in-vivo-proximity-biotinylation-for-protein-interaction-studies

<em>In Vivo</em> Proximity Biotinylation for Protein Interaction Studies in <em>Paramecium tetraurelia</em>

Paramecium is a major model organism for the study of molecular mechanisms involved in programmed genomic rearrangements and transposon elimination during ...

University of Bern

Human physiology is highly susceptible to frameshift mutations within coding regions, and many hereditary diseases and cancers are caused by such indels. Presently, therapeutic options to counteract them are limited and, in the case of direct genome editing, risky. Here, we show that release factor 1 (eRF1) from Euplotes, an aquatic protist known for frequent +1 frameshifts in its coding regions, can enhance +1 ribosomal frameshifting at slippery heptameric sequences in human cells without an apparent requirement for an mRNA secondary structure. We further show an increase in frameshifting rate at the premature termination sequence found in the HEXA gene of Tay-Sachs disease patients, or a breast cancer cell line that harbors a tumor-driving frameshift mutation in GATA3. Although the overall increase in frameshifting would need further improvement for clinical applications, our results underscore the potential of exogenous factors, such as Eu eRF1, to increase frameshifting in human cells.

Eukaryotic release factor 1 from Euplotes promotes frameshifting at premature stop codons in human cells.

Many eukaryotic organisms, from ciliates to mammals, employ programmed DNA elimination during their postmeiotic reproduction. The process removes specific regions from the somatic DNA and has broad functions, including the irreversible silencing of genes, sex determination, and genome protection from transposable elements or integrating viruses. Multiple mechanisms have evolved that explain the sequence selectivity of the process. In some cases, the eliminated sequences lack centromeres and are flanked by conserved sequence motifs that are specifically recognized and cleaved by designated nucleases. Upon cleavage, all DNA fragments that lack centromeres are lost during the following mitosis. Alternatively, specific sequences can be destined for elimination by complementary small RNAs (sRNAs) as in some ciliates. These sRNAs enable a PIWI-mediated recruitment of chromatin remodelers, followed up by the precise positioning of a cleavage complex formed from a transposase like PiggyBac or Tc1. Here, we review the known molecular interplay of the cellular machinery that is involved in precise sRNA-guided DNA excision, and additionally, we highlight prominent knowledge gaps. We focus on the modes through which sRNAs enable the precise localization of the cleavage complex, and how the nuclease activity is controlled to prevent off-target cleavage. A mechanistic understanding of this process could enable the development of novel eukaryotic genome editing tools.

Article	Year
Eukaryotic release factor 1 from Euplotes promotes frameshifting at premature stop codons in human cells. iScience\| PubMed ID: 38510117	2024
Functions and mechanisms of eukaryotic RNA-guided programmed DNA elimination. Biochemical Society transactions\| PubMed ID: 40305257	2025

Bozhidar-Adrian Stefanov

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