Processing of mRNA

There are significant differences in the ways that messenger RNAs (mRNAs) for protein-coding genes are produced and processed in prokaryotic and eukaryotic cells.

Prokaryotes - Prokaryotic mRNAs are synthesized on the bacterial nucleoid in direct contact with the cytosol and are immediately available for translation. The Shine-Dalgarno sequence (see here) near the 5' end of the mRNA binds to a site on the prokaryotic ribosomal RNA (rRNA), allowing attachment of the ribosome and initiation of translation, often even before transcription is completed.

Eukaryotes - In eukaryotes, the mRNA is produced in the nucleus and must be exported into the cytosol for translation. Furthermore, the initial product of transcription (pre-mRNA) may include introns, which must be removed before translation can occur. There is no ribosomal attachment sequence like the Shine - Dalgarno sequence in prokaryotes. For all these reasons, eukaryotic mRNA requires extensive processing before it can be used as a protein template. This processing takes place while mRNA is still in the nucleus.

> Capping - The first modification occurs at the 5' end of the pre-mRNA. A GTP residue is added in reverse orientation and forms, together with the first two nucleotides of the chain, a structure known as a cap (Figure 28.30). The cap is "decorated" by the addition of methyl groups to the N-7 position of the guanine and to one or two sugar hydroxyl groups of the cap nucleotides. The cap structure serves to position the mRNA on the ribosome for translation.

> Splicing - After being capped, the pre-mRNA becomes complexed with a number of small nuclear ribonucleoprotein particles (snRNPs), which are themselves complexes of small nuclear RNAs (snRNAs) and special splicing enzymes. The snRNP--pre--mRNA complex is called a spliceosome. snRNAs recognize and bind intron--exon splice sites by means of complementary sequences (Figure 28.31). Table 28.6 shows some representative splice site sequences and the consensus sequences common to most introns. Excision of a single intron involves assembling and disassembling a spliceosome. Figure 28.32 and Figure 28.33 depict possible chemical and molecular aspects of the process. The sequence of reactions can be summarized as follows:

> > 1. It begins with the attachment of the U1 snRNP to the G site at the 5' end of the intron.

> > 2. The U2 snRNP then attaches at the branch site.

> > 3. Assembly of the spliceosome continues, including the addition of several more snRNPs,

> > 4. The lariat loop in the intron is formed and the two exons are joined. Splicing has now been accomplished, and the products--a ligated mRNA and a looped intron--are released. As the spliceosome disintegrates, the looped intron is degraded, and the mRNA is exported from the nucleus.

> Alternative Splicing - Some gene transcripts may be spliced in different ways, in different tissues of an organism or at different developmental stages. Alternative splicing of the heavy chains of immunoglobulins (see here) results in proteins that may or may not carry a hydrophobic membrane-binding domain. Another example of alternative splicing is shown in Figure 28.34. The protein -tropomyosin is used in different kinds of contractile systems in various cell types (see here) . A single gene is transcribed, but the specific splicing patterns in different tissues provide a variety of -tropomyosins. There are three positions at which alternative choices can be made for which exon to splice in. The choice of splice site appears to be determined by a cell-specific protein that interacts with the spliceosome. The economy of alternative splicing, given the size of the genome is significant.

> RNA editing - RNA editing is a process affecting mitochondrial mRNAs of some unicellular eukaryotes, which involves insertion or deletion of uridine residues into messages during the processing steps. Apparently, the insertions are made by a kind of reverse splicing mechanism, and only at certain points. Small RNAs, called guide RNAs, are required for the process. A quite different type of editing involves deamination of adenosine to inosine in mRNA. Neither alternative splicing nor editing formally contradicts the basic concept that gene sequence carries the cell's information, because the sites for splicing and editing are themselves contained in the DNA, as are the sequences of the small nuclear RNAs and the guide RNAs.

After capping, poly(A) tailing, and splicing are complete, the newly formed mRNA is exported from the nucleus, almost certainly through the nuclear pores. It is then attached to ribosomes for translation.

See also: Introns, Termination of Eukaryotic Transcription, Eukaryotic vs Prokaryotic Translation

INTERNET LINKS:

> 1. Small RNA Database

> 2. The RNA World

> 3. RNA Editing

> 4. RNA Modification Database

> 5. 'U' Insertion/Deletion Database

> 6. Spliceosome

> 7. Splice Sites