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Species-specific differential cleavage and polyadenylation of plasminogen activator inhibitor type 1 hnRNA
1Cardiovascular Division, Washington University School of Medicine, St Louis, MO 63110.
Abstract:
Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiologic inhibitor of the naturally occurring plasminogen activators. In higher primates two forms of mature PAI-1 mRNA (3.2 kb and 2.2 kb) arise by alternative cleavage and polyadenylation of PAI-1 hnRNA which is regulated in a tissue-specific fashion in humans. In other mammals only the 3.2 kb mRNA has been detected. The putative downstream polyadenylation site in humans that gives rise to the 3.2 kb PAI-1 mRNA consists of three overlapping copies of the consensus polyadenylation sequence while no consensus polyadenylation sequence is found upstream at a position that could generate the shorter mRNA species. To determine whether differential cleavage and polyadenylation of PAI-1 mRNA is due to species-specific differences in trans-acting factors that process PAI-1 mRNA or to the presence of a nonconsensus polyadenylation site acquired recently during primate evolution we prepared plasmids in which the 3' nontranslated region of the human PAI-1 gene or the mouse PAI-1 cDNA was inserted downstream of the neomycin gene in the plasmid pSV2neo. We show that the 3'-nontranslated region of the human PAI-1 gene but not the mouse PAI-1 cDNA conferred alternative cleavage and polyadenylation to the neomycin gene in transfected human Hep G2 cells as well as mouse NIH3T3 and rat L6 cells.
Insights
Human plasminogen activator inhibitor type 1 (PAI-1) mRNA exhibits alternative polyadenylation, producing distinct mRNA forms. This species-specific processing is linked to the human PAI-1 gene
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Plasminogen activator inhibitor type 1 (PAI-1) is a key regulator of fibrinolysis.
- Humans exhibit two mature PAI-1 mRNA forms (3.2 kb and 2.2 kb) due to alternative polyadenylation, unlike other mammals which only show the 3.2 kb form.
- The human PAI-1 gene possesses a unique downstream polyadenylation site, absent in other mammals, potentially explaining the shorter mRNA species.
Purpose of the Study:
- To investigate whether the differential cleavage and polyadenylation of PAI-1 mRNA is due to species-specific trans-acting factors or a primate-specific nonconsensus polyadenylation site.
- To determine the role of the 3' untranslated region of the human PAI-1 gene in alternative mRNA processing.
Main Methods:
- Constructed plasmids containing the 3' nontranslated region of the human PAI-1 gene or mouse PAI-1 cDNA downstream of the neomycin gene in pSV2neo.
- Transfected these plasmids into human Hep G2, mouse NIH3T3, and rat L6 cells.
- Analyzed the resulting neomycin gene mRNA products to assess alternative cleavage and polyadenylation.
Main Results:
- The 3'-nontranslated region of the human PAI-1 gene conferred alternative cleavage and polyadenylation to the neomycin gene in all tested cell lines (human, mouse, and rat).
- The mouse PAI-1 cDNA 3' nontranslated region did not induce alternative polyadenylation.
- This suggests the human PAI-1 gene's 3' region contains elements responsible for the observed species-specific mRNA processing.
Conclusions:
- The alternative cleavage and polyadenylation of human PAI-1 mRNA is primarily determined by features within the human PAI-1 gene's 3' untranslated region.
- This processing mechanism appears to be a recent evolutionary acquisition in primates.
- The findings highlight the role of specific gene sequences in generating transcript diversity across species.