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Gene and pseudogene of the mouse cation-dependent mannose 6-phosphate receptor. Genomic organization, expression, and
T Ludwig1, U Rüther, R Metzger
1European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Abstract:
The cation-dependent mannose 6-phosphate receptor (CD-MPR) is one of the two transmembrane proteins involved in transport of lysosomal enzymes. We have cloned the mouse CD-MPR gene and also a very unusual processed-type CD-MPR pseudogene. They are both present at one copy per haploid genome and map to chromosomes 6 and 3, respectively. Comparison of the complete 10-kilobase (kb) sequence of the functional gene with the cDNA indicates that it contains seven exons. Exon 1 encodes the 5'-untranslated region of the mRNA, the others (exons 2-7) encode the luminal, transmembrane, and cytoplasmic domains of the CD-MPR. Exon 7 also contains a 1.2-kb-long 3'-untranslated region of the mRNA. A unique transcription-initiation site was determined by primer extension of mouse liver mRNA. The promoter elements in the 5' upstream region of this site resemble those contained in genes constitutively transcribed. However, Northern blot analysis demonstrates that the CD-MPR is variably expressed in adult mouse tissues and during mouse development. The pseudogene, which is flanked by direct repeats, is almost colinear with the cDNA indicating that it presumably arose by reverse transcription of an mRNA. However, the pseudogene differs from the cDNA. It contains at its 5' end, an additional 340-nucleotide (nt) sequence homologous to the promoter region of the functional gene. This sequence exhibits some promoter activity in vitro. Furthermore, a 24-nt insertion interrupts the region homologous to the 5'-noncoding region of the cDNA. In the functional gene, this 24-nt sequence occurs between exon 1 and 2, where it is flanked by typical consensus sequences of exon/intron boundaries. Therefore, it may represent an additional exon of the functional gene. These two features of the pseudogene suggest that expression of the CD-MPR gene may be regulated by use of different promoters and/or alternative splicing.
Insights
Researchers cloned the mouse cation-dependent mannose 6-phosphate receptor (CD-MPR) gene and a pseudogene. Analysis suggests CD-MPR gene expression may be regulated by alternative promoters and splicing.
Area of Science:
- Genetics
- Molecular Biology
- Cell Biology
Background:
- The cation-dependent mannose 6-phosphate receptor (CD-MPR) is crucial for lysosomal enzyme transport.
- Understanding CD-MPR gene structure and regulation is key to comprehending lysosomal targeting.
Purpose of the Study:
- To clone and characterize the mouse CD-MPR gene and a related pseudogene.
- To investigate the structural features and potential regulatory mechanisms of CD-MPR gene expression.
Main Methods:
- Gene cloning and sequencing of the functional CD-MPR gene and its pseudogene.
- Primer extension analysis to identify the transcription-initiation site.
- Northern blot analysis to assess gene expression patterns.
- In vitro promoter activity assays.
Main Results:
- The functional CD-MPR gene comprises seven exons, encoding key receptor domains.
- A processed pseudogene was identified, containing promoter-like sequences and an insertion.
- The pseudogene's features suggest potential alternative promoter usage and alternative splicing for CD-MPR regulation.
- CD-MPR expression varies across mouse tissues and developmental stages.
Conclusions:
- The mouse CD-MPR gene structure was elucidated, revealing its exon-intron organization.
- The identified pseudogene provides insights into potential regulatory mechanisms of CD-MPR gene expression.
- Alternative promoter utilization and alternative splicing may play roles in regulating CD-MPR function.