Dissecting the role of RNA helicase DDX3X in mRNA translation and B-cell function during immune responses

During B-cell immune responses, germinal centre structures are the primary sites of antibody affinity maturation and a common site of B-cell lymphomagenesis. Mutations in DEAD-box helicase 3X (DDX3X) have previously been found in B-cell lymphomas driven by the transcription factor MYC. Reduced DDX3X function buffers the effect of MYC on the upregulation of global translation, ultimately lowering the levels of proteotoxic stress and enabling lymphoma development. These findings suggest the functions of MYC and DDX3X are related but how this occurs in the context of physiological B-cell responses has remained largely unexplored. Given the critical role for MYC in driving selective B-cell expansion in germinal centres, we hypothesise that DDX3X plays a role in antibody affinity maturation by promoting the increase in protein synthesis triggered downstream of MYC upregulation. Over the course of this project, we have developed a novel mouse model where we can acutely deplete DDX3X protein using a chemically inducible degron system. We have utilised this tool in vivo to demonstrate that when DDX3X is degraded, we observe a reduction in antigen-specific B-cell clones in germinal centres during a T-cell dependent immune response. Additionally, we degraded the protein ex vivo and established a role for DDX3X in protein synthesis during B-cell activation. To further characterise this process, we have compared time points after B-cell activation on both the RNA and protein level to markers of mRNA translation in germinal centre B-cells. Furthermore, we have performed ribosome profiling studies to establish which mRNAs are differentially translated between WT and DDX3X-depleted B-cells. Ultimately, our aim is to establish whether the 5’UTR structure of mRNA is playing a role in the translation of DDX3X-sensitive transcripts in B-cells, and we have begun work on this by implementing a state-of-the-art single-molecule methodology that allows RNA structures to be found in living cells using chemical modification and long-read sequencing. We have generated the first in cellulo RNA structure dataset in lymphocytes, and hope to utilise this in follow up work investigating DDX3X target genes with potential importance for germinal centre biology. These studies provide evidence that the RNA helicase DDX3X plays a crucial role in controlling mRNA translation during normal B-cell immune responses. Our work has the potential to shed light on mechanisms at play during B-cell lymphomagenesis and, more generally, in other conditions where DDX3X function is impaired.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Taylor, Ella

Advisor dc:contributor.advisor

• Ribeiro de Almeida, Claudia

• RNA helicases
• Humoral immune responses
• B-cells
• DDX3X

Licence

• https://www.repository.cam.ac.uk/bitstreams/7d3bb821-8cf7-4e92-bd74-ce51080fdcbd/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.127877

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/399296