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8.3Post-transcriptional Modification

8.3.1

RNA Cleavage

Various types of rRNA are transcribed as a single strand of precursor RNA (Fig. 8-7A). These rRNA are produced through post-transcriptional cleavage of it. This is also called trimming. This is a fitting method because all types of rRNA are required when needed. Some tRNA is also included in this precursor RNA and synthesized together with rRNA.

In the same manner, multiple tRNA molecules are synthesized by cleaving a single strand of precursor RNA (Fig. 8-7B). However, this does not mean that all types of tRNA are synthesized from a single strand. RNaseP is an enzyme involved in trimming. In this enzyme containing RNA, the RNA, rather than the protein, is responsible for its activity. Furthermore, detailed trimming occurs in addition to the cleaving depicted in Fig. 8-7.

Fig. 8-7 Cleavage of precursor RNA into rRNA and tRNA

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8.3.2

Base Modification

rRNA and tRNA undergo base modification once RNA strands have been produced. mRNA also receives base modification, but to a lesser extent. Methylation is the main form of modification to which rRNA is subjected. In this process, a methyl group is transferred from S-adenosylmethionine. A substantial amount of base modification occurs in tRNA, producing minor bases including pseudouridine, 4-thiouridine, thymidine, dihydrouridine, and 1-methylguanosine.*5 Minor bases are important for the functioning of tRNA. Another important modification in eukaryotes is the appending of three bases, CCA, to the 3′ terminus by a specific enzyme. In eubacteria, CCA is attached from the beginning, and thus, the 3′ terminus of mature tRNA has CCA in both prokaryotes and eukaryotes.

*5 In addition to the five main types of bases contained in high-molecular-weight DNA and RNA, minor bases are believed to play important roles despite their small quantities.

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8.3.3

mRNA Processing

Fig. 8-8 Modification resulting in the mature mRNA in eukaryotic cells

While eubacterial mRNA is used without receiving post-synthesis modification, mRNA of eukaryotes and archaea is transcribed from DNA as the precursor pre-mRNA (Fig. 8-8). After undergoing the three types of changes (processing) discussed below, pre-mRNA becomes mRNA. The mature mRNA includes coding regions that determine the primary structure of proteins (Fig. 8-8). We examine this phenomenon in eukaryotes.

Fig. 8-9 Cap structure

■Capping (Cap Formation)
7-Methylguanosine with 5′–5′ phosphate bonds is added to the 5′ terminus of mRNA. No other instance of 5′–5′ bonding is observed in nucleotide bonding. This is known as the cap structure (Fig. 8-9). This structure is essential when mRNA is used for protein synthesis because it binds to ribosomes via special proteins attached to this cap. Because prokaryotic mRNA does not have this cap structure, it would not function when introduced into the eukaryotic protein synthesis apparatus.

Fig. 8-10 Splicing from pre-mRNA to mRNA

■Poly(A) Addition
A poly(A) addition signal (AAUAAA…) sequence is located near the 3′ terminus of pre-mRNA, and after enzymatic cleavage at a site approximately 20 bases downstream from this location, dozens to more than 1000 A’s (adenylic acid) are added by poly(A) polymerase. This synthesis does not require a template. The size of mature mRNA is not fixed because the length of the poly(A) chain differs even among the same type of mRNA. Poly(A) chains are probably important in the initiation of protein synthesis and inhibition of mRNA degradation.
As an experimental method, mRNA with poly(A) chains can be concentrated or purified by complementary binding with oligo dT that has been bound to a fine resin surface. This method cannot be used to concentrate prokaryotic mRNA because it does not contain poly(A) chains.

■Splicing
Splicing is perhaps the most remarkable aspect of processing in eukaryotes. Genes in eukaryotes consist of exon regions with amino acid sequence information (code) and intron regions without this information. Pre-mRNA is synthesized in a form that it contains both. Splicing involves removing only the intron regions by excision from transcribed pre-mRNA and connecting only the exon regions to produce mRNA (Fig. 8-8). Spliceosomes, which are complexes containing ncRNA called small nuclear RNA (snRNA), are involved in the linking of two distant exons after excision of introns. They bring together two breakpoints by binding to the breakpoint regions in pre-mRNA (Fig. 8-10A). This complex reaction proceeds in the following manner: the 5′ splice site is cleaved, and after a phosphate on the 5′ terminus of intron binds to the 2′ hydroxy group of an A near the 3′ terminus, a hydroxy group on the 3′ terminus of exon attacks the 3′ splice site, excising intron and linking exons (Fig. 8-10B). The excised intron is in a lasso-like shape termed “lariat.”

Many types of mRNA can be produced during splicing, including those in cases when some introns remain because of not being excised or two introns are excised along with exon between them. This is termed alternative splicing. As a result, multiple types of proteins with differing amino acid sequences can be synthesized from this mRNA and thus they have differing functions. Thus, many types of proteins can be synthesized from only one gene and that gene can function as multiple genes (see selection 2 of Chapter24, Fig. 24-2B). This is one of the reasons why while there are only 25,000 genes in humans, they are estimated to be virtually equivalent to 100,000. In a special case, a single individual has hundreds of millions of types of antibody genes (see Column Selection 1 of Chapter 10).

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mRNA Editing

Editing is a phenomenon whereby the mRNA sequence is changed. Base conversions occur in terrestrial plants and mammals and base insertions and deletions occur frequently in trypanosomes.

As opposed to base substitution, base conversion involves interconversion between cytidine and uridine by base modification (terrestrial plants) and a change from adenosine to inosine (mammals). In humans, editing occurs in mRNA for apoB protein, which is involved in lipid transportation and metabolism. It involves change of the codon for glutamine to a termination codon. While apoB protein is synthesized in the small intestine and liver, editing occurs in the small intestine but not in the liver. Editing also occurs in mRNA for glutamate receptors, with the codon for glutamine changing to that for arginine resulting in a change in protein function.

When base insertion and deletion occurs, the reading frame for the mRNA produced often changes through the intervention of guide RNA (gRNA). As a result, the genetic code may not correspond to any amino acid of the protein, and the reason for this phenomenon is not yet completely understood.

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8.3.4

RNA Transport to the Cytoplasm

In eukaryotes, RNA is synthesized in the nucleus, and after being modified, the mature rRNA, tRNA, and mRNA are exported through nuclear pores to the cytoplasm for protein synthesis. rRNA synthesized in the nucleolus is modified. It binds to ribosomal proteins and is then transported. These types of RNA are transported by binding with specific transport proteins that function in transport from the nucleus to the cytoplasm. After being transported to the cytoplasm, RNA dissociates from the transport proteins (see Selection 3 of Chapter 12, Fig. 12-11).

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