Descripción del proyecto
Estudio estructural de la biogénesis del espliceosoma
Los genes eucariotas se transcriben en pre-ARNm, que posteriormente sufre un proceso de corte y empalme para producir ARNm maduro. El proceso de corte y empalme se lleva a cabo mediante un complejo dinámico de múltiples subunidades llamada espliceosoma. El objetivo del proyecto StructuRNP, financiado con fondos europeos, es investigar la biogénesis de los componentes básicos del espliceosoma, las denominadas ribonucleoproteínas nucleares pequeñas (snRNP). Los investigadores emplearán bioquímica, criomicroscopía electrónica y espectrometría de masas con entrecruzamiento para determinar las estructuras de las proteínas participantes y su papel en la biogénesis del espliceosoma. Los resultados esperados revelarán cómo se produce el ensamblaje regulado del espliceosoma.
Objetivo
The spliceosome is a multi-megadalton enzyme that catalyses the excision of non-coding introns from nuclear pre-mRNA. It is assembled from five small nuclear ribonucleoprotein particles (snRNPs) and non-snRNP factors, while each snRNP contains a unique small nuclear RNA (snRNA, U1-U5) and is bound by the heptameric Sm-ring. Acquisition of the Sm-ring is a key quality control step in snRNP biogenesis, defects of which are linked with several human diseases. In humans, snRNP biogenesis requires at least 15 proteins, including the 9-subunit survival of motor neuron (SMN) complex. The SMN complex carries out the regulated and specific loading of the Sm ring onto snRNA. Recent studies have revealed the structure of several key proteins that play a role in snRNP assembly, however the complete structures of either the SMN complex alone or with interacting Sm ring and or the snRNP as a whole, are lacking and culminate to a big gap in our knowledge of the snRNP assembly. In this proposal, I will reconstitute key steps in snRNP biogenesis in humans and will determine their structures using cryo-electron microscopy and cross-linking mass spectrometry. I will study how the SMN complex specifically loads the Sm-ring onto U1 snRNA, as a model snRNP. Additionally, purification and structural analysis of endogenous SMN complex will reveal SMN complex stoichiometry and validate the in vitro work. The structures will reveal the interacting partners and the structural transitions within the assembled snRNP and will enable structure-guided biochemical and tissue culture validation of SMN complex function, while my background in protein biochemistry and human tissue culture makes me ideally suited to carry out this high-impact project. StructuRNP will provide first insights into the molecular events and choreography of the spliceosome, providing a leap in our understanding of spliceosome function and thus will have far reaching implications for RNA and spliceosome fields.
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MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Coordinador
1030 Wien
Austria