Exploring the link between RAG1 Cellular Distribution and Components of the CRL4DCAF1 E3 Ligase
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Authors
Erickson, Lukas
Issue Date
2025
Volume
Issue
Type
Thesis
Language
en_US
Keywords
DCAF1 , Degradation , Localization , RAG1
Alternative Title
Abstract
Prior work in the Swanson lab showed the turnover of RAG1 is dependenton DCAF1, the CRL4DCAF1 E3 ubiquitin ligase, and the 26S proteasome. RAG1
sequestration in nucleoli and nuclear puncta have been implicated in regulating its
activity. RAG1 nucleolar egress and puncta formation are dependent on the same
domain as DCAF1 association. We investigated whether DCAF1 or the CRL4
ligase is responsible for regulating RAG1 localization within HEK293T cells.
Treating transfected cells expressing RAG1 with inhibitors of the ubiquitin-
proteasome pathway did not change RAG1 localization during a time-lapse. The
distribution pattern of cells transfected with full-length RAG1 (FLRAG1) or core
RAG1 (cRAG1) and DCAF1 after incubation with the proteasome inhibitor,
bortezomib, revealed significantly reduced puncta compared to non-treated cells.
Unlike FLRAG1, cRAG1 half-life is not extended by proteasome inhibition, so we
assume this effect is indirect.
Co-expression of DCAF1 with FLRAG1 caused a decrease in its expression
and a non-significant trend toward an increased frequency of cells with diffuse
RAG1 distribution. The D1-DCAF1 mutant induced prominent nuclear puncta of
FLRAG1, an effect that was consistent but non-significant. D1-DCAF1 does not
associate with RAG1, so we hypothesize its effects were due to interference with
CRL4DCAF1 and RAG1. These data suggest DCAF1 encourages RAG1 nucleolar
egress and CRL4 association with the RAG1-DCAF1 complex serves to limit the
formation of RAG1 nuclear puncta.
Description
2025
Citation
Publisher
Creighton University
License
Copyright is retained by the Author.
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