Abstract
The primary mRNAs or the pre-mRNAs frequently undergo splicing that involves excision of
intron sequences and ligation of the coding exon sequences. Across tissues and conditions,
within an individual plant and animal, the splicing can occur in more than one way for a pre-
mRNA giving rise to alternative mRNA transcripts (isoforms/splice forms). It is known that such
alternative splicing is the major mechanism of producing the diversity across the transcriptomes
and/or proteomes of tissues using the same genome (Nilsen and Graveley, 2010).
Specific short sequences of nucleotides forming the splice sites and regulatory motifs are located
in the introns and exons and such 'motifs' can be used to select or de-select a specific type of
intron-excision and exon-ligation. Thus, the motifs can act as enhancers or silencers of splicing.
A number of studies revealed that pre-mRNA sequences also harbor structurally identifiable
elements in vivo that may regulate splicing. Using such motifs and structural features, the cells
seem to splice the pre-mRNAs in multiple ways. Various splice-events seem to have evolved to
increase the protein repertoire of the cell. Conventional types of alternative splicing mechanisms
are exon skipping, alternative 3‘and 5‘ splice sites, intron retention, mutually exclusive exons,
alternative promoters and alternative poly adenylation (A) sites. While many studies have
indicated the relative frequency of different types of splice events, more specific studies are
warranted, specifically to study their frequency across differently expressed tissues in mammals,
even under normal conditions. The analysis of motifs in the context of different type of splice
events is particularly at a very preliminary stage for every species/group. Hence, the current
study attempts to exploit recently established sets of tissue-specific and ubiquitously transcribed
genes to compare the types of alternative splicing-events, and then explore the possible sequence
and structural features across the major types of events.
The presence of these RNA secondary structures is usually predicted in
silico by computational programs like Mfold, RNAfold, RNAStructure, Sfold, etc and are
confirmed by in vitro (by biochemical methods) and in vivo (by small scale experiments and
mutational analysis) studies. Though very little is understood regarding their role in alternative
splicing, the studies indicate that pre-mRNAs, being highly flexible in nature forms secondary
structures (e.g. stem loop structures), whichare likely to play role in inclusion/exclusion of a
particular exon(s), or in selection of 5‘ splice sites or 3‘ splice sites (donor or acceptor) and thus
determine the variety of transcript-isoforms. In our attempt to understand structures of pre-
mRNAs that play a role in alternative splicing events of human, mouse and rat species, we have extracted published information on 30 mammalian genes for which pre-mRNA secondary
structural analysis was done either in silico or in vitro or in vivo.
Exon skipping events involving cassette exons are the predominant type of alternative splicing
(Ast, 2004). Earlier studies showed that 74% alternative splicing events are tissue specific
(Wanget al, 2008). However, evaluating the distribution of tissue specific/cell
type/developmental stage specific splice-events has not been easy for researchers despite the
huge amount of genome- & transcriptome-wide experimental data. With the aim to understand
tissue specific splice-events, a comparative analysis was performed in the current study on
alternative splice events of 200 genes belonging to testis exclusive and ubiquitously expressed
sets, obtained from MGEx-Tdb (Acharya et al, 2010). This analysis showed that apart from exon
skipping, alternative promoter driven splice events are enriched in the testis exclusive genes.
Alternative 5‘ and 3‘ splice sites comprise moderately abundant splice-events. Thus the results
indicate that at genes expressed preferentially in different tissues show varied frequencies of
splice-events and thereby generate the protein diversity. Our data also reveals that intra-exon
deletions, along with intron retention, mutually exclusive events and alternative poly adenylation
(A) sites, also form the minor splice-events across the tissues.
With the aim to identify splice-event specific motifs, sequence analysis was performed. A
distinct set of 3‘ ends of introns of cassette exons and constitutive exons were used for this
purpose. Occurrence and distribution of experimentally proven human branch point motifs were
analyzed across the intron-exon junctions. Though there is no significant difference in the
arrangement of branch point motifs, the study discovered a few novel motifs found within 100
nucleotide length intron-exon junctions of cassette and constitutive exons. A specific novel motif
of 19 bases length is particularly interesting as it i