Exploring the link between RAG1 Cellular Distribution and Components of the CRL4DCAF1 E3 Ligase

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.

Grantor dc:publisher

Creighton University

Year dc:date.issued

2025

Author dc:creator

• Erickson, Lukas

Advisor dc:contributor.advisor

• Swanson, Patrick

• DCAF1
• Degradation
• Localization
• RAG1

Statement dc:rights

• Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.

Language dc:language.iso

en_US

Repository record dc:identifier.uri

https://cdr.creighton.edu/handle/10504/158610

OAI identifier oai:identifier

oai:cdr.creighton.edu:10504/158610