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Alternatively spliced mRNAs encoding soluble isoforms of the erythropoietin receptor in murine cell lines and bone
C Barron1, A R Migliaccio, G Migliaccio
1Dipartimento di Genetica e di Biologia dei Microrganismi, Università degli Studi di Milano, Italy.
Abstract:
32D Epo and 32D GM cells are subclones of the murine 32D cell line which are selectively dependent for proliferation and survival on erythropoietin (Epo) or granulocyte/macrophage colony-stimulating factor (GM-CSF), respectively. 32D GM cells were previously shown to express significant levels of the Epo receptor mRNA and protein which was retained intracellularly and did not appear on the cell surface. We have now analyzed the EpoR mRNA from the 32D GM line, using PCR followed by direct sequencing. Several alternatively spliced products were detected. In some molecules, intron 5 (I5) or part of I6 or both were retained. Retention of I5 results in a mRNA potentially encoding an almost complete extracellular domain, while retention of I6 gives rise to a mRNA encoding the complete extracellular and transmembrane domains. A different type of splicing results in the loss of exon 5 (E5), giving rise to a sequence encoding a truncated extracellular domain. These alternatively spliced sequences are differentially represented in 32D Epo versus 32D GM cells. All are additionally present in normal bone marrow cells. Apart from these alternatively spliced EpoR RNAs, no other abnormalities were detected in EpoR RNA from 32D GM cells that could account for the intracellular retention of EpoR in the non-erythroid subclones of 32D.
Insights
Murine 32D GM cells exhibit intracellular erythropoietin receptor (EpoR) retention. Analysis revealed alternatively spliced EpoR mRNA variants, but no direct cause for this intracellular localization was found in these cells.
Area of Science:
- Cell Biology
- Molecular Biology
- Hematopoiesis
Background:
- 32D Epo and 32D GM cells are murine cell lines dependent on erythropoietin (Epo) or granulocyte/macrophage colony-stimulating factor (GM-CSF) respectively.
- 32D GM cells express Epo receptor (EpoR) mRNA and protein, but EpoR is retained intracellularly, not on the cell surface.
Purpose of the Study:
- To analyze the EpoR mRNA from 32D GM cells using PCR and direct sequencing.
- To identify alternative splicing products of EpoR mRNA and their differential representation in 32D Epo versus 32D GM cells.
Main Methods:
- Polymerase Chain Reaction (PCR) amplification of EpoR mRNA.
- Direct sequencing of PCR products.
- Analysis of alternatively spliced EpoR mRNA variants.
Main Results:
- Several alternatively spliced EpoR mRNA products were detected in 32D GM cells.
- These included retention of intron 5 (I5), part of intron 6 (I6), or both.
- Loss of exon 5 (E5) also occurred, leading to a truncated extracellular domain.
- These alternatively spliced sequences were differentially represented in 32D Epo and 32D GM cells and also present in normal bone marrow cells.
Conclusions:
- Alternative splicing of EpoR mRNA occurs in 32D GM cells, producing various isoforms.
- Despite the presence of alternatively spliced EpoR RNAs, no specific abnormality was identified to fully explain the intracellular retention of EpoR in 32D GM cells.