# Exploring the Role of RAG1 in Post-transcriptional Gene Regulation

> **NIH NIH R21** · CHILDREN'S HOSP OF PHILADELPHIA · 2020 · $220,000

## Abstract

PROJECT SUMMARY/ABSTRACT
RAG1/RAG2 (RAG) endonuclease-mediated assembly of antigen receptor genes in developing B and T cells
is essential for adaptive immunity. Yet, this process has fatal risks as evidenced by generation of self-reactive
receptors and formation of oncogenic antigen receptor locus translocations. The RAG proteins are comprised
of core domains essential for recombination and non-core regions that control the induction and repair of RAG
DNA double strand breaks (DSBs). In 2000, mutations of the RAG1 N-terminus non-core region were found to
cause Omenn Syndrome, a B and T lymphocyte immunodeficiency linked with autoimmunity. Since then, the
RAG1 N-terminus has been discovered to contain an ubiquitin ligase and interact with another ubiquitin ligase
and a kinase. However, it remains unknown how the RAG1 N-terminus promotes lymphocyte differentiation
and tolerance by mechanisms beyond modulating RAG activity. Igκ and Igλ B cells develop from pre-B cells.
The applicant has established mouse models and a primary pre-B cell culture system to elucidate functions of
the RAG1 N-terminus in B cell development. RAG DSBs induced during Igκ recombination in pre-B cells signal
transcriptional activation of the cytoplasmic Pim2 kinase, which promotes survival of pre-B cells with RAG Igκ
DSBs to promote Igλ B cell development. The applicant now demonstrates that Rag1C/C mice lacking the Rag1
N-terminus have impaired development of Igλ B cells and decreased stability and expression of Pim2 protein
induced by RAG Igκ DSBs. He shows that Rag1PG/PG mice lacking Rag1 ubiquitin ligase activity have a similar
defect in Igλ B cell development, implying that RAG1-mediated protein ubiquitination is required for RAG DSBs
to inhibit proteasome-mediated Pim2 degradation. The PI hypothesizes that RAG1 ubiquitin ligase activity at
RAG Igκ DSBs triggers intracellular signals that post-transcriptionally shape the pre-B cell DSB response and
thereby orchestrate B cell development. The applicant proposes to test fundamental aspects of this hypothesis
by determining how the RAG1 N-terminus stabilizes Pim2 protein (Aim 1) and post-transcriptionally shapes the
pre-B cell proteome to modulate expression of proteins that control B cell development (Aim 2). This study will
cement the innovative concept that RAG1 has critical functions that transcend its role in cleaving and helping
repair DNA as a RAG subunit. The results should yield novel insights into mechanisms that coordinate Igκ and
Igλ recombination, pre-B cell survival, and B cell receptor signaling to create broad protective antigen receptor
repertoires and prevent autoimmunity. Considering that autoimmunity in Omenn Syndrome mainly manifests in
the T cell compartment, knowledge and approaches from this project will lay the strong foundation for a future
RO1 grant and other avenues of basic and translational immunology research.

## Key facts

- **NIH application ID:** 9831122
- **Project number:** 5R21AI135435-02
- **Recipient organization:** CHILDREN'S HOSP OF PHILADELPHIA
- **Principal Investigator:** CRAIG H BASSING
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $220,000
- **Award type:** 5
- **Project period:** 2018-12-04 → 2020-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9831122

## Citation

> US National Institutes of Health, RePORTER application 9831122, Exploring the Role of RAG1 in Post-transcriptional Gene Regulation (5R21AI135435-02). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/9831122. Licensed CC0.

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