In Vivo Recognition of a Vertebrate Mini-Exon as an Exon-Intron-Exon Unit

Abstract

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

Selection of Olduvai Domains during Evolution: A Role for Primate-Specific Splicing Super-Enhancer and RNA Guanine Quadruplex in Bipartite NBPF Exons

“…Small exons (~60 nts or less) have been associated with poor inclusion in messenger RNAs [ 16 , 17 , 18 , 19 ]. This length limit also applies to constitutively spliced internal exons, which have an optimal size range between 60 and 200 nts [ 19 ].…”

“…This length limit also applies to constitutively spliced internal exons, which have an optimal size range between 60 and 200 nts [ 19 ]. Although smaller exons may lack cross-exon interactions with spliceosomal components, many mini- or even micro-exons are efficiently recognised in vivo in a constitutively or alternatively spliced manner [ 17 , 20 ]. Such exons are often located very close to additional exons upstream or downstream that are separated by short introns withregulatory functions [ 17 ].…”

“…Although smaller exons may lack cross-exon interactions with spliceosomal components, many mini- or even micro-exons are efficiently recognised in vivo in a constitutively or alternatively spliced manner [ 17 , 20 ]. Such exons are often located very close to additional exons upstream or downstream that are separated by short introns withregulatory functions [ 17 ]. The requirement for flanking exons disappeared when mini-exons were expanded [ 17 ].…”

“…Such exons are often located very close to additional exons upstream or downstream that are separated by short introns withregulatory functions [ 17 ]. The requirement for flanking exons disappeared when mini-exons were expanded [ 17 ]. The splicing of an intron was also enhanced when coupled with the splicing of a downstream intron, possibly through a mechanism independent of exon junction complex depositions [ 19 ], further adding to the evidence that individual exons are not selected independently.…”

Microexon alternative splicing of small GTPase regulators: Implication in central nervous system diseases

“…Additional pioneering work contributed to the microexon splicing network and this consisted of the work of Sterner and Berget (1993), who initially utilized microexon (7 nt) from Troponin I gene and identified that the upstream exon was required for inclusion of microexon (Sterner & Berget, 1993). In subsequent studies, they used the microexon (6 nt) from the chicken cardiac troponin gene and showed that an intron splicing enhancer element (ISE) containing G‐rich sequence was essential for microexon inclusion.…”