Evolutionarily Conserved Intronic Splicing Regulatory Elements in the Human Genome


Splicing is the process by which introns are removed from premature messenger ribonucleic acid (pre‐mRNA), and exons are ligated to form mature mRNA before translation into protein products. Aside from the consensus splice signals such as the branch point, acceptor and donor splice sites, additional cis‐regulatory elements embedded within the exons and introns control the recognition of exonic (approximately 150 bases) from intronic sequence (approximately 100–100 000 bases). Intronic splicing elements that are evolutionarily conserved across multiple species play important roles in the regulation of constitutive splicing, where a single mRNA is produced, and alternative splicing, where multiple mRNA isoforms are generated.

Keywords: alternative splicing; intronic splicing regulatory elements; evolution; bioinformatics; comparative genomics

Figure 1.

Various methods through which conserved (or nonconserved) intronic splicing regulatory elements can be identified. (a) Early work focused on identifying AS events for a specific gene of interest. Once an event was identified, the researchers could mutate or delete various intronic regions to identify specific regulatory regions that were critical for regulation of splicing of that specific exon. (b) Systematic evolution of ligands by exponential enrichment (SELEX) can be performed to identify the most preferred binding site of a splicing factor. A consensus binding site can be generated from the enriched RNAs after several rounds of SELEX. (c) Experimental screens for splicing regulatory elements can be conducted by inserting random k‐mers into a splicing sensitive reporter, which enables the selection of k‐mers that caused exon‐skipping or inclusion. The list of k‐mers can then be clustered to identify core regulatory elements. (d) The sequencing of mature mRNA transcripts isolated from various human tissues, either by full‐length sequencing (cDNAs) or by sequencing expressed sequence tags (ESTs), alternatively spliced events can be identified in an unbiased genome‐wide manner. These events could then either be directly analysed for enrichment of specific intronic motifs, or could be used to identify smaller subsets of apparent tissue‐specific events that could then yield tissue‐specific intronic elements. (e) Recent microarray technologies have allowed high‐throughput identification of AS events either by probing for the presence of specific exon junctions (top) that would indicate skipping of an internal exon, or by probing for the expression of each individual exon (bottom), where exon skipping will cause a single exon to have far lower expression than other exons in this gene. By repeating this analysis on multiple tissue samples tissue‐specific AS events can thus be identified in an automated and high‐throughput matter, and can then be analysed for intronic regulatory motifs in the same manner described in (d).



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Further Reading

Ladd AN and Cooper TA (2002) Finding signals that regulate alternative splicing in the post‐genomic era. Genome Biology 3(11): reviews0008.1–reviews0008.16.

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Van Nostrand, Eric L, and Yeo, Gene W(Jul 2008) Evolutionarily Conserved Intronic Splicing Regulatory Elements in the Human Genome. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021005]