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
After the start of transcription, the 5' ends of eukaryotic mRNA molecules are modified by the addition of a guanylyl residue to form a cap structure, G(5')ppp(5')N. The guanylyltransferase (GTP:mRNA guanylyltransferase, EC 2.7.7.50) reaction responsible for cap formation usually proceeds via a covalent enzyme-GMP intermediate. We have studied the alphavirus-specific guanylyltransferase by incubating lysates from Semliki Forest and Sindbis virus-infected cells with [alpha-32P]GTP, using vaccinia virus and mock-infected cells as controls. One additional 32P-labeled protein was detected in alphavirus-infected cells but only in the presence of S-adenosylmethionine. This protein was identified as the nonstructural protein nsP1. The properties of the covalent enzyme-guanylate complex were studied with Semliki Forest virus nsP1 expressed in recombinant baculovirus-infected cells. S-Adenosylmethionine and divalent cations were required for the complex formation. The reaction was specific for guanylate nucleotides (GTP, dGTP) and was inhibited by pyrophosphate. nsP1 could be labeled with S-adenosyl[methyl-3H]methionine but only under conditions in which the nsP1-guanylate complex was formed. 7-Methyl-GMP was released from the nsP1-guanylate complex by treatment with acid or acidic hydroxylamine. Similar treatment of vaccinia virus capping enzyme released GMP. These findings suggest that in the capping of alphavirus mRNAs the guanine is methylated before linkage to the mRNA molecule.
Subject(s)
Alphavirus/enzymology , Methyltransferases/metabolism , RNA Cap Analogs/metabolism , RNA Caps/biosynthesis , RNA, Viral/metabolism , Cations, Divalent , Cells, Cultured , Diphosphates , Guanosine Triphosphate/metabolism , Methylation , Methyltransferases/chemistry , RNA Cap Analogs/isolation & purification , Recombinant Proteins/metabolism , S-Adenosylmethionine/metabolism , Substrate SpecificityABSTRACT
Trypanosoma brucei mRNA is discontinuously synthesized via the 5' addition of a "mini-exon" sequence. The mini-exon-specific cap structure was purified from a complete RNase T2 and phosphatase digest of in vivo 32P-labeled poly(A)+RNA. The purified cap structure was sequenced by a series of partial and complete enzymatic digests by nuclease P1 and venom phosphodiesterase. This approach demonstrated that the T. brucei mini-exon cap structure consists of N7-methylguanosine linked in a conventional 5'-5' triphosphate bond to five nucleotides, in the sequence A*A*C(2'-O)mU*A (asterisks denote modifications that were not fully characterized in this work). 2'-O-methylations and other modifications appear to be present in this novel cap structure, which could have a functional role in the metabolism of the mini-exon.
