RNA modifications are emerging as an epigenetic pathway for regulation of gene expression. More than 100 types of post-transcriptional modifications have been identified in cellular RNA. Pseudouridine is the most abundant modified base in cells. It has been found in tRNA, rRNA, snRNA and other non-coding RNAs. These modifications are essential for biological functions such as spliceosome biogenesis. It is also required for the progression of the apoptotic signal through intrinsic mitochondrial cell death. Recent studies have shown detection of pseudouridine in yeast and human transcriptome and the level of pseudouridylation in mRNA can be induced by environmental cues such as stress, but the functional role of pseudouridylation in mRNA is still unclear. Pseudouridine has been linked to human diseases. An increased level of oxidized pseudouridine has been associated with neurodegenerative conditions such as Alzheimer’s and Parkinson’s diseases. Mutations in pseudouridine synthase box H/ACA RNP have been linked to the X-linked form of the bone marrow failure syndrome dyskeratosis congenita. Formation of pseudouridine involves the detachment, flipping and reattachment of U base to the ribose. This process is catalyzed by a family of pseudouridine synthases.
RNA Pseudouridylate Synthase Domain Containing 4 (RPUSD4) belongs to the RluA sub-family of pseudouridine synthases. In bacteria, RluA is a dual-specificity enzyme responsible for pseudouridylating 23S rRNA and several tRNAs. We have solved the crystal structure of human RPUSD4 at 1.54 Å resolution. RPUSD4 adopts a conserved pseudouridine synthase fold, characterized by an eight-stranded β sheet core. Additional β strands extend the core β sheet. The core sheet is also decorated by helices and loops on one side, where the catalytic cleft locates.
RPUSD4 protein was expressed in E.coli BL21 (DE3) codon plus RIL in TB medium in the presence of 50 μg/mL of kanamycin. Cell were grown at 37oC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM and incubated overnight at 15oC.
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80˚C. For purification, the cell paste was thawed and resuspended in lysis buffer buffer (50 mM HEPES, pH 7.4, 0.5 M NaCl, 5 mM imidazol, 2 mM β-mercaptoethanol, 5% glycerol) with protease inhibitor (1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
The crude extract was cleared by centrifugation. The lysate was loaded onto 5 mL HiTrap column (GE Healthcare), charged with Ni2+. The column was washed with 10 CV of 20 mM HEPES pH 7.4, containing 500 mM NaCl, 50 mM imidazole, 5% glycerol, and the protein was eluted with elution buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 250 mM imidazole, 5% glycerol). The protein was loaded onto Superdex200 column (26x60) (GE Healthcare), equilibrated with 20 mM PIPES, pH 6.5, 250 mM NaCl. The fractions containing RPUSD4 were pooled and further purified to homogeneity by ion-exchange chromatography on Source 30S column (10x10) (GE Healthcare), equilibrated with buffer containing 20 mM PIPES, pH 6.5, and eluted with linear gradient of NaCl up to 500 mM concentration (20 CV).
Protein stock concentration
Mass spec characterization: The expected mass for RPUSD4 is 33412.98 Da, measured mass is 33411.64 Da.
Purified RPUSD4 protein (9.2 mg/mL) was crystallized using sitting drop vapor diffusion method at 20 °C by mixing 1 μl of the protein solution with 1 μl of the reservoir solution containing 20% PEG3350, 0.2 M CaCl2.