Approximately 80 percent of the total RNA in rapidly growing mammalian cells (e.g., cultured HeLa cells) is rRNA, and 15 percent is tRNA; protein-coding mRNA thus constitutes only a small portion of the total RNA. The primary transcripts produced from most rRNA genes and from tRNA genes, like pre-mRNAs, are extensively processed to yield the mature, functional forms of these RNAs.
Pre-rRNA Genes Are Similar in All Eukaryotes and Function as Nucleolar Organizers
The 28S and 5.8S rRNAs associated with the large (60S) ri-bosomal subunit and the 18S rRNA associated with the small (40S) ribosomal subunit in higher eukaryotes (and the smaller, functionally equivalent rRNAs in all other eukary-otes) are encoded by a single type of pre-rRNA transcription unit. Transcription by RNA polymerase I yields a 45S primary transcript (pre-rRNA), which is processed into the mature 28S, 18S, and 5.8S rRNAs found in cytoplasmic ribosomes. Sequencing of the DNA encoding pre-rRNA from many species showed that this DNA shares several properties in all eukaryotes. First, the pre-rRNA genes are arranged in long tandem arrays separated by nontranscribed spacer regions ranging in length from =2 kb in frogs to =30 kb in hu-
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▲ 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.]
mans (Figure 12-32). Second, the genomic regions corresponding to the three mature rRNAs are always arranged in the same 5'^3' order: 18S, 5.8S, and 28S. Third, in all eu-karyotic cells (and even in bacteria), the pre-rRNA gene codes for, and the corresponding primary transcript contains, regions that are removed during processing and rapidly degraded. The general structure of pre-rRNAs is diagrammed in Figure 12-33.
Both the synthesis and processing of pre-rRNA occurs in the nucleolus. When pre-rRNA genes initially were identified in the nucleolus by in situ hybridization, it was not known whether any other DNA was required to form the nucleolus. Subsequent experiments with transgenic Drosophila strains demonstrated that a single complete pre-rRNA transcription unit induces formation of a small nucleolus. Thus a single pre-rRNA gene is sufficient to be a nucleolar organizer, and all the other components of the ribosome diffuse to the newly formed pre-rRNA. The structure of the nucleolus observed by light and electron microscopy results from the processing of pre-RNA and the assembly of ribosomal subunits.
▲ FIGURE 12-33 General structure of eukaryotic pre-rRNA transcription units. The three coding regions (blue) encode the 18S, 5.8S, and 28S rRNAs found in ribosomes of higher eukaryotes or their equivalents in other species. The order of these coding regions in the genome is always 5' ^ 3'. Variations in the lengths of the transcribed spacer regions (tan) account for the major difference in the lengths of pre-rRNA transcription units in different organisms.
Small Nucleolar RNAs Assist in Processing Pre-rRNAs and Assembling Ribosome Subunits
Following their synthesis in the nucleolus, nascent pre-rRNA transcripts are immediately bound by proteins, forming pre-ribosomal ribonucleoprotein particles (pre-rRNPs). The largest of these (80S) contains an intact 45S pre-rRNA molecule, which is cut in a series of cleavage and exonucleolytic steps that ultimately yield the mature rRNAs found in ribo-somes (Figure 12-34). During processing, pre-rRNA also is extensively modified, mostly by methylation of the 2'-hydroxyl group of specific riboses and conversion of specific uridine residues to pseudouridine. Some of the proteins in the pre-rRNPs found in nucleoli remain associated with the mature ribosomal subunits, whereas others are restricted to the nu-cleolus and assist in assembly of the subunits.
The positions of cleavage sites in pre-rRNA and the specific sites of 2'-0-methylation and pseudouridine formation are determined by approximately 150 different small nucleolus-restricted RNA species, called small nucleolar RNAs (snoRNAs), which hybridize transiently to pre-rRNA molecules. Like the snRNAs that function in pre-mRNA processing, snoRNAs associate with proteins, forming ribonucleoprotein particles called snoRNPs. One large class of snoRNPs helps position a methyltransferase enzyme near methylation sites in the pre-mRNA. Another positions the enzyme that converts uridine to pseudouri-dine. Others function in the cleavage reactions that remove transcribed spacer regions. Once cleaved from pre-rRNAs, these sequences are degraded by the same exosome-associated 3' ^ 5' nuclear exonucleases that degrade in-trons spliced from pre-mRNAs.
Some snoRNAs are expressed from their own promoters by RNA polymerase II or III. Remarkably, however, the large majority of snoRNAs are spliced-out introns of genes encoding functional mRNAs encoding proteins involved in ribosome synthesis or translation. Some snoRNAs are introns spliced from apparently nonfunctional mRNAs. The genes encoding these mRNAs seem to exist only to express snoRNAs from excised introns.
Unlike pre-rRNA genes, 5S rRNA genes are transcribed by RNA polymerase III in the nucleoplasm outside the nucle-olus. Without further processing, 5S RNA diffuses to the nucleolus, where it assembles with the 28S and 5.8S rRNAs and proteins into large ribosomal subunits (see Figure 12-34 and Figure 4-28). When assembly of ribosomal subunits in the nucleolus is complete, they are transported through nuclear pore complexes to the cytoplasm, where they appear first as free subunits. The ribosomal subunits are the largest cellular structures known to be transported through nuclear pore complexes.
▲ FIGURE 12-34 Processing of pre-rRNA and assembly of ribosomes in higher eukaryotes. Ribosomal and nucleolar proteins associate with 45S pre-RNA as it is synthesized, forming an 80S pre-rRNP Sites of cleavage and chemical modifications are determined by small nucleolar RNAs (not shown). Note that synthesis of 5S rRNA occurs outside the nucleolus.
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