Ribosomal RNA Genes and the Organization of the Nucleolus

The nucleolus, which is not surrounded by a membrane, is organized around the chromosomal regions that contain the genes for the 5.8S, 18S, and 28S rRNAs. Eukaryotic ribosomes contain four types of RNA, designated the 5S, 5.8S, 18S, and 28S rRNAs (see Figure 7.4). The 5.8S, 18S, and 28S rRNAs are transcribed as a single unit within the nucleolus by RNA polymerase I, yielding a 45S ribosomal precursor RNA (Figure 8.22). The 45S pre-rRNA is processed to the 18S rRNA of the 40S (small) ribosomal subunit and to the 5.8S and 28S rRNAs of the 60S (large) ribosomal subunit. Transcription of the 5S rRNA, which is also found in the 60S ribosomal subunit, takes place outside of the nucleolus and is catalyzed by RNA polymerase III.

Figure 8.22

Ribosomal RNA genes. Each rRNA gene is a single transcription unit containing the 18S, 5.8S, and 28S rRNAs and transcribed spacer sequences. The rRNA genes are organized in tandem arrays, separated by nontranscribed spacer DNA.

To meet the need for transcription of large numbers of rRNA molecules, all cells contain multiple copies of the rRNA genes. The human genome, for example, contains about 200 copies of the gene that encodes the 5.8S, 18S, and 28S rRNAs and approximately 2000 copies of the gene that encodes 5S rRNA. The genes for 5.8S, 18S, and 28S rRNAs are clustered in tandem arrays on five different human chromosomes (chromosomes 13, 14, 15, 21, and 22); the 5S rRNA genes are present in a single tandem array on chromosome 1.

The importance of ribosome production is particularly evident in oocytes, in which the rRNA genes are amplified to support the synthesis of the large numbers of ribosomes required for early embryonic development. In Xenopus oocytes, the rRNA genes are amplified approximately 2000-fold, resulting in about 1 million copies per cell. These amplified rRNA genes are distributed to thousands of nucleoli (Figure 8.23), which support the accumulation of nearly 10¹² ribosomes per oocyte.

Figure 8.23

Nucleoli in amphibian oocytes. The amplified rRNA genes of Xenopus oocytes are clustered in multiple nucleoli (darkly stained spots). (From D. D. Brown and I. B. Dawid, 1968. Science 160: 272.)

Morphologically, nucleoli consist of three distinguishable regions: the fibrillar center, dense fibrillar component, and granular component (Figure 8.24). These different regions are thought to represent the sites of progressive stages of rRNA transcription, processing, and ribosome assembly. The rRNA genes are located in the fibrillar centers, with transcription occurring primarily at the boundary of the fibrillar centers and dense fibrillar component. Processing of the pre-rRNA is initiated in the dense fibrillar component and continues in the granular component, where the rRNA is assembled with ribosomal proteins to form nearly completed preribosomal subunits, ready for export to the cytoplasm.

Figure 8.24

Structure of the nucleolus. An electron micrograph illustrating the fibrillar center (FC), dense fibrillar component (DFC), and granular component (G) of a nucleolus. (Courtesy of David L. Spector, Cold Spring Harbor Laboratory.)

Following each cell division, nucleoli form around the chromosomal regions that contain the 5.8S, 18S, and 28S rRNA genes, which are therefore called nucleolar organizing regions. The formation of nucleoli requires the transcription of 45S pre-rRNA, which appears to lead to the fusion of small prenucleolar bodies that contain processing factors and other components of the nucleolus. In most cells, the initially separate nucleoli then fuse to form a single nucleolus. The size of the nucleolus depends on the metabolic activity of the cell, with large nucleoli found in cells that are actively engaged in protein synthesis. This variation is due primarily to differences in the size of the granular component, reflecting the levels of ribosome synthesis.

Transcription and Processing of rRNA

Each nucleolar organizing region contains a cluster of tandemly repeated rRNA genes that are separated from each other by nontranscribed spacer DNA. These genes are very actively transcribed by RNA polymerase I, allowing their transcription to be readily visualized by electron microscopy (Figure 8.25). In such electron micrographs, each of the tandemly arrayed rRNA genes is surrounded by densely packed growing RNA chains, forming a structure that looks like a Christmas tree. The high density of growing RNA chains reflects that of RNA polymerase molecules, which are present at a maximal density of approximately one polymerase per hundred base pairs of template DNA.

Figure 8.25

Transcription of rRNA genes. An electron micrograph of nucleolar chromatin, showing three rRNA genes separated by nontranscribed spacer DNA. Each rRNA gene is surrounded by an array of growing RNA chains, resulting in a Christmas tree appearance. (Courtesy (more...)

