ABSTRACT
Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, (7Me)G5'-ppp5'-A(2'OMe). The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and (7Me)GpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC(n) and (7Me)GpppAC(n) (1 < or = n < or = 9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1 < or = n < or = 7) in quantities of up to 100 nmol starting from 200 nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2'-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.
Subject(s)
Methyltransferases/metabolism , Oligoribonucleotides/biosynthesis , RNA Cap Analogs/biosynthesis , Adenosine/metabolism , Chromatography, High Pressure Liquid , Cytidine Triphosphate/metabolism , DNA Primase , Guanine/metabolism , Humans , Oligoribonucleotides/isolation & purification , Oligoribonucleotides/metabolism , RNA Cap Analogs/chemistry , RNA Cap Analogs/isolation & purification , Templates, GeneticABSTRACT
The m(7)G caps of most spliceosomal snRNAs and certain snoRNAs are converted posttranscriptionally to 2,2,7-trimethylguanosine (m(3)G) cap structures. Here, we show that yeast Tgs1p, an evolutionarily conserved protein carrying a signature of S-AdoMet methyltransferase, is essential for hypermethylation of the m(7)G caps of both snRNAs and snoRNAs. Deletion of the yeast TGS1 gene abolishes the conversion of the m(7)G to m(3)G caps and produces a cold-sensitive splicing defect that correlates with the retention of U1 snRNA in the nucleolus. Consistently, Tgs1p is also localized in the nucleolus. Our results suggest a trafficking pathway in which yeast snRNAs and snoRNAs cycle through the nucleolus to undergo m(7)G cap hypermethylation.
Subject(s)
Cell Nucleolus/enzymology , Guanosine/metabolism , Hydro-Lyases , Methyltransferases/physiology , RNA Cap Analogs/biosynthesis , RNA Caps/metabolism , RNA, Fungal/metabolism , RNA, Small Nuclear/metabolism , RNA, Small Nucleolar/metabolism , Ribonucleoproteins, Small Nucleolar , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/genetics , Amino Acid Motifs , Autoantigens/physiology , Cold Temperature , Evolution, Molecular , Guanosine/analogs & derivatives , Methylation , Methyltransferases/genetics , Microtubule-Associated Proteins/metabolism , Nuclear Proteins/metabolism , Phenotype , Protein Structure, Tertiary , RNA Cap Analogs/physiology , RNA Splicing/genetics , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Ribonucleoproteins, Small Nuclear/physiology , S-Adenosylmethionine/metabolism , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid , Substrate Specificity , Two-Hybrid System TechniquesABSTRACT
There are more than twenty capped small nuclear RNAs characterized in eukaryotic cells. All the capped RNAs appear to be involved in the processing of other nuclear premessenger or preribosomal RNAs. These RNAs contain either trimethylguanosine (TMG) cap structure or methylated gamma phosphate (Mppp) cap structure. The TMG capped RNAs are capped with M7G during transcription by RNA polymerase II and trimethylated further post-transcriptionally. The Mppp-capped RNAs are transcribed by RNA polymerase III and also capped post-transcriptionally. The cap structures improve the stability of the RNAs and in some cases TMG cap is required for transport of the ribonucleoproteins from cytoplasm to the nucleus. Where tested, the cap structures were not essential for their function in processing other RNAs.
Subject(s)
Eukaryotic Cells/physiology , RNA Cap Analogs/physiology , RNA, Nuclear/physiology , Animals , DNA-Directed RNA Polymerases/physiology , Eukaryotic Cells/metabolism , Humans , Methylation , RNA Cap Analogs/biosynthesis , RNA, Nuclear/biosynthesisABSTRACT
The mechanism of ethionine carcinogenesis and more generally the relationship between alkylation of nucleic acids by chemical carcinogens and oncogenesis still remain obscure. In the present study the rat liver tRNA ethylation by L-[ethyl-1-3H]ethionine was reinvestigated by examining in particular the highly radioactive 'pyrimidine-nucleotide-like' fraction found earlier in acid hydrolysates of hepatic tRNA from ethionine-treated rats. The following results were obtained: (1) ultraviolet-spectral and chromatographic analyses showed the presence of 1,7-diethylguanosine in this 'pyrimidine-nucleotide-like' fraction; (2) the dialkyl compound was recovered exclusively in the form of imidazole-ring-opened derivatives. When [1-14C]ethylnitrosourea was used as alkylating agent, the in vivo ethylation pattern of tRNA from various organs of rat showed an analogous radioactive 'pyrimidine-nucleotide-like' fraction as main radioactive product. On the contrary, tRNA ethylation pattern after in vitro reaction with [1-14C]ethylnitrosourea exhibited a main radioactivity peak (85% of the total radioactivity recovered) in coincidence of the chromatographic area of 1,7-diethylguanine. The 1,7-diethylguanosine moieties of tRNA were extremely labile both under physiological and alkaline conditions. The 1,7-diethylguanine-associated radioactivity was completely lost from [14C]ethyl-tRNA after only 7 h incubation at 37 degrees C and pH 7.3, while at pH 11.4 this process was preceded by the conversion of the 1,7-diethylguanosine residues into imidazole-ring-opened derivatives.
