RNA splicing

Self splicing of group I and-group II introns, by two transesterification reactions
Fig. 33.3. Self splicing of group I and-group II introns, by two transesterification reactions.

Common secondary structure of a group I intron in Tetrahymena, facilitated by internal pairing due to consensus sequences (P, Q and R, S) and the pairing at the ends due to internal guide sequence (IGS)
Fig. 33.4. Common secondary structure of a group I intron in Tetrahymena, facilitated by internal pairing due to consensus sequences (P, Q and R, S) and the pairing at the ends due to internal guide sequence (IGS).
In eukaryotes, the RNA transcribed from DNA almost invariably undergoes RNA splicing to yield mature RNA sequences (rRNA, mRNA, tRNA). It involves removal of sequences mainly corresponding to introns in split genes, although, other examples of RNA splicing may also be available. The mechanisms available for this purpose include the following : (i) self splicing of fungal mitochondrial and other group I introns, (ii) self splicing of mitochondrial group II introns through lariat formation without assistance from any proteins or spliceosomes, (iii) splicing of higher eukaryotic nuclear introns, through the formation of spliceosomes at intron-exon junctions, (iv) splicing of yeast tRNA precursor molecules by cleavage due to endonuclease followed by fusion due to ligase.

Self-splicing of group I introns
Self-splicing of RNA molecules involving group I introns, found in rRNA genes of Tetrahymena and Physarum nuclei, in fungal mitochondria and in phage T4, takes place through two transesterification reactions (exchange of phosphate esters, which leave the total number of phosphodiester bonds unchanged). Group I introns are characterized by (i) the absence of conserved sequences at splicing junction, and by (i) presence of short conserved consensus sequences internally. In the first transesterification, the 5' splice site is cleaved, as guanosine is added to the 5' end of intervening sequence (IVS) to be cleaved. In the second transesterification step, the 3' splice site is cleaved as the exons are joined (Fig. 33.3a). The excised IVS or intron can form a circle by cyclization reaction and these circles can again regenerate linear molecules due to autocatalysis. It means that excised IVS RNA and truncated versions of the IVS RNA also act as ribozymes to cleave, join or de-phosphorylate RNA substrates.

As indicated earlier, all group I introns have some short consensus sequences, which allow intramolecular base pairing to generate a characteristic secondary structure within an intron. A characteristic feature of this secondary structure, is the pairing between two ends of an intron, with the help of an internal guide sequence (IGS), as shown in Figure 33.4. Internal base pairing is facilitated by the presence of two pairs of elements : (i) P and Q each 10 bases long, of which 6-7 bases are involved in pairing; (ii) R and S elements, each 12 bases long, of which only 5 are involved in pairing (Fig. 33.4). Any mutations in this consensus sequence have been shown to stop RNA self splicing, indicating the significance of consensus sequence in splicing.
Self splicing of group I and-group II introns, by two transesterification reactions
Fig. 33.3. Self splicing of group I and-group II introns, by two transesterification reactions.

Common secondary structure of a group I intron in Tetrahymena, facilitated by internal pairing due to consensus sequences (P, Q and R, S) and the pairing at the ends due to internal guide sequence (IGS)
Fig. 33.4. Common secondary structure of a group I intron in Tetrahymena, facilitated by internal pairing due to consensus sequences (P, Q and R, S) and the pairing at the ends due to internal guide sequence (IGS).

In the presence of group I introns, self splicing of RNA can be achieved even in bacterial cells. Tfiis has been shown by inserting a group I intron interrupting the coding sequence of β-galactosidase enzyme. When such an interrupted gene for β-galactosidase is inserted in E. coli, the latter was found to be capable of synthesizing functional β-galactosidase enzyme. This suggested that group I intron in the RNA transcribed within E. coli was capable of self splicing. However, introns with conserved splicing junctions, found in higher eukaryotes, need spliceosomes formed by snRNA molecules and therefore can not be spliced out in bacterial cells.

A hammerhead secondary structure in viroids and virusoids showing site for self cleavage by arrow
Fig. 33.5. A hammerhead secondary structure in viroids and virusoids showing site for self cleavage by arrow.

Self splicing of group I and-group II introns, by two transesterification reactions
Fig. 33.3. Self splicing of group I and-group II introns, by two transesterification reactions.
Splicing of group II introns
Mitochondrial group II introns resemble nuclear introns and are excised as lariats like nuclear introns. They have consensus sequences at the splicing junctions, GT and APy and a branch sequence resembling TACTAAC box. The splicing reaction is autonomous unlike that in the nuclear introns, where trans-acting snRNPs take part in the formation of spliceosomes. Splicing in these group II introns gives rise to lariat directly, which then gives rise to a-linear form having no activity. This self splicing reaction may be regarded as an intermediate step between RNA mediated self splicing in group I introns and protein dependent RNA splicing of nuclear introns (Fig. 33.3 a).

Self splicing through formation of hammer-head
Incase of viroids (naked infectious RNA molecules capable of replicating) and virusoids = satellite RNA (encapsidated with plant viruses, and incapable of replicating independently), a consensus sequence forms a 'hammerhead' secondary structure, which helps in self cleavage (Fig. 33.5).
A hammerhead secondary structure in viroids and virusoids showing site for self cleavage by arrow
Fig. 33.5. A hammerhead secondary structure in viroids and virusoids showing site for self cleavage by arrow.

Self splicing of group I and-group II introns, by two transesterification reactions
Fig. 33.3. Self splicing of group I and-group II introns, by two transesterification reactions.