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