Comprehending Prokaryotic Argonaute Systems

Descripción del proyecto

El papel de las proteínas argonauta en las procariotas

Las proteínas argonauta son componentes del silenciamiento de genes inducido por el ARN conocido como ribointerferencia. Se unen a diferentes tipos de ARN no codificantes pequeños que las guían hacia objetivos concretos, lo que conduce a la escisión del ARNm o la inhibición de la traducción. Sin embargo, su origen evolutivo está en las procariotas. El proyecto COMPASS mapeará las funciones de sistemas de proteínas argonauta procariota (pAgo, por sus siglas en inglés) no estudiados en los que las pAgo se asocian con proteínas auxiliares. La hipótesis es que las pAgo se unen con secuencias de ADN exógenas, lo que resulta en la incorporación de las proteínas auxiliares y desempeña funciones totalmente nuevas que van desde la estimulación de la transferencia horizontal de genes hasta el desencadenamiento de la muerte celular programada. Los resultados del proyecto permitirán el rediseño de sistemas pAgo programables para el desarrollo de herramientas genéticas.

Objetivo

Eukaryotic Argonaute proteins are known for their central role in RNA interference pathways. Yet, the evolutionary origin of Argonaute proteins lies in prokaryotes, where other proteins essential for RNA interference are absent. Therefore, prokaryotic Argonaute proteins (pAgos) must have distinct ancestral functions. Although a handful of closely related pAgos interfere with exogenous DNA invaders such as plasmids, pAgos are extremely diverse in terms of sequence conservation and domain architecture. In addition, many pAgos genetically associate with various putative enzyme domains, which suggests that they are functionally interdependent. As such, different pAgos must rely on distinct mechanism and are expected to fulfil a range of different roles. Therefore, the function of most pAgos remains completely unknown.

The COMPASS project will map the function of unexplored pAgo systems, in which pAgos associate with auxiliary proteins. I hypothesize that in these systems, pAgo binds exogenous DNA sequences in a guide-dependent manner. This can result in the recruitment and/or activation of the auxiliary proteins. As pAgo-associated auxiliary proteins are homologous to proteins involved in DNA recombination, NAD+ turnover, or protein deacetylation, these pAgo systems are expected to fulfil completely novel roles ranging from stimulating horizontal gene transfer to triggering programmed cell death.

The uncharacterized roles of these pAgo systems and the mechanisms underlying their functionality will be elucidated by a multidisciplinary approach combining microbiology, protein biochemistry, and X-ray crystallography techniques. Not only will the results facilitate a deeper understanding of the evolutionary diversification of pAgos, it will also enable the repurposing of programmable pAgo systems for the development of genetic tools that facilitate guide sequence-directed DNA recombination and high-sensitivity detection of target DNA sequences.