Irmantas Mogila has defended his thesis entitled "Mechanistic studies of type III CRISPR-Cas activity regulation and characterization of associated translation-inhibiting effectors" for the Doctor of Science in Biochemistry degree.
Scientific supervisors: Dr. Gintautas Tamulaitis (Vilnius University, Natural Sciences, Biochemistry);
Prof. Dr. Virginijus Šikšnys (Vilnius University, Natural Sciences, Biochemistry); Dr. Giedrė Tamulaitienė (Vilnius University, Natural Sciences, Biochemistry).
Composition of the Dissertation Defense Board: Chairperson - Prof. Dr. Saulius Serva (Vilnius University, Natural Sciences, Biochemistry); Dr. Vasili Hauryliuk (Lund University, Sweden, Natural Sciences, Biochemistry); Dr. Patrick Pausch (Vilnius University, Natural Sciences, Biochemistry); Prof. Dr. Edita Sužiedėlienė (Vilnius University, Natural Sciences, Biochemistry); Dr. Marijonas Tutkus (Vilnius University, Natural Sciences, Biophysics).
Prokaryotic CRISPR-Cas systems provide adaptive protection against viral nucleic acids. Well-characterized Type III-A CRISPR-Cas complexes, known as Csm, degrade foreign RNA. This study aimed to expand the current understanding of Csm activity by introducing new insights into Type III-A system deactivation and identifying the functions of auxiliary proteins.
Fluorescence correlation spectroscopy (FCS) measurements revealed that the products of rapid RNA hydrolysis remain sequestered within the complex for over an hour. The retained RNA stimulates the DNase activity of the Cas10 subunit, which is inhibited upon RNA dissociation.
Cas10 proteins also synthesize cyclic oligoadenylates (cAn), which activate auxiliary CARF effectors. Sequence analysis revealed that CARF domains evolved through fusion with RelE domains, which exhibit mRNA interferase activity, forming Cami1 proteins. This study sought to characterize these effectors.
Biochemical assays and in vivo toxicity tests demonstrated that Cami1 hydrolyzes mRNA and inhibits bacterial growth when activated by cA4. Cami1 CARF domains also hydrolyze their own activators. Structural analysis of Cami1 proteins revealed that these proteins enter ribosomes via the stalk protein bL12. This interaction between the ribosomal stalk and a translation-inhibiting toxin has been observed in prokaryotes for the first time.
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| Photo by Raimundas Šuika |