 proteins, many of which participate in synthesis of ribosomes, have been detected in the nucleolus.5313 Some of these proteins, acting together with the snoRNAs discussed in the next section, catalyze hydrolytic cleavage of the pre-rRNA molecules. For completion of pre-ribosomal particles additional protein molecules enter the nucleolus and associate with the pre-rRNA particles, then diffuse out of the nucleus. 

Figure 4.4 Nucleolar localized anti-HIV-1 ribozyme. The anti-HIV-1 ribozyme was inserted within the apical loop of the small nucleolar RNA U16. The chimeric U16-ribozyme was placed behind the Pol III U6 snRNA promoter. Transcripts of the chimeric RNA localize to the nucleolus, as shown by the in situ hybridization and co-localization with the small nucleolar RNA U3. The in situ hybrids were performed in transfected 293 cells. The ribozyme probe was fluorescein labeled (green), whereas the U3 probe was labeled with Cy3 (red). Reproduced with permission from Michienzi et al. (2000). (see Color Plate 3)...

Most of the RNA made in the cell is ribosomal RNA. The large and small subunit RNAs are synthesized by RNA polymerase I. Ribosomal RNA is made in a specialized organelle, the nucleolus, which contains many copies of the rRNA genes, a correspondingly large number of RNA polymerase I molecules, and the cellular machinery that processes the primary transcripts into mature... 

As mentioned above, one consequence of stalled RNA polymerase II at a DNA adduct is activation of transcription-coupled repair [27], This effect may depend on the type of polymerase, however, since the removal of some types of DNA damage is slower from RNA-polymerase I transcribed ribosomal DNA than from a nuclear gene [160], The lower level of repair in the nucleolus could also reflect the influence of other transcription factors, such as the HMG-domain protein UBF, which bind to cisplatin-mod-ified DNA [145]. When HeLa cells were exposed to cisplatin at concentrations which did not seem to affect nuclear transcription, inhibition of rDNA gene expression was associated with the redistribution of UBF, along with other factors responsible for rRNA transcription [138], These observations indicate how cisplatin might exert a combination of effects. Transcription is stopped due to titration of essential factors by the platinum-DNA adducts, and the same proteins could shield the lesions from the repair activity. 

Proteins are part of a dynamic network of biomolecules that interact to regulate their localization and function within the cell. Disruption of this physical and chemical system of interactions has become the first paramount step in performing protein analysis by shotgun proteomics. Protein isolation techniques, for instance, have allowed understanding of the complex dynamics of proteins among cellular subcompartments, such as the nucleolus or the mitochondrion (6). Membrane-embedded proteins (7) and DNA-binding transcription factors (8) are two other prominent examples where inadequate protein extraction may hamper further analysis by LC-MS. 

Nucleolar organizing region (NOR) Area of the nucleolus where a great deal of rRNA transcription and synthesis occurs. 

Finally, a large variety of proteins contribute to the assembly and maturation of the telomerase RNP, and these vary much more in evolution than TERT and the other proteins listed above. Budding yeast telomerase RNA is an RNA polymerase 11 transcript, and its intracellular transport and assembly are mediated by the same Sm proteins found in the small nuclear RNPs involved in RNA splicing [27]. Current evidence suggests that the RNA may be made in the nucleus, exported to the cytoplasm to pick up protein components, and then reimported into the nucleus where it functions [28, 29]. Human telomerase RNA, also a pol II transcript, has a snoRNP (small nucleolar RNP) domain, appears to be matured in the nucleolus, and binds dyskerin and other snoRNP proteins [30, 31]. Defects in the RNA or the dyskerin protein that interrupt this maturation can lead to a human disease, dyskeratosis congenita [30, 32]. Ciliate telomerase RNA is transcribed instead by pol III and, at least in Euplotes, is bound by a... 

A FIGURE 12-32 Electron micrograph of pre-rRNA transcription units from nucleolus of a frog oocyte. Each "feather" represents a pre-rRNA molecule associated with protein in a pre-ribonucleoprotein particle (pre-RNP) emerging from a transcription unit. Pre-rRNA transcription units are arranged in tandem, separated by nontranscribed spacer regions of nucleolar chromatin. [Courtesy of Y. Osheim and O. J. Miller, Jr.]... 

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