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Regulated tissue-specific alternative splicing of enhanced green fluorescent protein transgenes conferred by alpha-tropomyosin regulatory elements in transgenic mice.
J Biol Chem. 2004 Aug 27; 279(35):36660-9.JB

Abstract

The mutually exclusive exons 2 and 3 of alpha-tropomyosin (alphaTM) have been used as a model system for strictly regulated alternative splicing. Exon 2 inclusion is only observed at high levels in smooth muscle (SM) tissues, whereas striated muscle and non-muscle cells use predominantly exon 3. Experiments in cell culture have shown that exon 2 selection results from repression of exon 3 and that this repression is mediated by regulatory elements flanking exon 3. We have now tested the cell culture-derived model in transgenic mice. We show that by harnessing the intronic splicing regulatory elements, expression of an enhanced green fluorescent protein transgene with a constitutively active promoter can be restricted to SM cells. Splicing of both endogenous alphaTM and a series of transgenes carrying regulatory element mutations was analyzed by reverse transcriptasePCR. These studies indicated that although SM-rich tissues are equipped to regulate splicing of high levels of endogenous or transgene alphaTM RNA, other non-SM tissues such as spleen, which express lower amounts of alphaTM, also splice significant proportions of exon 2, and this splicing pattern can be recapitulated by transgenes expressed at low levels. We confirm the importance in vivo of the negatively acting regulatory elements for regulated skipping of exon 3. Moreover, we provide evidence that some of the regulatory factors responsible for exon 3 skipping appear to be titratable, with loss of regulated splicing sometimes being associated with high transgene expression levels.

Authors+Show Affiliations

Department of Biochemistry, Tennis Court Road, University of Cambridge, Cambridge CB2 1QW, United Kingdom.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15194683

Citation

Ellis, Peter D., et al. "Regulated Tissue-specific Alternative Splicing of Enhanced Green Fluorescent Protein Transgenes Conferred By Alpha-tropomyosin Regulatory Elements in Transgenic Mice." The Journal of Biological Chemistry, vol. 279, no. 35, 2004, pp. 36660-9.
Ellis PD, Smith CW, Kemp P. Regulated tissue-specific alternative splicing of enhanced green fluorescent protein transgenes conferred by alpha-tropomyosin regulatory elements in transgenic mice. J Biol Chem. 2004;279(35):36660-9.
Ellis, P. D., Smith, C. W., & Kemp, P. (2004). Regulated tissue-specific alternative splicing of enhanced green fluorescent protein transgenes conferred by alpha-tropomyosin regulatory elements in transgenic mice. The Journal of Biological Chemistry, 279(35), 36660-9.
Ellis PD, Smith CW, Kemp P. Regulated Tissue-specific Alternative Splicing of Enhanced Green Fluorescent Protein Transgenes Conferred By Alpha-tropomyosin Regulatory Elements in Transgenic Mice. J Biol Chem. 2004 Aug 27;279(35):36660-9. PubMed PMID: 15194683.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Regulated tissue-specific alternative splicing of enhanced green fluorescent protein transgenes conferred by alpha-tropomyosin regulatory elements in transgenic mice. AU - Ellis,Peter D, AU - Smith,Christopher W J, AU - Kemp,Paul, Y1 - 2004/06/11/ PY - 2004/6/15/pubmed PY - 2004/10/7/medline PY - 2004/6/15/entrez SP - 36660 EP - 9 JF - The Journal of biological chemistry JO - J Biol Chem VL - 279 IS - 35 N2 - The mutually exclusive exons 2 and 3 of alpha-tropomyosin (alphaTM) have been used as a model system for strictly regulated alternative splicing. Exon 2 inclusion is only observed at high levels in smooth muscle (SM) tissues, whereas striated muscle and non-muscle cells use predominantly exon 3. Experiments in cell culture have shown that exon 2 selection results from repression of exon 3 and that this repression is mediated by regulatory elements flanking exon 3. We have now tested the cell culture-derived model in transgenic mice. We show that by harnessing the intronic splicing regulatory elements, expression of an enhanced green fluorescent protein transgene with a constitutively active promoter can be restricted to SM cells. Splicing of both endogenous alphaTM and a series of transgenes carrying regulatory element mutations was analyzed by reverse transcriptasePCR. These studies indicated that although SM-rich tissues are equipped to regulate splicing of high levels of endogenous or transgene alphaTM RNA, other non-SM tissues such as spleen, which express lower amounts of alphaTM, also splice significant proportions of exon 2, and this splicing pattern can be recapitulated by transgenes expressed at low levels. We confirm the importance in vivo of the negatively acting regulatory elements for regulated skipping of exon 3. Moreover, we provide evidence that some of the regulatory factors responsible for exon 3 skipping appear to be titratable, with loss of regulated splicing sometimes being associated with high transgene expression levels. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/15194683/Regulated_tissue_specific_alternative_splicing_of_enhanced_green_fluorescent_protein_transgenes_conferred_by_alpha_tropomyosin_regulatory_elements_in_transgenic_mice_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=15194683 DB - PRIME DP - Unbound Medicine ER -