RNA processing reactions

Eukaryotic mRNA processing is much more complex and has many consequences for gene expression. Most obviously, many eukaryotic genes contain introns, which are found in the primary transcript. For the mRNA to be translated into a useful protein, some way to remove them from the transcript and still preserve the coding 

one phosphate of the 5 -terminal phosphate of the pre-mRNA transcript is cleaved to yield a diphosphate, which then attacks GTP, releasing inorganic pyrophosphate and making a 5 -5 phosphate bond. The immature cap is then methylated. The G is methylated to make N7-methyl G, while the first and (sometimes) the second nucleotides of the transcript are methylated on their 2 -hydroxyl groups. The cap performs two functions First, it is usually required for binding the mRNA to the ribosome, and it also seems to allow the recognition of introns. 

Both capping and poly A formation precede intron removal. Thus, the first steps of processing result in a pre-mRNA that has a 5 cap and a 3 polyA tail, but with all its introns present. 

The term splicing refers to the process by which introns are removed and the mRNA put back together to form a continuous coding sequence in the 5 -3 direction. Remembering how accurate this process must be is important. If only a single nucleotide of an intron were left in the processed mRNA, the protein made from that mRNA would be non-functional, because the ribosome would read the wrong codons. The cellular machinery that splices pre-mRNAs uses information at the splice junctions to determine where to cut and where to rejoin the mRNA. Removal of introns from transcripts containing more than one intron usually occurs in a preferred but not exclusive order. Several pathways are used. 

In summary, mRNA processing requires an interconnected set of reactions Cap and polyA synthesis, followed by intron removal. Soluble enzymes catalyze the former two reactions, while the latter set of reactions involves both RNA and protein components. 

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The primary RNA transcript from a protein-coding gene in a eukaryotic cell must be modified by several RNA processing reactions in order to become a functional mRNA molecule. The 5 end is modified to form a 5 cap structure. Most pre-mRNAs are then cleaved near the 3 end and a poly(A) tail is added. Intron sequences are removed by RNA splicing. 

L. Minvielle-Sebastia and W. Keller. 1999. mRNA polyadenylation and its coupling to other RNA processing reactions and to transcription Curr. Opin. Cell Biol. 11 352-357. (PuhMed)... 

FIG. 1.1 Biogenesis of trypanosomatid mRNA. A schematic view of trypanosome gene expression. Mature mRNAs are generated from polycistronic pre-mRNAs via two RNA processing reactions, trans-splicing and polyadenylation. For details, see text. 

Ullu, E., Matthews, K. R. and Tschudi, C. (1993) Temporal order of RNA process reactions in trypanosomes rapid trans-splicing precedes polyadenylation of newly-synthesized tubulin transcripts. Mol. Cell Biol. 13 720 725. 

Fluorouracil (5-FU) is inactive in its parent form and requires activation via a complex series of enzymatic reactions to ribosyl and deoxyribosyl nucleotide metabolites. One of these metabolites, 5-fluoro-2 -deoxyuridine-5 -monophosphate (FdUMP), forms a covalently ternary complex with the enzyme thymidylate synthase and the reduced folate 5,10-methylenetetrahydrofolate, a reaction critical for the de novo synthesis of thymidylate. This results in inhibition of DNA synthesis through "thymineless death." 5-FU is converted to 5-fluorouridine-5 -triphosphate (FUTP), which is then incorporated into RNA, where it interferes with RNA processing and mRNA translation. 5-FU is also converted to 5-fluorodeoxyuridine-5 -triphosphate (FdUTP), which can be incorporated into cellular DNA, resulting in inhibition of DNA synthesis and function. Thus, the cytotoxicity of 5-FU is thought to be the result of combined effects on both DNA- and RNA-mediated events. 

The recombination process is not as precise as the site-specific recombination described earlier, so additional variation occurs in the sequence at the V-J junction. This increases the overall variation by a factor of at least 2.5, thus the cells can generate about 2.5 X 1,200 = 3,000 different V-J combinations. The final joining of the V-J combination to the C region is accomplished by an RNA-splicing reaction after transcription, a process described in Chapter 26. 

RNA phosphodiesler backbone, they have also been shown lo participate in cleavage of DNA. replication of RNA, and reactions with phosphate monocstcrs, Other RNAs arc associated with enzymes to form riboprotein complexes involved iit many biological processes. The multifunctional character of RNA, particularly the involvement of RNA ill enzymatic processes, has led to the hypothesis that life on earth evolved from RNA. and that RNA had both the genetic and catalytic functions commonly associated with DNA and proteins, respectively. 

Elongation continues until transcription comes to a halt at varying distances downstream of the gene, releasing the primary RNA transcript, pre-mRNA. This molecule then undergoes processing reactions to yield mRNA. 

These enzymes are not classified as nucleotidyltransferases, although they catalyze nucleotidyl group transfers in the course of activating the S -phosphoryl groups for the ligation process. The activation mechanism involves a covalent adenylyl-enzyme as an intermediate and a double displacement on of ATP (or NAD+). The chemical mechanism of the RNA ligase reaction is similar. The stereochemistry of these reactions is known for RNA ligase and is consistent with the mechanism as formulated above (81, 82). 

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