136 7 Splicing and Alternative Splicing Impact on Gene Design
contain introns [30]. Furthermore, the location of introns within a gene is
strongly biased toward its 5′ end [31]. This bias is thought to arise due to homol-
ogous recombination of a gene’s cDNA with its genomic copy and could simulta-
neously explain how introns might have been lost during yeast evolution. cDNA
arises by reverse transcription of mRNA, which does not contain introns and is
a by-product of the activity of retrotransposons. Reverse transcription starts at
the 3′ of the mRNA and often terminates prematurely, which leads to a 3′ bias in
cDNAs and, as a result, to preferential loss of introns at the 3′ end of genes after
recombination of the cDNA with the genomic copy.
Furthermore, S. cerevisiae introns tend to be located in highly expressed
mRNAs. 27% of all mRNAs produced per hour are generated from the 5%
intron-containing genes [36]. Genome-wide analyses of mRNA [37] and protein
[38] levels showed that, on average, intron-containing genes produce ~3.9-fold
more RNA and 3.3-fold more protein than intronless genes.7.4 Splicing without the Spliceosome
7.4.1 Group I and Group II Self-Splicing Introns
Interrupted genes are found not only in the genomes of yeast and metazoan, but
are present in all classes of organisms. The majority of introns are spliced out by
the spliceosome (nuclear pre-mRNA introns). Besides this, self-splicing introns
(group I and group II) exist, in which the intervening sequences can excise them-
selves from the RNA in an autocatalytic manner [39].
Group I and II introns are found in the DNA of organelles, bacteria, and the
nucleus of lower eukaryotes (group I only). Their occurrence is more sporadic in
bacteria than in lower eukaryotes and is most common in the organelles of higher
plants. Whereas group II introns are mainly found in organelles, group I introns
interrupt rRNA, mRNA, and tRNA in bacteria, as well as in the organelles of
lower eukaryotes, and some plants. In addition, they have been found in several
bacteriophages.
Nuclear pre-mRNA introns are defined by cis-acting sequence elements that
are recognized by the spliceosome. Group I and group II introns, in contrast,
adopt a typical secondary structure that contains distinct domains, which then
folds into a highly complex tertiary structure. As a consequence, the catalytic
mechanism of this splicing reaction solely depends on the sequence and the cor-
rect folding of the intron. The RNA tertiary structure brings the 5′ and the 3′
splice sites in close proximity and generates a catalytic site. The fold is stabilized
by several magnesium ions, allowing the RNA to perform the splicing reaction in
vitro by itself, without any enzymatic activities provided by proteins. Proteins are
required only to assist correct folding of the complex structure in vivo. For this
fundamental discovery that RNA can harbor catalytic function, Tom R. Cech
(together with Sidney Altman) was awarded with the Nobel Prize in Chemistry
in 1989 [40].
For group I introns, the only factors required for autosplicing are monovalent
and divalent cations and a guanine nucleotide cofactor. The 3′ hydroxyl group of