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Multiple mRNA species are generated by alternate polyadenylation from the human calmodulin-I gene
S Senterre-Lesenfants1, A S Alag, M E Sobel
1Molecular Pathology Section, Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland 20892, USA.
Abstract:
Three distinct genes encode an identical calmodulin protein in mammalian cells. In addition, multiple mRNA transcripts, with approximate sizes of 1.6 kb and 4.4 kb, are visualized on Northern blots hybridized to calmodulin-I cDNA probes. To elucidate the mechanism generating multiple calmodulin mRNAs, the complete sequence of the 4194 base human calmodulin-I mRNA was determined from cDNA clones and 3' rapid amplification of complementary ends (3' RACE). The 5' untranslated region of calmodulin-I mRNA contains a GC-rich domain containing multiple repeats of GGC interrupted by a GCA sequence, as well as a tandem repeat sequence of eight GCA triplets. The 3' untranslated region of calmodulin-I mRNA contains two canonical and one aberrant (ATTAAA) polyadenylation signal, consistent with the sizes of 1.6 kb and 4.4 kb mRNAs visualized on Northern blots, and a potential minor 4.2 kb mRNA detected by 3' RACE. Hybridization experiments using specific probes upstream and downstream of the polyadenylation signals demonstrated that alternate use of polyadenylation signals is the molecular mechanism for multiple calmodulin-I mRNA transcripts in human cells. Thirteen adenine rich elements with the motif AUUUA were detected in the 3' untranslated region. Three such motifs are embedded in regions that are conserved with the rat 3' untranslated region of calmodulin-I mRNA. One of these is surrounded by an adenine-uridine rich region that can form an 11-base pair stem structure. We propose that sequences in the 3' untranslated region of calmodulin-I mRNA may play a role in the regulation of calmodulin expression.
Insights
Multiple calmodulin messenger RNA (mRNA) transcripts arise from alternative polyadenylation signal usage in human cells. This mechanism explains the different mRNA sizes observed, potentially regulating calmodulin gene expression.
Area of Science:
- Molecular Biology
- Gene Expression Regulation
- Genomics
Background:
- Mammalian cells express an identical calmodulin protein from three distinct genes.
- Multiple calmodulin mRNA transcripts of approximately 1.6 kb and 4.4 kb are observed.
- The mechanism generating these multiple calmodulin mRNA transcripts remains unclear.
Purpose of the Study:
- To elucidate the molecular mechanism responsible for generating multiple calmodulin mRNA transcripts.
- To determine the complete sequence of the human calmodulin-I mRNA.
- To investigate the role of 3' untranslated regions in calmodulin gene expression regulation.
Main Methods:
- Sequencing of the human calmodulin-I mRNA (4194 bases) using cDNA clones and 3' rapid amplification of complementary ends (3' RACE).
- Northern blot analysis to visualize mRNA transcripts.
- Hybridization experiments with specific probes to analyze polyadenylation signal usage.
Main Results:
- The 3' untranslated region of calmodulin-I mRNA contains two canonical and one aberrant polyadenylation signal (ATTAAA).
- Alternate use of these polyadenylation signals is the molecular mechanism generating 1.6 kb and 4.4 kb calmodulin-I mRNA transcripts.
- Thirteen AUUUA motifs, potential regulatory elements, were identified in the 3' untranslated region, some conserved across species.
Conclusions:
- Alternative polyadenylation signal usage is the primary mechanism for generating multiple calmodulin-I mRNA transcripts in human cells.
- The 3' untranslated region of calmodulin-I mRNA, with its AUUUA motifs, likely plays a significant role in regulating calmodulin expression.
- Understanding these regulatory mechanisms is crucial for comprehending calmodulin's role in cellular functions.