Biology subjects

Buchumenski, I.

Publications and source records attributed to Buchumenski, I..

3 recordsLinked to original sources

bioRxiv2024

PACT prevents aberrant activation of PKR by endogenous dsRNA without sequestration

The innate immune sensor PKR for double-stranded RNA (dsRNA) is critical for antiviral defense, but its aberrant activation by cellular dsRNA is linked to various diseases. The dsRNA-binding protein PACT plays a critical yet controversial role in the PKR pathway. We demonstrate that PACT is a direct and specific suppressor of PKR against endogenous dsRNA ligands like inverted-repeat Alu RNAs, which robustly activate PKR in the absence of PACT. PACT-mediated inhibition does not involve competition for dsRNA binding. Instead, PACT impairs PKRs ability to scan along dsRNA--a process necessary for PKR molecules to encounter and autophoshorylate each other for activation. By scanning along dsRNA and directly interacting with PKR, PACT restricts PKRs movement on dsRNA, reducing the likelihood of PKR molecular collisions and subsequent autophosphorylation, effectively inhibiting PKR without sequestering dsRNA. Consequently, PKR inhibition is more robust with longer and less abundant dsRNA, and minimal with abundant or short dsRNA. Thus, PACT functions to adjust the PKR activation threshold for long endogenous dsRNA without altering its inherent activity, revealing new mechanisms for establishing self-tolerance.

biochemistry↗

bioRxiv2024

The prevalent NR2E3 c.932G>A mutation induces aberrant splicing that can be rescued using splice-shifting antisense oligonucleotides

Mutations in NR2E3 have been implicated in several progressive retinal disease phenotypes such as enhanced S-cone syndrome, Goldmann-Favre syndrome and retinitis pigmentosa. One of the most frequent mutations in NR2E3 is c.932G>A (p.R311Q), where pathogenicity is thought to stem from the resulting amino acid substitution. However, multiple studies that evaluated the effect of this substitution on the protein, did not elucidate the molecular basis underlying the pathogenicity. Primed by bioinformatic analyses, we hypothesized and experimentally validated that the NR2E3 c.932G>A mutation leads to aberrant splicing which results in a short, non-functional protein isoform. Using cell models expressing WT and mutant constructs of the full NR2E3 sequence (including exonic and intronic regions), we observed that the mutated transcript exhibits a high level (75%) of aberrant splicing through gain of a novel splice acceptor site within exon 6. This mis-splicing results in the in-frame loss of 186 base pairs that code for a portion of the protein ligand binding domain. We further designed and evaluated splice-shifting antisense oligonucleotides (ASOs), that circumvented the aberrant splicing. The best performing ASO successfully restored 70% of the total NR2E3 full-length isoform levels and demonstrated rescue of nuclear localization and rhodopsin transcriptional activation. This study demonstrates the importance of understanding splicing consequences of pathogenic mutations, allowing the design and development of ASO-based therapies. Our findings set the stage for the potential treatment of NR2E3-related retinal degeneration caused by the c.932G>A mutation using splice-shifting ASOs.

genomics↗

bioRxiv2017

A-to-I RNA editing uncovers hidden signals of adaptive genome evolution in animals

In animals, the most common type of RNA editing is the deamination of adenosines (A) into inosines (I). Because inosines base-pair with cytosines (C), they are interpreted as guanosines (G) by the cellular machinery and genomically encoded G alleles at edited sites mimic the function of edited RNAs. The contribution of this hardwiring effect on genome evolution remains obscure. We looked for population genomics signatures of adaptive evolution associated with A-to-I RNA edited sites in humans and Drosophila melanogaster. We found that single nucleotide polymorphisms at edited sites occur 3 (humans) to 15 times (Drosophila) more often than at unedited sites, the nucleotide G is virtually the unique alternative allele at edited sites and G alleles segregate at higher frequency at edited sites than at unedited sites. Our study reveals that coding synonymous and nonsynonymous as well as silent and intergenic A-to-I RNA editing sites are likely adaptive in the distantly related human and Drosophila lineages.

evolutionary biology↗