Reina-San-Martin, B. | Search bioRxiv

bioRxiv2024

Gene-specific RNA homeostasis revealed by perturbation of the NuA4/Tip60 acetyltransferase complex

Transcript buffering entails the reciprocal modulation of mRNA synthesis and degradation rates to maintain stable RNA levels under varying cellular conditions. Current research supports a global, non-sequence-specific connection between mRNA synthesis and degradation, but the underlying mechanisms are still unclear. In this study, we investigated changes in RNA metabolism following acute depletion of TIP60/KAT5, the acetyltransferase subunit of the NuA4 transcriptional coactivator complex, in mouse embryonic stem cells. By combining RNA sequencing of nuclear, cytoplasmic, and newly synthesised transcript fractions with biophysical modelling, we demonstrate that TIP60 predominantly enhances transcription of numerous genes, while a smaller set of genes undergoes TIP60-dependent transcriptional repression. Surprisingly, transcription changes caused by TIP60 depletion were offset by corresponding changes in RNA nuclear export and cytoplasmic stability, indicating gene-specific buffering mechanisms. Similarly, disruption of the unrelated ATAC coactivator complex also resulted in gene-specific transcript buffering. These findings reveal that transcript buffering functions at a gene-specific level and suggest that cells dynamically adjust RNA splicing, export, and degradation in response to individual RNA synthesis alterations, thereby sustaining cellular homeostasis.

genomics↗

bioRxiv2020

Fam72a controls the balance between error-prone and error-free DNA repair during antibody diversification.

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. B cell receptor diversification through somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by cytidine deamination in DNA mediated by activation induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by Uracil DNA glycosylase (UNG) and by mismatch repair (MMR) proteins2-4. Although uracils arising in DNA are faithfully repaired2-7, it is not known how these pathways are co-opted to generate mutations and double stranded DNA breaks (DSBs) in the context of SHM and CSR2,4,8. Here we have performed a genome-wide CRISPR/Cas9 knockout screen for genes involved in CSR. The screen identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)9 and that is overexpressed in several cancers9. We show that the FAM72A-UNG2 interaction controls the protein levels of UNG2 and that CSR is defective in Fam72a-/- B cells due to the specific upregulation of UNG2. Moreover, we show that in Fam72a-/- B cells SHM is reduced by 5-fold and that upregulation of UNG2 results in a skewed mutation pattern. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation. Consequently, deficiency in Fam72a leads to supraphysiological levels of UNG2 and enhanced uracil excision, shifting the balance from error-prone to error-free DNA repair. Our findings have potential implications for tumorigenesis, as Fam72a overexpression would lead to reduced UNG2 levels, shifting the balance toward mutagenic DNA repair and rendering cells more prone to acquire mutations.

immunology↗