Abstract
At least four alternatively spliced mRNAs can be synthesized from the human AT1R (hAT1R) gene that differ only in the inclusion or exclusion of exon 2 and/or 3. RT-PCR experiments demonstrate that splice variants harboring exon 2 accounts for at least 30% of all the hAT1R mRNA transcripts expressed in the human tissues investigated. Since exon 2 contains two upstream AUGs or open reading frames (uORFs), we hypothesized that these AUGs would inhibit the translation of the downstream hAT1R protein ORF harbored in exon 4. This study demonstrates that the inclusion of exon 2 in hAT1R mRNA transcripts dramatically reduces hAT1R protein levels (nine-fold) and significantly attenuates Ang II responsiveness (∼four-fold). Interestingly, only when both AUGs were mutated in combination were the hAT1R density and Ang II signaling levels comparable with those values obtained using mRNA splice variants that did not include exon 2. This observation is consistent with a model where the majority of the ribosomes likely translate uORF#1 and are then unable to reinitiate at the downstream hAT1R ORF, in part due to the presence of AUG#2 and to the short intercistronic spacing. Importantly, TGF-β1 treatment (4 ng/ml for 4 h) of fibroblasts up-regulated hAT1R mRNA splice variants, which harbored exon 2, six-fold. Since AT1R activation is closely associated with cardiovascular disease, the inclusion of exon 2 by alternative splicing represents a novel mechanism to reduce the overall production of the hAT1R protein and possibly limit the potential pathological effects of AT1R activation.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 21-31 |
| Number of pages | 11 |
| Journal | Molecular and Cellular Endocrinology |
| Volume | 249 |
| Issue number | 1-2 |
| DOIs | |
| State | Published - Apr 25 2006 |
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Keywords
- Alternative splicing
- Angiotensin II receptors
- Translational regulation
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Endocrinology
Cite this
TGF-β1 regulation of human AT1 receptor mRNA splice variants harboring exon 2. / Martin, Mickey M.; Buckenberger, Jessica A.; Knoell, Daren L.; Strauch, Arthur R.; Elton, Terry S.
In: Molecular and Cellular Endocrinology, Vol. 249, No. 1-2, 25.04.2006, p. 21-31.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - TGF-β1 regulation of human AT1 receptor mRNA splice variants harboring exon 2
AU - Martin, Mickey M.
AU - Buckenberger, Jessica A.
AU - Knoell, Daren L.
AU - Strauch, Arthur R.
AU - Elton, Terry S.
PY - 2006/4/25
Y1 - 2006/4/25
N2 - At least four alternatively spliced mRNAs can be synthesized from the human AT1R (hAT1R) gene that differ only in the inclusion or exclusion of exon 2 and/or 3. RT-PCR experiments demonstrate that splice variants harboring exon 2 accounts for at least 30% of all the hAT1R mRNA transcripts expressed in the human tissues investigated. Since exon 2 contains two upstream AUGs or open reading frames (uORFs), we hypothesized that these AUGs would inhibit the translation of the downstream hAT1R protein ORF harbored in exon 4. This study demonstrates that the inclusion of exon 2 in hAT1R mRNA transcripts dramatically reduces hAT1R protein levels (nine-fold) and significantly attenuates Ang II responsiveness (∼four-fold). Interestingly, only when both AUGs were mutated in combination were the hAT1R density and Ang II signaling levels comparable with those values obtained using mRNA splice variants that did not include exon 2. This observation is consistent with a model where the majority of the ribosomes likely translate uORF#1 and are then unable to reinitiate at the downstream hAT1R ORF, in part due to the presence of AUG#2 and to the short intercistronic spacing. Importantly, TGF-β1 treatment (4 ng/ml for 4 h) of fibroblasts up-regulated hAT1R mRNA splice variants, which harbored exon 2, six-fold. Since AT1R activation is closely associated with cardiovascular disease, the inclusion of exon 2 by alternative splicing represents a novel mechanism to reduce the overall production of the hAT1R protein and possibly limit the potential pathological effects of AT1R activation.
AB - At least four alternatively spliced mRNAs can be synthesized from the human AT1R (hAT1R) gene that differ only in the inclusion or exclusion of exon 2 and/or 3. RT-PCR experiments demonstrate that splice variants harboring exon 2 accounts for at least 30% of all the hAT1R mRNA transcripts expressed in the human tissues investigated. Since exon 2 contains two upstream AUGs or open reading frames (uORFs), we hypothesized that these AUGs would inhibit the translation of the downstream hAT1R protein ORF harbored in exon 4. This study demonstrates that the inclusion of exon 2 in hAT1R mRNA transcripts dramatically reduces hAT1R protein levels (nine-fold) and significantly attenuates Ang II responsiveness (∼four-fold). Interestingly, only when both AUGs were mutated in combination were the hAT1R density and Ang II signaling levels comparable with those values obtained using mRNA splice variants that did not include exon 2. This observation is consistent with a model where the majority of the ribosomes likely translate uORF#1 and are then unable to reinitiate at the downstream hAT1R ORF, in part due to the presence of AUG#2 and to the short intercistronic spacing. Importantly, TGF-β1 treatment (4 ng/ml for 4 h) of fibroblasts up-regulated hAT1R mRNA splice variants, which harbored exon 2, six-fold. Since AT1R activation is closely associated with cardiovascular disease, the inclusion of exon 2 by alternative splicing represents a novel mechanism to reduce the overall production of the hAT1R protein and possibly limit the potential pathological effects of AT1R activation.
KW - Alternative splicing
KW - Angiotensin II receptors
KW - Translational regulation
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UR - http://www.scopus.com/inward/citedby.url?scp=33646474556&partnerID=8YFLogxK
U2 - 10.1016/j.mce.2006.01.009
DO - 10.1016/j.mce.2006.01.009
M3 - Article
C2 - 16504375
AN - SCOPUS:33646474556
VL - 249
SP - 21
EP - 31
JO - Molecular and Cellular Endocrinology
JF - Molecular and Cellular Endocrinology
SN - 0303-7207
IS - 1-2
ER -