RNA splicing involves cleavages of the primary RNA transcripts to generate exon sequences that are then stitched together. Most protein-coding genes undergo RNA splicing, and so do many RNA genes. But additional types of cleavage occur in the processing of most types of noncoding RNA including ribosomal RNAs, tRNAs, miRNAs, and so on.
Ribosomal RNA synthesis provides exceptional examples of RNA cleavages. Four major classes of eukaryotic ribosomal RNA (rRNA) have been identified: 28S, 18S, 5.8S, and 5S rRNA (S is the Svedberg coefficient, a measure of how fast large molecular structures sediment in an ultracentrifuge, corresponding directly to size and shape). 18S rRNA is found in the small subunits of ribosomes; the other three are components of the large subunit. Very large amounts of rRNA are required for cells to carry out protein syn thesis, and many genes are devoted to making rRNA in the nucleolus, a visibly distinct compartment of the nucleus.
In human cells, a cluster of about 250 genes synthesizes 5S rRNA using RNA polymerase III. However, the 28S, 18S, and 5.8S rRNAs are encoded by consecutive genes on a common 13 kilobase (kb) transcription unit (Figure 1) that is transcribed by a dedicated polymerase, RNA polymerase I. A compound unit of the 13 kb transcription unit and an adjacent 27 kb nontranscribed spacer is tandemly repeated about 30–40 times at the nucleolar organizer regions on the short arms of each of the five human acrocentric chromosomes (13, 14, 15, 21, and 22). Although on different chromosomes, these five clusters of rRNA genes, each about 1.5 Mb long, are brought into close proximity within nucleoli, where they are transcribed in concert; the aggregate cluster of rRNA genes is sometimes referred to as ribosomal DNA (rDNA).
Fig1. Three major rRNA classes are synthesized by cleavage of a shared primary transcript. (A) In human cells, the 18S, 5.8S, and 28S rRNAs are encoded by a single transcription unit that is 13 kb long. It occurs within tandem repeat units of about 40 kb that also include a roughly 27 kb nontranscribed (intergenic) spacer. (B) Transcription by RNA polymerase I produces a 13 kb primary transcript (45S rRNA) that then undergoes a complex series of post-transcriptional cleavages. (C–E) Ultimately, individual 18S, 28S, and 5.8S rRNA molecules are released. The 18S rRNA will form part of the small ribosomal subunit. The 5.8S rRNA binds to a complementary segment of the 28S rRNA; the resulting complex will form part of the large ribosomal subunit. The latter also contains 5S rRNA, which is encoded separately by dedicated genes transcribed by RNA polymerase III.
In the case of transfer RNAs, cleavages are required to remove both a 5′ leader sequence of eight nucleotides and a 3′ trailer sequence of four nucleotides, prior to addition of the terminal CCA sequence at the 3′ end. When we consider the details of gene regulation in Chapter 10, we will also illustrate how miRNAs are formed from larger pre cursors by RNA cleavage.
Chemically modified nucleosides in RNA
In humans (and other vertebrates), chemical modification of nucleotides in DNA is limited to methylation at the 5′ carbon of certain cytosines, as described above. However, maturation of RNA involves frequent and highly varied modification of nucleotides (more than 100 different types of modification are recorded in the RNA Modification Database). The modifications occur at the level of nucleosides and they can be of three types: base modifications, sugar modifications, and altered glycosidic bonding (see Figure 2 for examples).
Fig2. Examples of modified nucleosides in RNA. Shown here are two uridine modifications and one each for adenosine and guanosine. Two of the examples are base modifications: dihydrouridine (extra hydrogen atoms at carbons 5 and 6 of uracil) and inosine (replacement of the 6-amino group by a carbonyl group). 2-O-methylguanosine is an example of a sugar modification (the hydrogen of the 2′ hydroxyl [OH] group of the ribose is replaced by a methyl group). Pseudouridine is an isomer of uridine and is produced by altering the glycosidic bond that connects the base to the sugar (in the normal N-glycosidic bond uracil is attached through nitrogen 1, but in pseudouridine the glycosidic bond forms with carbon 5 of uracil instead of nitrogen 1).
At least 16 different modified nucleosides occur naturally in mRNA (see Figure 1 of Li & Mason [2014], PMID 24898039; Further Reading). 7-Methylguanosine and 2′-O-ribosyl methylations occur at the 5′ end of vertebrate mRNAs (see Figure 3). In addition, various modifications can occur in internal nucleotides in mRNA. The most common is N6-methyladenosine (a methyl group is attached to the N6 position of adenine); the methylated adenosine often occurs in the sequence (A/G) mAC the methylated adenine is immediately preceded by a purine and followed by a cytosine. Other internal modified nucleosides in mRNA include 5-methylcytidine and N6,2′-O-dimethyladenosine.
Fig3. The 5′ cap of a eukaryotic mRNA. The 5′ end of a eukaryotic mRNA has a specialized cap that provides protection against exonucleases and has various other functions, including assisting initiation of translation (see text). The capping process involves: (i) removal of the gamma (γ) phosphate of the original terminal 5′ nucleotide, which is normally a purine (Pu); (ii) addition of a GMP (derived from a GTP precursor) through a 5′-5′ triphosphate linkage (gray shading); (iii) methylation of nitrogen atom 7 of the new 5′ terminal G to produce 7-methylguanosine (m7G). In mRNAs synthesized in vertebrate cells, the 2′ carbon atom of the ribose of each of the two adjacent nucleotides is also methylated, as illustrated by pink shading. N, any nucleotide.
Nucleoside modifications are especially prevalent in stable noncoding RNAs, including rRNAs (where the RNA modifications are undertaken using different snoRNAs) and particularly so in transfer RNAs. Approximately 12% of the nucleotides in tRNAs are modified, mostly as a result of some type of methylation (in the tRNA illustrated in Figure 4, nine out of the 74 nucleotides show chemical modifications, and seven of the nine involve methylation). The patterns of nucleotide modification are highly conserved in different tRNAs. That is, the same types of modification tend to occur at specific nucleotide positions—the TψC and D arms of a tRNA are even defined by the occurrence of pseudouridine (ψ) and dihydrouridine (D) at defined nucleotide positions (see Figure 4).
Fig4. Extensive intramolecular base pairing and nucleoside modification in transfer RNA. The tRNAGly shown here has the classical cloverleaf tRNA structure, with three stem-loops (the D arm, the anticodon arm, and the TψC arm) plus a stretch of base pairing between the 5′ and 3′ terminal sequences (called the acceptor arm because the 3′ end is where an amino acid is attached). Note that different tRNAs tend to have the same number of base pairs in the stems of the different arms of their cloverleaf structure and that the anticodon at the center of the middle loop identifies the tRNA according to the amino acid it will bear. Nine of the 74 nucleotides have been subjected to a nucleoside modification, including four 5-methylcytidines (m5C) and one each of 1-methyladenosine (m1A), 2′-O-methyluridine (Um), 5,6-dihydrouridine (D), and pseudouridine (ψ). In addition, a uracil was methylated at its carbon 5 to give thymine (T). See Figure 2 for the structures of some of the modified nucleotides.
The precise purpose of the nucleotide modifications remains to be clarified. Many of the modifications in the main body of tRNA are known to affect tRNA folding and stability, and several of the modifications around the anticodon loop can affect translation or cell growth (for example, in wobble base pairing and stabilization of codon– anticodon interactions). In mRNA, the comparatively abundant 6-methyladenosine has been implicated in regulating alternative splicing, and the same modification is used to signal that precursor miRNAs are ready to be processed into miRNAs.
