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An altered intracellular distribution of the autoantigen La/SS-B when translated from a La mRNA isoform
1Institut für Physiologische Chemie, Johannes-Gutenberg Universitat, Mainz, Germany.
Abstract:
Transcription of the gene encoding for the nuclear autoantigen La resulted in La mRNA isoforms. A promoter switching combined with an alternative splicing pathway replaced exon 1 with exon 1'. Similar to mRNAs encoding for ribosomal proteins, exon 1' started with a pyrimidine-rich 5'-terminus. Moreover, exon 1' contained 5'-GC-rich regions and an oligo(U)-tail of 23 uridine residues. Exon 1' encoded for three open reading frames upstream of the La protein reading frame. In spite of this unusual structure, exon 1' La mRNAs were translated not only in vitro but also in transiently transfected cells. The translational efficiency of exon 1' La mRNA was about 14% of exon 1 La mRNA using rabbit reticulolysate for in vitro translation. Finally, we established permanently transfected mouse cell lines expressing the human exon 1 or exon 1' La mRNA isoform. In all cell lines the respective La mRNAs were translated to La protein. The exon 1 La mRNA-expressing cell lines displayed a mostly nuclear staining pattern. In contrast, a major portion of La protein was found in the cytoplasm of cell lines expressing exon 1' La mRNA.
Insights
Novel La mRNA isoforms, generated through promoter switching and alternative splicing, are translated into functional La protein. The exon 1
Area of Science:
- Molecular biology
- Gene regulation
- Protein synthesis
Background:
- The nuclear autoantigen La protein plays a role in cellular processes.
- La mRNA isoforms arise from alternative splicing and promoter usage.
- Understanding La mRNA structure and function is crucial for comprehending gene regulation.
Purpose of the Study:
- To investigate the structural and functional characteristics of novel La mRNA isoforms.
- To determine the translational efficiency and protein localization of different La mRNA variants.
- To establish cell models for studying the impact of La mRNA isoforms on protein expression and localization.
Main Methods:
- Analysis of mRNA structure, including 5'-terminus and oligo(U)-tail identification.
- In vitro translation assays using rabbit reticulolysate.
- Transient and stable transfection of mouse cell lines with human La mRNA isoforms.
- Immunofluorescence microscopy to determine protein localization.
Main Results:
- A novel La mRNA isoform (exon 1') was identified, featuring a pyrimidine-rich 5'-terminus, GC-rich regions, and an oligo(U)-tail.
- Exon 1' La mRNA contains upstream open reading frames but is still translated.
- The translational efficiency of exon 1' La mRNA is approximately 14% of the exon 1 isoform.
- Cell lines expressing exon 1' La mRNA show predominantly cytoplasmic localization of La protein, unlike the nuclear localization seen with exon 1 La mRNA.
Conclusions:
- Alternative splicing and promoter switching generate functional La mRNA isoforms with distinct structural features.
- The exon 1' La mRNA isoform, despite its unusual structure, is translated into La protein.
- The alternative exon 1' influences the subcellular localization of the La protein, with a shift towards the cytoplasm.