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Mouse peripherin isoforms
F Landon1, A Wolff, B de Néchaud
1Université Paris 6, faculté de médecine Pitié-Salpêtrière, CNRS UMR 7000, France. landon@idf.ext.jussieu.fr
Abstract:
Three distinct mRNAs have been shown to be produced by alternative splicing from the unique mouse peripherin gene. They generate three translation products, one major form, Pe-58, and two minor forms, Pe-56 which possess a shorter C-terminal sequence, and Pe-61 in which an additional sequence has been inserted in the central rod domain (Landon et al., 1989, EMBO J. 8, 1719-1726). In this study, the simultaneous occurrence of multiple transcripts in murine nervous tissues and neuroblastoma cell lines was shown by PCR amplification of fragments overlapping the sites of alternative splicing. Recombinant peripherin isoforms were purified from E. coli expressing full-length cDNAs. Rabbit antisera were raised against synthetic peptides mimicking parts of the two C-terminal sequences and of the inserted sequence of Pe-61 and were immunoadsorbed until they became monoreactive. By western blot analysis, the peripherin isoforms were localised in neuroblastoma NB2a cell lysates and detergent insoluble fractions separated by two-dimensional electrophoresis. In addition, each isoform was resolved into several charge variants. At the cellular level, each antibody decorated the filament array of the NB2a cells, suggesting the participation of the minor peripherin isoforms in the intermediate filament network.
Insights
Mouse peripherin gene alternative splicing produces multiple mRNA transcripts and protein isoforms. These peripherin isoforms, including minor ones, are present in nervous tissues and contribute to intermediate filament networks.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- The mouse peripherin gene undergoes alternative splicing to produce distinct mRNAs.
- These mRNAs translate into three protein isoforms: Pe-58 (major), Pe-56 (shorter C-terminus), and Pe-61 (inserted central domain).
Purpose of the Study:
- To investigate the simultaneous occurrence of multiple peripherin transcripts in murine nervous tissues and neuroblastoma cell lines.
- To characterize the peripherin isoforms and their cellular localization within intermediate filament networks.
Main Methods:
- Polymerase Chain Reaction (PCR) amplification to detect alternative splicing events.
- Recombinant expression of peripherin isoforms in E. coli.
- Generation and immunoadsorption of specific rabbit antisera against peripherin sequences.
- Western blot analysis and two-dimensional electrophoresis for isoform localization and charge variant identification.
Main Results:
- Multiple peripherin transcripts were confirmed in murine nervous tissues and neuroblastoma cell lines.
- Peripherin isoforms were localized in neuroblastoma cell lysates and detergent-insoluble fractions.
- Each isoform exhibited several charge variants.
- Antibodies against peripherin isoforms decorated the intermediate filament network in NB2a cells.
Conclusions:
- Minor peripherin isoforms, previously less characterized, are present and functional in neuronal cells.
- These isoforms actively participate in the formation and maintenance of the intermediate filament network.