Coding Base Altered

Base inserted or removed

FIGURE 12.01 Types of RNA Processing

RNA processing can be divided into cutting/joining or base alteration strategies.

RNA is Processed in Several Ways

Many RNA molecules are modified in a variety of ways after being synthesized.

RNA is made by RNA polymerase, using a DNA template, in the process known as transcription (see Ch. 6). Sometimes the RNA molecule is ready to function immediately after it has been transcribed (e.g. most bacterial mRNAs). However, in many cases, the RNA needs further processing before it is functional. In these cases, the original RNA molecule, before any further processing occurs, is known as the primary transcript. For specific classes of RNA, the precursor (i.e., primary transcript) may be referred to as pre-mRNA, pre-tRNA etc. The term, hnRNA (heterogeneous nuclear RNA) was also used previously, before the relationship of precursor RNA to the final processed RNA product was understood.

Three major types of processing, base modification, cleavage and splicing (Fig. 12.01) apply to all classes of RNA. In addition, eukaryotic mRNA undergoes capping and tailing as well as splicing. Like tRNA, which contains modified bases that are made after transcription (Chapter 8), rRNA, especially from higher organisms, contains modified bases. Certain RNA molecules are made as longer precursors that are trimmed to the correct length. In other, related cases, several RNA molecules are included in the same primary transcript, which is then cleaved into several parts. This applies to the rRNA molecules of both prokaryotes and eukaryotes. Splicing involves the removal of long segments from an RNA molecule by cleavage and rejoining of the ends. This is characteristic of eukaryotic mRNA due to the presence of non-coding introns in many eukaryotic genes (see Ch. 4). The primary primary transcript The original RNA molecule obtained by transcription from a DNA template, before any processing or modification has occurred splicing Removal of intervening sequences and re-joining the ends of a molecule;usually refers to removal of introns from RNA

The processing of RNA sometimes involves other RNA molecules, either as guides or as actual enzymes— ribozymes.

transcript still contains the introns, which must be removed at the RNA level by splicing.

In the simpler cases, mRNA processing relies on typical enzymes consisting of proteins. However, as shown below, more complex RNA processing involves other RNA molecules. These RNAs are involved both in sequence recognition and in the actual chemical reactions of cutting and splicing. In fact, certain introns are self-splicing, that is they cut themselves out in a reaction that does not require any protein components (see below). Such RNA enzymes are known as ribozymes. As already mentioned in Chapter 8, the transpeptidation reaction in protein synthesis is catalyzed by ribosomal RNA, not by ribosomal proteins. The involvement of RNA in such fundamental processes as protein synthesis and RNA processing has led to the idea that ribozymes were more common in early life. Indeed the "RNA world" hypothesis suggests that the original enzymes were all RNA and that protein only assumed this role later in evolution. The RNA world scenario is discussed in more detail in Chapter 20, "Molecular Evolution".

Coding RNA (i.e. mRNA) is used on ly to carry information whereas non-coding RNA is not translated but performs a variety of active roles as RNA.

Many non-coding RNA molecules are found inside the eukaryotic nucleus.

Coding and Non-Coding RNA

In bacterial cells, RNA makes up about 20% of the organic material. In eukaryotes, it only accounts for about 3-4%.Although most genes are transcribed to give the mRNA that encodes proteins, this mRNA is only a small fraction of the total RNA. RNA may be divided into coding RNA (i.e. mRNA) and non-coding RNA, which includes tRNA, rRNA and a variety of other RNA molecules that function directly as RNA and are not translated into protein.

Although there are many different molecules of mRNA, each is only present in relatively few copies. In E. coli there are an average of 3-4 copies of about 400 different mRNAs. In contrast, there are many copies of rRNA and tRNA. For example, E. coli contains 10-20 thousand ribosomes each associated with one copy of each rRNA. Ribosomal RNA thus accounts for about 80% of the total RNA and tRNA for 14-15%. The mRNA only makes up 4-5% by weight of the RNA.

Ribosomal RNA and transfer RNA are found in all living cells. The other types of non-coding RNA vary from organism to organism. Bacteria contain several small regulatory RNAs (see Ch. 9) as well as tmRNA (transfer and messenger RNA in a single unit) that rescues ribosomes trapped by defective messages (see Ch. 8). In eukaryotes we find small nuclear RNA (snRNA), small nucleolar RNA (snoRNA) and small cytoplasmic RNA (scRNA) molecules. The snRNA and snoRNA (sometimes called U-RNA as they are rich in U) are involved in processing other RNA molecules in the eukaryotic nucleus (see below). The scRNA is a miscellaneous group that comprises molecules with various functions. An increasing number of small regulatory RNA molecules are being found in eukaryotes and, to a lesser extent, in prokaryotes. In eukaryotes the two major classes are siRNA (short interfering RNA), involved in RNA interference, and miRNA (microRNA), short RNA molecules involved in regulating gene expression (see Ch. 11).

non-coding RNA RNA molecule that functions without being translated into protein;includes tRNA, rRNA, snRNA, snoRNA, scRNA, tmRNA and some regulatory RNA molecules ribozyme An RNA molecule that shows enzymatic activity

RNA world Theory that early life depended largely or entirely on RNA for both enzyme activity and for carrying genetic information and that

DNA and protein emerged later in evolution small cytoplasmic RNA (scRNA) Small RNA molecules of varied function found in the cytoplasm of eukaryotic cells small nuclear RNA (snRNA) Small RNA molecules that are involved in RNA splicing in the nucleus of eukaryotic cells small nucleolar RNA (snoRNA) Small RNA molecules that are involved in ribosomal RNA base modification in the nucleolus of eukaryotic cells U-RNA Uracil-rich small RNA (includes snRNA and snoRNA)

Eukaryotic Messenger RNA Contains a Cap and a Tail 305

A) PRE-rRNA

16S rRNA sequence

16S rRNA 23S rRNA 5S rRNA

sequence sequence sequence

Ribonucleases cleave at arrows

FIGURE 12.02 Cleavage of rRNAs From Their Precursor In Prokaryotes

The pre-rRNA contains sequences for all three rRNA molecules as well as one or two tRNA molecules. Initial processing involves ribonucleases that cut the primary transcript at the sites shown by arrows. The ends must then be further trimmed. (Only the processing of the rRNA molecules is shown in full here; the tRNA is also trimmed after release.)

