A novel experimental system in yeast to determine the minimal level of translated mRNA transcripts required to support the function of conserved cellular pathways.

Adenosine-to-Inosine (A-to-I) is the most common RNA modification,
mediated by the adenosine deaminase acting on RNA enzyme (ADAR).
ADAR acts on double-stranded RNA (dsRNA) and edits A-to-I by
deamination, which is read as guanosines during translation. Such editing
may lead to recoding events resulting in missense mutations, and even the
correction of nonsense mutations. Advances made in understanding ADAR
protein`s mode of action enabled the development of several site-directed
RNA editing technologies as a therapeutic tool, that compared to CRISPR-
Cas-based genome editing technologies provides a safe, reversible
treatment that can be also used to fine-tune protein function. Using different
strategies, several groups have sought to guide editing machinery towards
pre-designed targets. However, these endeavors resulted in low editing
efficiency, and thus, not all mRNA transcripts are corrected. In most cases, it
is unclear which fraction of these mRNA molecules should be edited and
produce the protein levels required to support normal functionality. To address
this issue, we designed a model experimental system using the yeast
Saccharomyces cerevisiae, that exogenously expresses the human ADAR,
and carries an editable nonsense mutation, at the 5’ end of the selected
essential genes, thereby precluding their translation, and resulting in cell
death. Our preliminary studies demonstrate the levels of cell growth
restoration combined with NGS analysis can be used as a testing ground for
understanding the minimal level of translated transcripts required to support
the function of conserved cellular pathways.