Ring finger protein 121 is a potent regulator of adeno-associated viral genome transcription.

PLoS Pathogens
Victoria J MadiganAravind Asokan

Abstract

Adeno-associated viruses (AAV) are Dependoparvoviruses that have shown promise as recombinant vectors for gene therapy. While infectious pathways of AAV are well studied, gaps remain in our understanding of host factors affecting vector genome expression. Here, we map the role of ring finger protein 121 (RNF121), an E3 ubiquitin ligase, as a key regulator of AAV genome transcription. CRISPR-mediated knockout of RNF121 (RNF121 KO) in different cells markedly decreased AAV transduction regardless of capsid serotype or vector dose. Recombinant AAV transduction is partially rescued by overexpressing RNF121, but not by co-infection with helper Adenovirus. Major steps in the AAV infectious pathway including cell surface binding, cellular uptake, nuclear entry, capsid uncoating and second strand synthesis are unaffected. While gene expression from transfected plasmids or AAV genomes is unaffected, mRNA synthesis from AAV capsid-associated genomes is markedly decreased in RNF121 KO cells. These observations were attributed to transcriptional arrest as corroborated by RNAPol-ChIP and mRNA half-life measurements. Although AAV capsid proteins do not appear to be direct substrates of RNF121, the catalytic domain of the E3 ligase appears es...Continue Reading

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Citations

Apr 19, 2020·Human Gene Therapy·Anna C Maurer, Matthew D Weitzman
Oct 20, 2020·Trends in Molecular Medicine·Bijay P DhungelJohn E J Rasko
Jul 8, 2021·Journal of Virology·L Patrick HavlikAravind Asokan

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Methods Mentioned

BETA
Immunoprecipitation
ubiquitination
transfection
flow cytometry
acetylation
confocal microscopy
pull down
RNA seq
affinity-purification
pull-down

Software Mentioned

IDT
R
DeSeq2
StringDB
MiST ( mass spectrometry interaction statistics )
SubRead
GraphPad Prism
BBMAP

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