Identification and comparative analysis of Pax5 C-terminal isoforms expressed in human cord blood-derived B cell progenitors.
We identified three Pax5 isoforms due to alternative splicing of the C-terminal exons of its gene in cord blood (CB)-derived B cell progenitors cultivated on the murine bone marrow stromal (HESS-5) cells. Apart from wild type (wt), one isoform skips exon 9 without subsequent frameshift (del9), while the other has a frameshift insert between exons 8 and 9, resulting in novel C-terminal sequences (ins8'). Quantitative reverse transcription-polymerase chain reaction analysis revealed that wt mRNA could be detected in CB CD34(+) cells, but that del9 and ins8' isoforms only appeared after 1 or 3 weeks of co-culture, respectively. Expression of each isoform mRNA was markedly upregulated during B cell differentiation in vitro, and wild type continued to be the most abundant isoform. In a luciferase reporter assay using a synthetic CD19 enhancer, del9 isoform revealed slightly lower activity and ins8' isoform showed much lower activity, compared with Pax5-wt. Furthermore, retroviral expression of each Pax5 isoform in CB CD34(+) cells induced aberrant CD19 expression in a fraction of immature myeloid cells after 1 week of culture, although del9 and ins8' isoforms showed much less potent activity than Pax5-wt. These results suggest that Pax5-wt is quantitatively and qualitatively dominant over other C-terminal isoforms during human B cell differentiation.
Sekine
Rieko
R
Division of Molecular Therapy, Institute of Medical Science, University of Tokyo, Tokyo, Japan.
Kitamura
Toshio
T
Tsuji
Takashi
T
Tojo
Arinobu
A
D017398:Alternative Splicing; D018952:Antigens, CD34 / Q000378:metabolism; D001402:B-Lymphocytes / Q000166:cytology* / Q000276:immunology; D001483:Base Sequence; D002454:Cell Differentiation / Q000276:immunology; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D005260:Female; D005312:Fetal Blood / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D051757:PAX5 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D011247:Pregnancy; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018952:Antigens, CD34; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human; D020033:Protein Isoforms