2015 Theses Doctoral
RNA Exosome Regulated Antisense and Divergent Noncoding RNA Facilitate AID Targeting Throughout the B Cell Genome
Vertebrate immune systems are armed with the ability to generate highly specific immune responses capable of responding to nearly any foreign molecular threat. One of the major mediators of this response is immunoglobulins (Igs) produced by B lymphocytes. The specificity of individual Igs is created through a tightly orchestrated series of somatic DNA manipulations at Ig encoding loci resulting in functional gene rearrangements and nucleotide substitutions. These events serve to create a pool of naive B cells expressing Igs with distinct specificities, capable of expansion in response to antigen specific selection. Affinity of Ig towards antigen is enhanced through nucleotide substitutions introduced at the antigen binding variable region gene segments through the enzyme activation induced cytidine deaminase (AID) during the process of somatic hypermutation (SHM). AID also generates point mutations within noncoding DNA segments of the Ig heavy chain locus that are processed into double strand breaks leading to constant region isotype switching during class switch recombination (CSR).
The Ig diversification processes of SHM and CSR critically depend upon transcriptional activation of the relevant DNA segments. Transcription is thought to facilitate single strand DNA substrate recognition by AID during unwinding of the DNA duplex. The 3'-5' exoribonuclease RNA exosome serves as a transcription dependent cofactor of AID. RNA exosome is comprised of multiple structurally integral core subunits
and associated nuclease subunits. In this work, RNA exosome core subunit Exosc3 and nuclease Exosc10 have been targeted for conditional mutagenesis and loss of function analysis in mouse cells. RNA exosome deficient B cells were significantly impaired in AID dependent SHM and CSR Ig diversification processes. Transcriptome analyses revealed a striking accumulation of promoter proximal antisense divergent noncoding transcripts (xTSS-RNA) at a subset of genes upon loss of RNA exosome function. xTSS-RNAs mark regions of chromatin containing RNA exosome activity. Multiple known AID target sites including IgH and Myc were observed to express xTSS-RNA. Furthermore, genomic sites of recurrent AID dependent chromosomal translocations were enriched for xTSS-RNA. In addition to promoter proximal xTSS-RNA, cryptic intragenic antisense noncoding transcripts were found to accumulate at many genomic loci. In fact, multiple translocation hotspots precisely overlap regions of RNA exosome sensitive antisense transcription. AID targeted divergently transcribed promoters containing RNA exosome substrates possessed greater amounts of RNA:DNA hybrids, indicative of frequent transcriptional arrest. Lastly, RNA exosome deficient transcriptomes have revealed a substantial number of novel long intergenic noncoding RNAs and enhancer RNAs, indicating a hidden layer of cellular transcriptional activity.
A model of AID targeting utilizing transcriptional arrest is becoming increasingly apparent. Transcribed chromatin prone to undergo transcriptional arrest, such as Ig loci or xTSS-RNA expressing regions, frequently undergoes premature transcription termination coupled to RNA exosome mediated degradation of the nascent transcript. This process results in the creation of AID substrates and serves to stabilize its association with chromatin through multiple interactions involving RNA exosome and transcription complex subunits.
- Pefanis_columbia_0054D_12447.pdf binary/octet-stream 22.8 MB Download File