Related Experiment Videos
Differential splicing and alternative polyadenylation generate multiple mimecan mRNA transcripts
E S Tasheva1, L M Corpuz, J L Funderburgh
1Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901, USA. est@ksu.edu
Abstract:
We previously showed the 25-kDa corneal keratan sulfate proteoglycan to be a translation product of the gene producing osteoglycin and proposed the name mimecan for this gene and its product. We also demonstrated three mimecan RNA transcripts using Northern blot analysis. In this report, we investigate the mechanisms accounting for these transcripts. Ribonuclease protection analysis and reverse transcription-polymerase chain reaction of bovine corneal mRNA detected a mimecan transcript that lacked 278 base pairs of the 5'-untranslated region between residues 62 and 340. This splice variant represents the predominant form of mimecan mRNA in bovine cornea and sclera. It was also detectable in other bovine tissues as a minor transcript. Two additional cDNA clones that were isolated contained 398 bases of nucleotide sequence at the 3'-end of mimecan cDNA, not present in the published sequence. Ribonuclease protection analyses with the 3'-probe, which included the new sequence, allow detection of three RNA transcripts while 5'-probes recognized only two. These results indicate that the three canonical polyadenylation sites in the 3'-untranslated region of mimican mRNA are alternatively selected. Possible roles for this previously undetected degree of diversity of mimecan RNA isoforms transcribed in the same tissue are discussed.
Insights
Researchers identified a novel splice variant of the mimecan (osteoglycin) gene, which is the predominant form in bovine cornea and sclera. Alternative polyadenylation also contributes to mimecan RNA diversity.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The 25-kDa corneal keratan sulfate proteoglycan, known as mimecan, is a translation product of the osteoglycin gene.
- Previous studies identified three mimecan RNA transcripts, but the mechanisms generating this diversity were unclear.
Purpose of the Study:
- To investigate the mechanisms responsible for the generation of multiple mimecan RNA transcripts.
- To characterize splice variants and alternative polyadenylation in mimecan gene expression.
Main Methods:
- Ribonuclease protection analysis (RPA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to analyze bovine corneal mRNA.
- cDNA cloning was employed to identify novel sequences in the mimecan gene.
Main Results:
- A predominant mimecan mRNA splice variant lacking 278 base pairs in the 5'-untranslated region was detected in bovine cornea and sclera.
- Two additional cDNA clones revealed novel sequences at the 3'-end of mimecan cDNA.
- Alternative selection of three canonical polyadenylation sites in the 3'-untranslated region was confirmed.
Conclusions:
- The diversity of mimecan RNA isoforms arises from both alternative splicing and alternative polyadenylation.
- These findings highlight a previously unrecognized level of complexity in mimecan gene expression within the same tissue.