Human putative methyltransferase C9orf114

Target ID:

C9orf114

Deposition Date:

Tuesday 30th September 2014

Families:

Methyltransferases

Related Diseases:

Chromatin and epigenetics

Structure type:

apo

Download Coordinates:

4RG1

Authors:

Zeng H, Dong A, Li Y, Bountra C, Arrowsmith CH, Edwards AM, Brown PJ, Wu H

Site:

Toronto

4RG1

tabs

Structure Details

The methylation of a variety of biologically active molecules is a common theme in biology and RNA methylation is one of them. RNA methylation has been observed in different species of RNA, including mRNAs, tRNAs, and non-coding RNAs. The functional roles of RNA methylation are not completely understood. Recent studies have indicated that RNA methylation could be an epigenetic mechanism for regulation of gene expression and other cellular process. The methylation of various RNA molecules is catalyzed by a variety of RNA mathyltransferases and the modification can occur at different positions in RNA molecules. N6-methyladenosine (m6A) and 5-methycytosine (5-mC) are the most common and abundant methylation modification in RNA molecules in eukaryotes. Studies have shown that m6A and 5-mC RNA methylation can affect RNA stability and mRNA translation.

Human chromosome 9 open reading frame 114 (C9orf114) encodes a 376-aa protein of unknown function. We have solved the crystal structure of C9orf114 in complex with SAH at 1.86 Å resolution. The structure of C9orf114 contains a TIM-barrel-like fold, with a deep trefoil knot near the C-terminus. This structural arrangement is the characteristic of a family of RNA methyltransferase. Structural analysis by using the program DALI also showed that C9orf114 shares remarkable structural homology with a number of RNA methyltransferases.

Materials and Methods

C9orf114

PDB:4RG1

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:GI:38679912
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: 6XHis-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQ*G
Host:E.coli BL21 (DE3) codon plus RIL (Stratagen).

Construct

Prelude:
Sequence:gAEKEDRGRPYTLSVALPGSILDNAQSPELRTYLAGQIARACAIFCVDEIVVFDEEGQDAKTVEGEFTGVGKKGQACVQLARILQYLECPQYLRKAFFPKHQDLQFAGLLNPLDSPHHMRQDEESEFREGIVVDRPTRPGHGSFVNCGMKKEVKIDKNLEPGLRVTVRLNQQQHPDCKTYHGKVVSSQDPRTKAGLYWGYTVRLASCLSAVFAEAPFQDGYDLTIGTSERGSDVASAQLPNFRHALVVFGGLQGLEAGADADPNLEVAEPSVLFDLYVNTCPGQGSRTIRTEEAILISLAALQPGLTQAGARHT
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:C9orf114 protein was expressed in E.coli BL21 (DE3) codon plus RIL in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin. Cells 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.

Purification

Procedure
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 Tris-HCl pH 8.0, containing 250 mM NaCl, 50 mM imidazole, 5% glycerol, and the protein was eluted with elution buffer (20 mM Tris-HCl pH 8.0, 250 mM NaCl, 250 mM imidazole, 5% glycerol). The protein was loaded on Superdex200 column (26x60) (GE Healthcare), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 150 mM NaCl. The fractions containing C9orf114 were pooled and TEV protease was added to remove His-tag. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30Q column (10x10) (GE Healthcare), equilibrated with buffer 20 mM Tris-HCl, pH 8.0, and eluted with linear gradient of NaCl up to 500 mM concentration (20 CV).

Extraction

Procedure
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 (1X PBS, 250 mM NaCl, 2 mM ß-mercaptoethanol, 5% glycerol, 0.1% CHAPS) with protease inhibitor (0.1 mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:13.1 mg/mL - Enzymatic treatment: TEV
Ligand
MassSpec:The expected mass for C9orf114 is 34299.7 Da, measured mass is 34376.48 Da.
Crystallization:Purified C9orf114 protein (10.2 mg/mL) was complexed with S-adenosyl-L-homocysteine (SAH) (Sigma) at 1:5 molar ratio of protein:SAH and 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 28% PEG 3350, 0.2M di-NH4tart.
NMR Spectroscopy:
Data Collection:
Data Processing: