Expression and analysis of the SAM-dependent RNA methyltransferase Rsm22 from Saccharomyces cerevisiae

Rsm22-family proteins are conserved putative SAM-dependent methyltransferases with important functions in mitochondrial translation. Here, the results of a comparative bioinformatics analysis of Rsm22-type proteins are presented, the expression, biophysical characterization and crystallization of Saccharomyces cerevisiae Rsm22 are reported, a low-resolution SAXS structure of the protein is revealed, and SAM-dependent RNA methyl transferase activity of the protein is demonstrated.

PTC-100 Peltier effect cycling in a thermal cycler (MJ Research). Isolated DNA from the WT yeast (W1365B) strain was used as a template for the PCR amplification of all mitochondrial tRNAs. The standard protocol from Thermo Scientific Phusion High-Fidelity PCR Kit (user manual no. MAN0013363) was followed for assembling the reaction. The PCR reaction was carried out in thin walled PCR tubes and the mixture contained the following components: 1X reaction buffer, 20 ng template DNA, 25 pmols of forward and reverse primers each, Phusion® High fidelity DNA polymerase, 10 nmol dNTPs and sterile distilled water to adjust the total volume of the reaction up to 50 µl. Special attention was paid while assembling the reaction, that all the reagents were assembled on ice and the enzyme was added in the last step.

S3. Purification of the PCR product
The obtained PCR products were purified using the NucleoSpin® Gel and PCR Clean-up kit, following the protocol in the user manual provided by the manufacturer and stored at -20 °C for forthcoming experiments.

S4. Agarose gel electrophoresis
Agarose gel electrophoresis was used to visualize and analyze the DNA fragments from PCR products, separated according to their molecular weight. Similarly, to identify and analyze the tRNAs obtained by in vitro transcription reactions, agarose gel electrophoresis experiments were performed. The DNA fragments from the PCR product was detected by running 5 µl of 1X loading dye on 2% agarose gel. Similarly, for identifying the tRNAs (length ranging from 70-90 bp) a concentration of 1.5-2% agarose gel was used.
Ethidium bromide to a final concentration of approximately 0.2-0.5 µg/ml was added to the gel. 100 ng of samples were loaded in the wells to visualize clean bands on the gel. For sizing, Thermo Scientific GeneRuler 1 kb Plus and 100 bp were used as DNA ladders or markers for the experiments. The gel was run in TAE running buffer (40 mM Tris-acetate, 1 mM EDTA) for 1 hour and 30 minutes at a voltage of 80V. The images of the gel were captured with Bio-Rad ChemiDoc XRS+ machine. Acta Cryst. (2021). D77, doi:10.1107/S2059798321004149 Supporting information, sup-2

S5. In vitro transcription of yeast mt-tRNAs
Transcription reaction was assembled following the MEGAscript® Kit (Ambion/Thermo Fisher). The reaction was assembled at room temperature. First, the frozen reagents, RNA polymerase Enzyme mix, 10X reaction buffer and the 4 ribonucleotides solutions (ATP, CTP, GTP and UTP) provided in the kit were thawed. The 10X reaction buffer was kept at room temperature so that the spermidine in the buffer cannot coprecipitate the template DNA. Equal volumes of the four ribonucleotide solutions were mixed to obtain a reaction volume of 20 µl. 300 ng of DNA template was used to achieve an optimal reaction. The amount and the composition of the reagents are listed in the Supplementary table T6. The transcription reaction assembly was mixed gently and incubated at 37 °C for 16 hours. After incubation, the completed transcription reaction was treated with 1 µl TURBO DNase enzyme and incubated for 15 minutes at 37°C for removal of the template DNA. The template is the purified PCR product with T7 promoter for high yield of tRNAs in vitro.

S6. Purification of the in vitro transcript tRNAs
The transcription reaction mixture was cleaned using the RNeasy® MinElute® Cleanup kit following the basic protocol provided by the manufacturer. In the last step of the cleanup, 14 µl of RNA elute was collected. 1 uL of the RNA sample diluted 10-fold was used to determine the concentration using NanoDrop. Acta Cryst. (2021). D77, doi:10.1107/S2059798321004149 Supporting information, sup-3

Figure S1
The online SEC-MALS analysis of monomeric (a) and dimeric (b) forms of Sc-Rsm22 using the Äkta system equipped with a Superdex200 10/300GL Increase column (GE Healthcare) and a Wyatt mini DAWN TREOS for recording the light scattering (LS) signal. In the panels a and b only the LS2 (LS signal measured at 90° angle) and UV signals are shown in red and green respectively. The UV signal shows that amount of aggregated protein is very less (less than 5%) in both runs, and the major fraction of the protein sample are either in the monomeric (panel a) or dimeric (panel b) forms. c) The plot of molar mass versus elution time for both monomeric (red) and dimeric (blue) Sc-Rsm22 is shown. Also shown are the corresponding LS signals measured at 90° angle (the same as in panels a and b. The molar masses were calculated from the refractive index (RI) and LS signals using the Astra software.