| Abstract [eng] |
miRNAs are 21-24 nt length double-stranded RNA molecules which do not encode anything. They play mayor role in post-transcriptional gene regulation level. These molecules are encoded by various organisms. miRNA maturation depends on the type of the cell (animal or plant). There is an additional step in plants when double stranded miRNA is methylated by HEN1 protein. HEN1 methyltransferase is a novel protein identified in plants. HEN1 protein has high molecular weight (106,6 kDa) and it is multistructural. Its features are not yet investigated. This is the only protein which modifies small RNA molecules by transferring methyl group from cofactor S-adenosil-L-methyonin to the 2′ -OH group of the last 3′ nt. HEN1 proteins is very specific to its substrate and recognizes it not by the nt sequence but by the molecule length and structure. After analysis of homologous proteins HEN1 conservative motives were identified. (Č. Venclovas) Two RNA binding motives (dsRNA-BM and La-type) were identified in the N-terminal region. By using shortened proteins and analyzing those by electrophoretic gel mobility in PAA gel shift it was find out that dsRNA-BM is responsible for binding miRNA. Theoretical structure model was made for dsRNA-BM. After analyzing it four amino acid residues (12K, 22K, 69K, and 70K) which can be important for the formation of protein and RNA complex were identified. After examination of mutant proteins with changed lysines it was determined that 12th lysine is important for protein and RNA interaction. The Comparison of the strength of HEN1 12 protein and RNA interaction it was noted that it is 88 times weaker than HEN1 FL protein. This shows the weak stability of the complex. After comparison of proteins’ specific activity it was determined that proteins with lysines mutations can transfer methyl group quite efficiently. Also it was identified that shortened protein HEN1-ΔR1 has quite high specific activity. All in all it can be concluded that dsRNA-BM is not necessary for methylation reaction. Analysis of 64 nt hairpin structure C/D guide RNA – sR47 shows that HEN1 protein can bind non-canonical structure RNA substrate. Comparison of shortened proteins features shows that not only dsRNA-BM but also methyltransferase domain can bind sR47. |
