RNA Editing and Guide RNA | Expression of Gene : Protein Synthesis RNA Processing (RNA Splicing, RNA Editing and Ribozymes) | Genetics, Biotechnology, Molecular Biology, Botany

⇒	Expression of Gene : Protein Synthesis 3. RNA Processing (RNA Splicing, RNA Editing and Ribozymes)
⇒	Addition of caps (m⁷G) and tails (polyA) for mRNA
	⇒	Addition of methylated cap at the 5' end
	⇒	Polyadenylation and the generation of 3' end
⇒	RNA splicing
	⇒	Self-splicing of group I introns
	⇒	Splicing of group II introns
	⇒	Splicing of eukaryotic hnRNA through spliceosomes/snurposomes
	⇒	Splicing of introns using RNA maturases or endonucleases
	⇒	Yeast tRNA splicing by cutting and rejoining
	⇒	Trans-splicing of transcripts in chloroplasts and mitochondria
	⇒	Constitutive vs. alternative Splicing
	⇒	RNA Editing and Guide RNA
⇒	Ribozymes

RNA Editing and Guide RNA
The phenomenon of RNA editing was discovered in mid 1980's, following some unexpected observations made by several groups of molecular biologists. These observations made particularly in the mitochondrial genes of Trypanosorria (which causes African sleeping sickness) and Leishmania (causing kala-azar), included the following : (i) Several genes having mutations, which were supposed to render these genes inactive (due to origin of premature stop signals, or due to loss of start signals), were still active, (ii) The sequences of mRNA molecules in several cases were such which could not have been derived from the DNA sequence of the corresponding genes. These DNA sequences have sometimes been described as nonsensical 'cryptogenes'. These observations were later explained by RNA editing, which sometimes included addition of upto 550 and deletion of upto 41 uridine residues. It was shown that RNA editing mainly involved addition and deletion of uridines or insertion of cytidines, and that these changes took place at very specific sites and not randomly.

Subsequently, both in Trypanosoma and Leishmania, mt-DNA sequences were found (mainly in minicircle mtDNA, but rarely also in maxicircle mt-DNA), that encode small RNA molecules, less than 40 nucleotides in length. This RNA apparently carried information for uridine insertions and deletions, and was therefore called guide RNA or gRNA. It was also shown that tails consisting of strings of uridine nucleotides are added to the gRNA and become the source of uridines inserted in mRNA. Later parallelism between RNA editing (done by gRNA) and self splicing of introns in hnRNA (done by ribozymes) was demonstrated by Thomas Cech (who shared 1989 Nobel Prize).

Like self splicing of introns, RNA editing also involves transesterification, but with the help of guide RNA or gRNA. In the first step, the guide RNA aligns itself (by base pairing) with the unedited RNA, splits it into two, and makes a new bond between one of the broken ends and the uridine at the tip of the tail of gRNA at its 3' end (Fig. 33.11). This reaction is facilitated by mitochondrial uridyl transferase activity. In the next step, the broken end of other RNA segment, which is not involved in addition of U, forms a bond with this RNA segment. The tail of gRNA is now released for another round of transesterification.

When a uridine needs to be deleted from mRNA, cleavage occurs at 5' end of uridine (to be deleted) which is mismatched during base pairing with gRNA. Subsequently 3' OH group in gRNA attacks 3' of the uridine to be deleted, leading to excision of uridine and production of gRNA that is one nucleotide longer.

Fig. 33.11. RNA editing using guide RNA (gRNA).

The above mechanism predicts an intermediate, in which gRNA is linked to mRNA. Such gRNA-mRNA chimaeric molecules were actually found and their presence verified by polymerase chain reaction (PCR), in which 5' primer specific to gRNA and 3' primer specific to mRNA were used. The PCR gave chimaeric molecules as amplification products (for PCR, consult Genetic Engineering and Biotechnology 1. Recombinant DNA and PCR (Cloning and Amplification of DNA)).





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