Mateja Jagić - Obrana doktorskog rada

Naslov:

Domain-specific interactions of BPM1 with DMS3 and RDM1 in RNA-directed DNA methylation

Interakcije domena proteina BPM1 s proteinima DMS3 i RDM1 u RNA-posredovanoj metilaciji DNA

Obrana rada održati će se u: 

Petak, 21. travnja 2023. godine u 12.00 sati

u predavaonici ZMB Seminar, PMF, Horvatovac 102a, Zagreb

Povjerenstvo za obranu imenovano je u sastavu:

Prof. dr. sc. Biljana Balen, PMF, Zagreb

Izv. prof. dr. sc. Petra Peharec Štefanić, PMF, Zagreb

Dr. sc. Zdravko Lorković, znanstveni savjetnik, Gregor Mendel Institute of Molecular Plant Biology GmbH, Vienna, Austria

Zamjena: izv. prof. dr. sc. Bojana Banović Đeri, Institute of molekular genetics and genetic engineering, University of Belgrade, Serbia

Mentor: prof. dr. sc. Dunja Leljak - Levanić, PMF, Zagreb

Abstract:

BPM1 protein belongs to the MATH-BTB protein family, characterized as adaptors of CUL3-dependent E3 ubiquitin ligase complex responsible for recognizing and directing substrates for proteasomal degradation. An initial mass spectrometry experiment identified a key component of the RdDM, the DMS3 protein, as a potential BPM1 partner. Since RdDM is the major epigenetic mechanism responsible for de novo DNA methylation in plants, the goal of this work was to further investigate the role of BPM1 in RdDM. By mass spectrometry, along DMS3, another component of RdDM, the RDM1, was determined in complex with BPM1. Their direct interactions with BPM1 were confirmed by Y2H, pull-down, microthermophoresis and FRET-FLIM. Colocalization of BPM1, DMS3, and RDM1 was observed in the nucleus, indicating the site of their common function. The reduced activity of the RdDM-controlled FWA gene promoter in the BPM1-overexpressing line together with protein-protein interaction data strongly suggest a role for BPM1 in controlling gene expression as part of RdDM.

Sažetak:

Protein BPM1 pripada porodici proteina MATH-BTB koji su opisani kao adapteri u CUL3-ovisnom kompleksu ubikvitinskih ligaza E3 odgovornih za prepoznavanje i usmjeravanje supstrata na proteasomalnu razgradnju. U preliminarnom eksperimentu spektrometrije masa kao potencijalni partner proteina BPM1 utvrđena je važna komponenta mehanizma RdDM, protein DMS3. Budući da je RdDM glavni epigenetski mehanizam odgovoran za metilaciju DNA de novo u biljkama, cilj ovog rada bio je detaljnije razjasniti ulogu BPM1 u sklopu RdDM-a. Spektrometrijom masa u kompleksu s proteinom BPM1 uz DMS3, utvrđena je i druga komponenta RdDM-a, protein RDM1. Njihove izravne interakcije s BPM1 potvrđene su metodama sustava dvaju kvašćevih hibrida, proteinske kopurifikacije, mikrotermoforeze te metodom FRET-FLIM. Kolokalizacija proteina BPM1, DMS3 i RDM1 utvrđena je u jezgri što ukazuje na mjesto njihove zajedničke funkcije. Smanjena aktivnost promotora gena FWA kontroliranog RdDM-om u liniji s prekomjernom ekspresijom gena BPM1 zajedno s rezultatima proteinskih interakcija snažno upućuju na ulogu proteina BPM1 u kontroli genske ekspresije u sklopu mehanizma RdDM.