The primary transcript of the rRNA genes is the large 45S pre-rRNA, which contains the 18S, 5.8S, and 28S rRNAs as well as transcribed spacer regions (Figure 8.26). External transcribed spacers are present at both the 5′ and 3′ ends of the pre-rRNAs, and two internal transcribed spacers lie between the 18S, 5.8S, and 28S rRNA sequences. The initial processing step is a cleavage within the external transcribed spacer near the 5′ end of the pre-rRNA, which takes place during the early stages of transcription. This cleavage requires the U3 small nucleolar RNP (see below) that remains attached to the 5′ end of the pre-rRNA, forming the characteristic knobs seen in Figure 8.25. Once transcription is complete, the external transcribed spacer at the 3′ end of the molecule is removed. In human cells, this step is followed by a cleavage at the 5′ end of the 5.8S region, yielding separate precursors to the 18S and 5.8S + 28S rRNAs. Additional cleavages then result in formation of the mature rRNAs. Processing follows a similar pattern in other species, although there are differences in the order of some of the cleavages.

Figure 8.26

Processing of pre-rRNA. The 45S pre-rRNA transcript contains external transcribed spacers (ETS) at both ends and internal transcribed spacers (ITS) between the sequences of 18S, 5.8S, and 28S rRNAs. The pre-rRNA is processed via a series of cleavages (more...)

In addition to cleavage, the processing of pre-rRNA involves a substantial amount of base modification resulting both from the addition of methyl groups to specific bases and ribose residues and from the conversion of uridine to pseudouridine (see Figure 6.38). In animal cells, pre-rRNA processing involves the methylation of approximately a hundred ribose residues and ten bases, in addition to the formation of about a hundred pseudouridines. Most of these modifications occur during or shortly after synthesis of the pre-rRNA, although a few take place at later stages of pre-rRNA processing.

The processing of pre-rRNA requires the action of both proteins and RNAs that are localized to the nucleolus. The involvement of small nuclear RNAs (snRNAs) in pre-mRNA splicing was discussed in Chapter 6. Nucleoli contain a large number (about 200) of small nucleolar RNAs (snoRNAs) that function in pre-rRNA processing. Like the spliceosomal snRNAs, the snoRNAs are complexed with proteins, forming snoRNPs. Individual snoRNPs consist of single snoRNAs associated with eight to ten proteins. The snoRNPs then assemble on the pre-rRNA to form processing complexes in a manner analogous to the formation of spliceosomes on pre-mRNA.

Some snoRNAs are responsible for the cleavages of pre-rRNA into 18S, 5.8S, and 28S products. For example, the most abundant nucleolar snoRNA is U3, which is present in about 200,000 copies per cell. As already noted, U3 is required for the initial cleavage of pre-rRNA within the 5′ external transcribed spacer sequences. Similarly, U8 snoRNA is responsible for cleavage of pre-rRNA to 5.8S and 28S rRNAs, and U22 snoRNA is responsible for cleavage of pre-rRNA to 18S rRNA.

The majority of snoRNAs, however, function to direct the specific base modifications of pre-rRNA, including the methylation of specific ribose residues and the formation of pseudouridines (Figure 8.27). Most of the snoRNAs contain short sequences of approximately 15 nucleotides that are complementary to 18S or 28S rRNA. Importantly, these regions of complementarity include the sites of base modification in the rRNA. By base pairing with specific regions of the pre-rRNA, the snoRNAs act as guide RNAs that target the enzymes responsible for ribose methylation or pseudouridylation to the correct site on the pre-rRNA molecule.

Figure 8.27

Role of snoRNAs in base modification of pre-rRNA. The snoRNAs contain short sequences complementary to rRNA. Base pairing between snoRNAs and pre-rRNA targets the enzymes that catalyze base modification (e.g., methylation) to the appropriate sites on (more...)

Ribosome Assembly

The formation of ribosomes involves the assembly of the ribosomal precursor RNA with both ribosomal proteins and 5S rRNA (Figure 8.28). The genes that encode ribosomal proteins are transcribed outside of the nucleolus by RNA polymerase II, yielding mRNAs that are translated on cytoplasmic ribosomes. The ribosomal proteins are then transported from the cytoplasm to the nucleolus, where they are assembled with rRNAs to form preribosomal particles. Although the genes for 5S rRNA are also transcribed outside of the nucleolus, in this case by RNA polymerase III, 5S rRNAs similarly are assembled into preribosomal particles within the nucleolus.

Figure 8.28

Ribosome assembly. Ribosomal proteins are imported to the nucleolus from the cytoplasm and begin to assemble on pre-rRNA prior to its cleavage. As the pre-rRNA is processed, additional ribosomal proteins and the 5S rRNA (which is synthesized elsewhere (more...)

The association of ribosomal proteins with rRNA begins while the pre-rRNA is still being synthesized, and more than half of the ribosomal proteins are complexed with the pre-rRNA prior to its cleavage. The remaining ribosomal proteins and the 5S rRNA are incorporated into preribosomal particles as cleavage of the pre-rRNA proceeds. The smaller ribosomal subunit, which contains only the 18S rRNA, matures more rapidly than the larger subunit, which contains 28S, 5.8S, and 5S rRNAs. Consequently, most of the preribosomal particles in the nucleolus represent precursors to the large subunit. The final stages of ribosome maturation follow the export of preribosomal particles to the cytoplasm, forming the active 40S and 60S subunits of eukaryotic ribosomes.