16S rRNA

sequence

23S rRNA sequence

5S rRNA sequence

Exonucleases start digestion at arrows

16S rRNA sequence

23S rRNA sequence

5S rRNA sequence

Ribosomal RNAs are transcribed together as one long transcript that must be cut apart and trimmed at the ends.

Transfer RNA precursors must be processed to give functional tRNA molecules.

Processing of Ribosomal and Transfer RNA

The three rRNA molecules of bacteria are transcribed together to give a single pre-rRNA. This contains 16S rRNA, 23S rRNA and 5S rRNA joined by linker regions (Fig. 12.02). In bacteria, this pre-rRNA transcript also includes some tRNAs. In most bacteria there are several copies of the rRNA genes, seven in E. coli, for example.

The mature rRNAs are made by cleavage of the precursor by ribonucleases. This occurs in two stages (Fig. 12.02). First, internal cuts are made, separating the three rRNAs. Ribonucleases III, P and F recognize sites where the pre-rRNA is folded into double-stranded regions held together by base pairing. After this cleavage, the ends are trimmed by several exonucleases.

In eukaryotes, there are four ribosomal RNAs. The 5S rRNA is made separately and does not need processing. The other three (18S rRNA, 28S rRNA and 5.8S rRNA) are made as a single pre-rRNA and processed much as in bacteria.

Transfer RNAs are transcribed as longer precursors that also need processing (Fig. 12.03). Some tRNAs are made singly, others are transcribed together and in bacteria, some are included in the pre-rRNA transcript.The 5'-end of bacterial tRNA is trimmed by ribonuclease P. This enzyme is of note because it is a ribozyme. Ribonuclease P consists of both an RNA molecule and a protein, but the catalytic activity is due to the RNA. The protein component merely modulates the activity of the RNA.

Eukaryotic Messenger RNA Contains a Cap and a Tail

In eukaryotic cells, transcription of genes to give messenger RNA is much more complex than in prokaryotes. First, eukaryotic genes are inside the nucleus, not free in the cytoplasm. Second, most eukaryotic genes are interrupted by segments of non-

ribonuclease P A ribonuclease involved in processing tRNA in bacteria that consists of an RNA ribozyme plus an accessory protein

FIGURE 12.03 Processing of Transfer RNA

Nucleotides shown in red are removed. First (1) ribonuclease E or F cleaves the precursor RNA near the 3' end. Second (2), ribonuclease D chews off bases from the new 3' end leaving the CCA at the end of the acceptor stem. Third (3), ribonuclease P cleaves the 5' end precisely.

coding DNA, the introns. As discussed previously (see Ch. 4) the DNA sequence of a eukaryotic gene consists of regions which code for part of the final protein, the exons, alternating with regions of non-coding DNA, the introns.

The RNA molecule resulting from transcription is known as the primary transcript. It is not yet genuine mRNA because it, too, has exons alternating with introns. If the primary transcript were translated it would result in a huge, dysfunctional protein containing many extra stretches of random amino acids due to the intron regions. The primary transcript is trapped inside the nucleus until the introns are removed. This process is known as splicing and involves cutting out the introns and joining the ends of the exons to generate an RNA molecule which has only the exons; i.e., it contains an uninterrupted coding sequence (Fig. 12.04).

In order to be recognized as a bona fide messenger RNA molecule, and allowed to exit the nucleus, two other modifications must be made. These are the addition of a cap structure to the front and a tail to the rear of the RNA molecule. In fact, these are added before splicing out the introns.

Capping is the First Step in Maturation of mRNA

Before leaving the nucleus, RNA molecules destined to become messenger RNA have a cap added to their 5'-ends and a tail added to their 3'-ends. This occurs inside the nucleus and before splicing. Shortly after transcription starts, the 5'-end of the growing RNA molecule is capped by the addition of a guanosine triphosphate (GTP) residue (Fig. 12.05). This is added in a backward orientation relative to the rest of the bases in the RNA. After addition of the GTP, the guanine base has a methyl group attached at the 7 position. This structure is known as a "capO" structure. Lower eukaryotes only proceed this far.

Further methyl groups may be added to the ribose sugars of the first one or two nucleosides of the original mRNA in some higher eukaryotes (Fig. 12.05). This gives respectively the "capl" and "cap2" structures. If the first base of the original mRNA

cap Structure at the 5'-end of eukaryotic mRNA consisting of a methylated guanosine attached in reverse orientation

FIGURE 12.03 Processing of Transfer RNA

Nucleotides shown in red are removed. First (1) ribonuclease E or F cleaves the precursor RNA near the 3' end. Second (2), ribonuclease D chews off bases from the new 3' end leaving the CCA at the end of the acceptor stem. Third (3), ribonuclease P cleaves the 5' end precisely.

In eukaryotes, the primary transcript is converted to mRNA in three steps:

1) adding a cap at the front

2) adding a tail to the end

3) removing the introns

The cap on a eukaryotic mRNA consists of GTP in reverse orientation.

Capping is the First Step in Maturation of mRNA 307

Gene

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