Basu Laboratory Research

Activation induced deaminase (AID) mediated mutations on Both strands of DNA substrate

Activation Induced cytidine Deaminase (AID) initiates Immunoglobulin (Ig) heavy chain (IgH) class switch recombination (CSR) and Ig variable region somatic hyper mutation (SHM) in B lymphocytes by deaminating cytidines on template and non-template strands of transcribed DNA substrates. However, the mechanism of AID access to the template DNA strand, particularly when hybridized to a nascent RNA transcript, has been an enigma. We now implicate the RNA exosome, a cellular RNA processing/degradation complex, in targeting AID to both DNA strands.

 In B lineage cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions in an AID dependent fashion, and is required for optimal CSR. Moreover, both the cellular RNA exosome complex and a recombinant RNA exosome core complex impart robust AID and transcription dependent DNA deamination of both strands of transcribed SHM substrates in vitro. Our findings reveal a role for non-coding RNA surveillance machinery in generating antibody diversity.

RNA exosome substrate antisense transcription, AID and B Cell genomic mutagenesis mechanisms

The immunoglobulin diversification processes of somatic hyper mutation and class switch recombination critically rely on transcription coupled targeting of AID to Ig loci in activated B lymphocytes. AID catalyzes deamination of cytidine deoxynucleotides on exposed single stranded DNA. In addition to driving immunoglobulin diversity, promiscuous targeting of AID mutagenic activity poses a deleterious threat to genomic stability. Recent genome wide studies have uncovered pervasive AID activity throughout the B cell genome.

It is increasingly apparent that AID activity is frequently targeted to genomic loci undergoing early transcription termination where RNA exosome promotes the resolution of stalled transcription complexes via co transcriptional RNA degradation mechanisms. In our laboratory, we study consequences of eukaryotic transcription that lead to RNA exosome recruitment, and ultimately AID mediated mutagenesis of the B cell genome.

Long non-coding RNA and enhancer RNA transcription that control distal DNA regulatory element interactions

We have ablated the cellular RNA degradation machinery in differentiated B cells and pluripotent embryonic stem (ES) cells by conditional mutagenesis of core (Exosc3) and nuclear RNase (Exosc10) components of RNA exosome and identified a vast  number of novel long noncoding RNAs (lncRNAs) and enhancer RNAs (eRNAs). Unexpectedly, eRNA expressing regions accumulate Rloop structures upon RNA exosome ablation, thus demonstrating the role of RNA exosome in resolving deleterious DNA/RNA hybrids arising from active enhancers. We have uncovered a distal divergent eRNAexpressing element (lncRNACSR) engaged in long range DNA interactions and regulating IgH 3’ regulatory region superenhancer function.

CRISPR-Cas9 mediated ablation of lncRNACSR transcription decreases its chromosomal looping mediated association with the IgH 3’regulatory region superenhancer and leads to decreased class switch recombination efficiency. We propose that the RNA exosome protects divergently transcribed lncRNA expressing enhancers, by resolving deleterious transcription coupled secondary DNA structures, while also regulating long range superenhancer chromosomal interactions important for cellular function.


RNA polymerase II stalling regulation

Activation Induced Deaminase (AID) function is essential for initiation of class switch recombination (CSR) and somatic hyper mutation (SHM) in B lymphocytes. AID deaminates cytidine residues on substrate sequences in the immunoglobulin(Ig) locus via a transcription dependent mechanism. This activity is stimulated by the eleven subunit cellular noncoding RNA 3’- 5’ exonucleolytic processing complex, RNA exosome. The mechanism by which the RNA exosome recognizes the free 3’end of immunoglobulin locus specific RNA substrates and stimulates AID DNA deamination activity on its in vivo substrate sequences is an important question.

 Here we report that the cellular HECT E3-ligase, Nedd4, supports AID function during CSR by controlling RNA polymerase II associated AID/RNA exosome complex function in B cells. We observe that Nedd4 ubiquitinates RNA pol II for degradation, thus providing a mechanism for generation of Ig locus specific 3’end free germline transcripts. In this study we link noncoding RNA processing following RNA polymerase I pausing with regulation of the mutator AID protein.