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Mutually exclusive splicing regulates the Nav 1.6 sodium channel function through a combinatorial mechanism that involves three distinct splicing regulatory elements and their ligands.

Zubović L, Baralle M, Baralle FE - Nucleic Acids Res. (2012)

Bottom Line: These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins.These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors.The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB) 34012, Trieste, Italy.

ABSTRACT
Mutually exclusive splicing is a form of alternative pre-mRNA processing that consists in the use of only one of a set of two or more exons. We have investigated the mechanisms involved in this process for exon 18 of the Na(v) 1.6 sodium channel transcript and its significance regarding gene-expression regulation. The 18N exon (neonatal form) has a stop codon in phase and although the mRNA can be detected by amplification methods, the truncated protein has not been observed. The switch from 18N to 18A (adult form) occurs only in a restricted set of neural tissues producing the functional channel while other tissues display the mRNA with the 18N exon also in adulthood. We demonstrate that the mRNA species carrying the stop codon is subjected to Nonsense-Mediated Decay, providing a control mechanism of channel expression. We also map a string of cis-elements within the mutually exclusive exons and in the flanking introns responsible for their strict tissue and temporal specificity. These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins. These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors. The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.

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Analysis of effect of the ESE and the ESS on the alternative splicing of SCN8A exon 18N and 18A. (A) Schematic representation of the minigenes based on the backbone of the SCN8A WT minigene that spans SCN8A exons 17 through to 19. Two slanted parallel lines indicate SCN8A break in endogenous intronic region. T7 and SP6 indicate primers, specifically to the plasmid, used to amplify specifically the transfected mRNA (B) RT–PCR analysis of the minigenes after transfection in HeLa cells. Right hand side of gel: indication of which exons are included in each amplicon.
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gks249-F5: Analysis of effect of the ESE and the ESS on the alternative splicing of SCN8A exon 18N and 18A. (A) Schematic representation of the minigenes based on the backbone of the SCN8A WT minigene that spans SCN8A exons 17 through to 19. Two slanted parallel lines indicate SCN8A break in endogenous intronic region. T7 and SP6 indicate primers, specifically to the plasmid, used to amplify specifically the transfected mRNA (B) RT–PCR analysis of the minigenes after transfection in HeLa cells. Right hand side of gel: indication of which exons are included in each amplicon.

Mentions: The analysis of the splicing regulatory elements was up to now performed in a reductive minigene context. It was then of interest to investigate any effect the ESE in exon 18N and the ESS in exon 18A may have on the ME-splicing outcome. Transfections were therefore performed with a SCN8A WT minigene that spans exons 17 to 19, encompassing both the 18N and 18A exons. In addition a series of related constructs were studied, in particular the SCN8A/ΔESE E18N minigene in which the ESE was deleted from exon 18N and the minigene SCN8A/ΔESS E18A in which the ESS in the exon 18A was deleted. Furthermore, in order to analyze the interplay between the two elements we also created the minigene SCN8A/ΔESE E18N/ΔESS E18A where both regulatory elements were deleted and the minigene SCN8A/+ ESS E18N in which the ESS from the exon 18A was created in exon 18N in an identical position to that found in exon 18A with minimal nucleotide changes and leaving the ESE intact (Figure 5A).Figure 5.


Mutually exclusive splicing regulates the Nav 1.6 sodium channel function through a combinatorial mechanism that involves three distinct splicing regulatory elements and their ligands.

Zubović L, Baralle M, Baralle FE - Nucleic Acids Res. (2012)

Analysis of effect of the ESE and the ESS on the alternative splicing of SCN8A exon 18N and 18A. (A) Schematic representation of the minigenes based on the backbone of the SCN8A WT minigene that spans SCN8A exons 17 through to 19. Two slanted parallel lines indicate SCN8A break in endogenous intronic region. T7 and SP6 indicate primers, specifically to the plasmid, used to amplify specifically the transfected mRNA (B) RT–PCR analysis of the minigenes after transfection in HeLa cells. Right hand side of gel: indication of which exons are included in each amplicon.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401437&req=5

gks249-F5: Analysis of effect of the ESE and the ESS on the alternative splicing of SCN8A exon 18N and 18A. (A) Schematic representation of the minigenes based on the backbone of the SCN8A WT minigene that spans SCN8A exons 17 through to 19. Two slanted parallel lines indicate SCN8A break in endogenous intronic region. T7 and SP6 indicate primers, specifically to the plasmid, used to amplify specifically the transfected mRNA (B) RT–PCR analysis of the minigenes after transfection in HeLa cells. Right hand side of gel: indication of which exons are included in each amplicon.
Mentions: The analysis of the splicing regulatory elements was up to now performed in a reductive minigene context. It was then of interest to investigate any effect the ESE in exon 18N and the ESS in exon 18A may have on the ME-splicing outcome. Transfections were therefore performed with a SCN8A WT minigene that spans exons 17 to 19, encompassing both the 18N and 18A exons. In addition a series of related constructs were studied, in particular the SCN8A/ΔESE E18N minigene in which the ESE was deleted from exon 18N and the minigene SCN8A/ΔESS E18A in which the ESS in the exon 18A was deleted. Furthermore, in order to analyze the interplay between the two elements we also created the minigene SCN8A/ΔESE E18N/ΔESS E18A where both regulatory elements were deleted and the minigene SCN8A/+ ESS E18N in which the ESS from the exon 18A was created in exon 18N in an identical position to that found in exon 18A with minimal nucleotide changes and leaving the ESE intact (Figure 5A).Figure 5.

Bottom Line: These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins.These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors.The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.

View Article: PubMed Central - PubMed

Affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB) 34012, Trieste, Italy.

ABSTRACT
Mutually exclusive splicing is a form of alternative pre-mRNA processing that consists in the use of only one of a set of two or more exons. We have investigated the mechanisms involved in this process for exon 18 of the Na(v) 1.6 sodium channel transcript and its significance regarding gene-expression regulation. The 18N exon (neonatal form) has a stop codon in phase and although the mRNA can be detected by amplification methods, the truncated protein has not been observed. The switch from 18N to 18A (adult form) occurs only in a restricted set of neural tissues producing the functional channel while other tissues display the mRNA with the 18N exon also in adulthood. We demonstrate that the mRNA species carrying the stop codon is subjected to Nonsense-Mediated Decay, providing a control mechanism of channel expression. We also map a string of cis-elements within the mutually exclusive exons and in the flanking introns responsible for their strict tissue and temporal specificity. These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins. These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors. The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1.

Show MeSH
Related in: MedlinePlus