Related Experiment Videos
Alternative processing of the human FMO6 gene renders transcripts incapable of encoding a functional
Ronald N Hines1, Kathleen A Hopp, Jose Franco
1Department of Pediatrics, Birth Defects Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226-4801, USA. rhines@mail.mcw.edu
Abstract:
The flavin-containing monooxygenases (FMOs) are a family of five microsomal enzymes important for the oxidative metabolism of environmental toxicants, natural products, and therapeutics. With the exception of FMO5, the FMO are encoded within a single gene cluster on human chromosome 1q23-25. As part of the human genome effort, an FMO-like gene, FMO6, was identified between FMO3 and FMO2 (GenBank accession no. AL021026). Sequence analysis of this putative FMO family member revealed nothing that would a priori argue against a functional gene and encoded protein. When FMO6 expression was examined by reverse transcriptase coupled polymerase chain reaction DNA amplification, transcripts were identified in 8 of 11 human liver samples, but 0 of 4 kidney biopsy samples. However, in all cases, the observed transcripts were shorter than predicted. Sequence analysis revealed skipping of exon 4, exons 3 and 4, and/or the use of alternative splice donor or acceptor sites in introns 3, 4, 6, and 8, resulting in nine unique transcripts. Based on an analysis of possible open reading frames, none of these transcripts would encode a functional FMO enzyme. Taking advantage of the high sequence identity between FMO3 and FMO6, it is posited that the loss of binding sites for the serine-arginine-rich splicing factor protein family within exons 3 and 4 contributes to the exon skipping events, although the most commonly observed alternative splice event results from a 21-bp insertion immediately 3' to the predicted FMO6 intron 8 splice acceptor site, diminishing the efficiency of this site.
Insights
The flavin-containing monooxygenase 6 (FMO6) gene, identified in the human genome, produces non-functional transcripts due to alternative splicing. These splicing variations prevent the encoding of a functional FMO enzyme.
Area of Science:
- Human molecular genetics
- Enzymology
- Genomics
Background:
- Flavin-containing monooxygenases (FMOs) are crucial microsomal enzymes involved in metabolizing xenobiotics and endogenous compounds.
- FMOs, except FMO5, are located in a gene cluster on human chromosome 1q23-25.
- A putative FMO-like gene, FMO6, was identified within this cluster.
Purpose of the Study:
- To investigate the expression and functional potential of the FMO6 gene.
- To characterize the transcripts produced by the FMO6 gene.
Main Methods:
- Reverse transcriptase coupled polymerase chain reaction (RT-PCR) DNA amplification was used to examine FMO6 expression in human liver and kidney samples.
- Sequence analysis was performed on identified FMO6 transcripts.
- Analysis of open reading frames was conducted to assess protein-coding potential.
Main Results:
- FMO6 transcripts were detected in human liver samples but not in kidney samples.
- All detected FMO6 transcripts were shorter than predicted, indicating aberrant splicing.
- Nine unique transcripts were identified, characterized by exon skipping and alternative splice site usage.
- Sequence analysis revealed that none of the identified transcripts could encode a functional FMO enzyme.
Conclusions:
- The FMO6 gene, despite initial sequence analysis suggesting potential functionality, produces only non-functional transcripts in human tissues examined.
- Alternative splicing events, including exon skipping and altered splice site recognition, are responsible for the lack of functional FMO6 protein.
- The loss of splicing factor binding sites and a specific insertion near an intron acceptor site are proposed mechanisms contributing to these splicing abnormalities.