Crystal structure of a fibrillarin homologue from Methanococcus jannaschii, a hyperthermophile, at 1.6 A resolution.
ABSTRACT Fibrillarin is a phylogenetically conserved protein essential for efficient processing of pre-rRNA through its association with a class of small nucleolar RNAs during ribosomal biogenesis. The protein is the antigen for the autoimmune disease scleroderma. Here we report the crystal structure of the fibrillarin homologue from Methanococcus jannaschii, a hyperthermophile, at 1.6 A resolution. The structure consists of two domains, with a novel fold in the N-terminal region and a methyltransferase-like domain in the C-terminal region. Mapping temperature-sensitive mutations found in yeast fibrillarin Nop1 to the Methanococcus homologue structure reveals that many of the mutations cluster in the core of the methyltransferase-like domain.
Full-textDOI: · Available from: David Boisvert, Jun 01, 2014
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ABSTRACT: Small nucleolar RNAs (snoRNAs) guide nucleotide modifications within ribosomal RNAs or spliceosomal RNAs by base-pairing to complementary regions within their RNA targets. The brain-specific snoRNA MBII-52 lacks such a complementarity to rRNAs or snRNAs, but instead has been reported to target the serotonin receptor 2C pre-mRNA, thereby regulating pre-mRNA editing and/or alternative splicing. To understand how the MBII-52 snoRNA might be involved in these regulatory processes, we isolated the MBII-52 snoRNP from total mouse brain by an antisense RNA affinity purification approach. Surprisingly, by mass spectrometry we identified 17 novel candidates for MBII-52 snoRNA binding proteins, which previously had not been reported to be associated with canonical snoRNAs. Among these, Nucleolin and ELAVL1 proteins were confirmed to independently and directly interact with the MBII-52 snoRNA by coimmunoprecipitation. Our findings suggest that the MBII-52 snoRNA assembles into novel RNA-protein complexes, distinct from canonical snoRNPs.RNA 07/2010; 16(7):1293-300. DOI:10.1261/rna.2109710 · 4.62 Impact Factor
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ABSTRACT: The rRNAs of Escherichia coli contain four 2'-O-methylated nucleotides. Similar to other bacterial species and in contrast with Archaea and Eukaryota, the E. coli rRNA modifications are catalysed by specific methyltransferases that find their nucleotide targets without being guided by small complementary RNAs. We show here that the ygdE gene encodes the methyltransferase that catalyses 2'-O-methylation at nucleotide C2498 in the peptidyl transferase loop of E. coli 23S rRNA. Analyses of rRNAs using MALDI mass spectrometry showed that inactivation of the ygdE gene leads to loss of methylation at nucleotide C2498. The loss of ygdE function causes a slight reduction in bacterial fitness. Methylation at C2498 was restored by complementing the knock-out strain with a recombinant copy of ygdE. The recombinant YgdE methyltransferase modifies C2498 in naked 23S rRNA, but not in assembled 50S subunits or ribosomes. Nucleotide C2498 is situated within a highly conserved and heavily modified rRNA sequence, and YgdE's activity is influenced by other modification enzymes that target this region. Phylogenetically, YgdE is placed in the cluster of orthologous groups COG2933 together with S-adenosylmethionine-dependent, Rossmann-fold methyltransferases such as the archaeal and eukaryotic RNA-guided fibrillarins. The ygdE gene has been redesignated rlmM for rRNA large subunit methyltransferase M.Molecular Microbiology 05/2009; 72(5):1147-58. DOI:10.1111/j.1365-2958.2009.06709.x · 5.03 Impact Factor
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ABSTRACT: Viral methyltransferases are involved in the mRNA capping process, resulting in the transfer of a methyl group from S-adenosyl-L-methionine to capped RNA. Two groups of methyltransferases (MTases) are known: (guanine-N7)-methyltransferases (N7MTases), adding a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), adding a methyl group to a ribose hydroxyl. We have expressed and purified two constructs of Meaban virus (MV; genus Flavivirus) NS5 protein MTase domain (residues 1–265 and 1–293, respectively). We report here the three-dimensional structure of the shorter MTase construct in complex with the cofactor S-adenosyl-L-methionine, at 2.9 Å resolution. Inspection of the refined crystal structure, which highlights structural conservation of specific active site residues, together with sequence analysis and structural comparison with Dengue virus 2′OMTase, suggests that the crystallized enzyme belongs to the 2′OMTase subgroup. Enzymatic assays show that the short MV MTase construct is inactive, but the longer construct expressed can transfer a methyl group to the ribose 2′O atom of a short GpppAC5 substrate. West Nile virus MTase domain has been recently shown to display both N7 and 2′O MTase activity on a capped RNA substrate comprising the 5′-terminal 190 nt of the West Nile virus genome. The lack of N7 MTase activity here reported for MV MTase may be related either to the small size of the capped RNA substrate, to its sequence, or to different structural properties of the C-terminal regions of West Nile virus and MV MTase-domains.Protein Science 01/2009; 16(6):1133 - 1145. DOI:10.1110/ps.072758107 · 2.86 Impact Factor