Development of an isoform-specific gene suppression system: the study of the human Pax-5B transcriptional element.
The transcription factor Pax-5, is vital during B lymphocyte differentiation and is known to contribute to the oncogenesis of certain cancers. The Pax-5 locus generates multiple yet structurally related mRNA transcripts through the specific activation of alternative promoter regions and/or alternative splicing events which poses challenges in the study of specific isoform function. In this study, we investigated the function of a major Pax-5 transcript, Pax-5B using an enhanced version of the Hepatitis Delta Virus ribozyme (HDV Rz) suppression system that is specifically designed to recognize and cleave the human Pax-5B mRNA. The activity of these ribozymes resulted in the specific suppression of the Pax-5B transcripts without altering the transcript levels of other closely related Pax-5 isoforms mRNAs both in vitro and in an intracellular setting. Following stable transfection of the ribozymes into a model B cell line (REH), we showed that Pax-5B suppression led to an increase of CD19 mRNA and cell surface protein expression. In response to this Pax-5B specific deregulation, a marked increase in apoptotic activity compared to control cell lines was observed. These results suggest that Pax-5B has distinct roles in physiological processes in cell fate events during lymphocyte development.
Robichaud
Gilles A
GA
Département de biochimie, RNA Group/Groupe ARN, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, J1H 5N4, Canada.
Perreault
Jean-Pierre
JP
Ouellette
Rodney J
RJ
D018941:Antigens, CD19 / Q000096:biosynthesis / Q000235:genetics; D017209:Apoptosis; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D005786:Gene Expression Regulation; D005818:Genetic Engineering; D003698:Hepatitis Delta Virus / Q000201:enzymology; D006801:Humans; D051757:PAX5 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D020871:RNA Stability; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
D018941:Antigens, CD19; D051757:PAX5 Transcription Factor; C497281:PAX5 protein, human; D020033:Protein Isoforms; D016337:RNA, Catalytic; D012333:RNA, Messenger; D014157:Transcription Factors