The overall objective of this proposal is to elucidate a critical process in immunoglobulin (Ig) isotype switching: the mechanism by which the specificity of Ig isotype switch is determined and ultimately achieved. We will test the hypothesis that chimeric Ig germline mRNA transcripts serve as the specific templates that determine the specificity of Ig class switch recombination with emphasis on switching to IgE. We have shown that such chimeric mRNA are derived from trans-splicing of germline Ig pre-mRNAs of C mu and the germline pre-mRNAs of downstream isotype(s). These chimeric Ig germline mRNAs, by Hoogsteen base-pairing to their corresponding double stranded DNA region, are ideal templates to function as """"""""bridging templates"""""""". This bridging will bring mu switch and the involved downstream isotype switch regions in close proximity and thereby direct Ig switch recombination. To test this hypothesis, we will determine in human primary B cells whether transfection of constructs producing specific chimeric Ig germline MRNA transcripts in anti-sense orientation blocks specific Ig switching recombination and isotype production and whether transfection of chimeric Ig germline MRNA in sense orientation specifically enhances switch recombination and Ig production. We will quantitatively determine the kinetics of appearance of chimeric Ig germline MRNA transcripts and their relationship to Ig isotype switching. To better define the role of chimeric Ig germline transcripts in Ig switching, we will determine the ability of known switch signals to regulate trans-splicing and the resulting production of chimeric Ig germline MRNAS. As the selection and pattern of switch recombination sites (i.e. primary vs. secondary recombination) has important implications for Ig isotype production and stabilization, we will extend our hypothesis by testing if the specific structure of chimeric Ig germline transcripts (3'-5' vs 5'-3' chimeras) have distinct abilities to mediate primary or secondary recombination predicting that 3'-5' chimeras (e.g. I epsilon-C mu transcripts) will mediate primary recombination whereas 5'-3' chimeras (e.g. I mu-C epsilon transcripts) will mediate secondary recombination. Finally, we will test the hypothesis that repetitive G bases in RNA (""""""""G4-RNA"""""""") as found in Ig switch regions plays a key role in promoting the trans-splicing of Ig germline pre-mRNA transcripts. Employing a cell free in vitro splicing system, we will analyze the effects of G4-RNA formation on Ig trans-splicing. Furthermore we will determine the ability of chimeric Ig germline mRNAs to pair in vitro to the corresponding region of double stranded DNA through Hoogsteen base pairing.
