Enzymatic conversion of 5'-phosphate-terminated RNA to 5'-di- and triphosphate-terminated RNA.

Abstract

We have isolated from vaccinia virus cores an enzyme, 5'-phosphate-polyribonucleotide kinase, that in the presence of ATP and Mg2+ catalyzes the conversion of 5'-phosphate and 5'-diphosphate termini of RNA to the 5'-triphosphate species. With the exception of dATP, other nucleoside triphosphates were inactive as hosphate donors; activity with dATP was 10%o of that observed with ATP The purified enzyme did not phosphorylate 5'-hydroxyl-or 5'-monophosphate-terminated polydeoxyribonucleotides, although a variety of 5'-monophosphateterminated RNA chains were active as phosphate acceptors. By using a coupled system of 5'-phosphatepolyribonucleotide kinase and guanylyltransferase in the presence of ATP, GTP, Mg2+, and S-adenosylmethionine, capping of 5'-P-,,5-PP-, and 5'-PPP-RNA was demonstrated; in the absence of 5'-phosphate-polyribonucleotide kinase only 5'-PPP-RNA was capped by guanylyltransferase. Vaccinia virus, a large DNA-containing virus that replicates in the cytoplasm of its host cells (1), contains a large number of enzymes, many of which are involved in the synthesis and processing of RNA (2). Virions made permeable in the presence of the four rNTPs synthesize 26S RNA that contains 5'-terminal cap structures and 3'-terminal poly(A). Current evidence suggests that this RNA is cleaved to yield 6-12S RNA products that are also capped and polyadenylylated (2).Previous studies showed that synthetic poly-and oligonucleotides were capped by purified guanylyltransferase, isolated from vaccinia virus cores, provided the 5'-ends of the RNA chains contained 5'-PPP termini (3, 4). RNA chains containing terminal 5'-OH, 5'-P (5), or 5'-PP (4) were not guanylylated.Thus, the viral guanylyltransferase provides a specific reagent for detecting small amounts of 5'-PPP-terminated RNA chains. Using this method, we observed that 5'-P-RNA was guanylylated after incubation with ATP, GTP, and crude fractions prepared from vaccinia virions. We have purified the enzyme activity responsible for the activation of 5' termini of RNA. This activity, 5'-phosphate-polyribonucleotide kinase (5'-P-PR kinase), catalyzes the ATP-dependent phosphorylation of 5'-P-RNA chains, yielding 5'-PP-and 5'-PPP-RNAs.MATERIALS AND METHODS Virus. Vaccinia virus (strain WR) was purified from infected HeLa cells by sedimentation through a sucrose cushion and two sucrose gradient sedimentations as described by Joklik (6).Enzyme Assay. Two methods were used interchangeably to measure 5'-P-PR kinase; the methods gave similar results. various amounts of 5'-P-PR kinase were incubated at 370 for 30 min. Reactions were halted by addition of 2 Mig of proteinase K in the presence of 0.1% sodium dodecyl sulfate; mixtures were incubated at 370 for 30 min, after which 0.2 ml of a solution containing bovine serum albumin (1 mg/ml) and denatured salmon sperm DNA (1 mg/ml) was added. The mixture was treated with 3 ml of 5% trichloroacetic acid; acid-insoluble material was collected by centrifugation, redissolved in 0.2 ml of a solution containing 0.1 M sodium pyr...

Identification of a Cytoplasmic Complex That Adds a Cap onto 5′-Monophosphate RNA

“…To be capped, the 5Ј-monophosphate end would have to be converted to a di-or triphosphate by a polynucleotide 5Ј-monophosphate kinase. The only reports of such an activity were published more than 30 years ago (23,27). Given these points, we suspected that the previous study identified some other type of modification that behaved like a cap.…”

“…These data also suggest that a kinase capable of converting 5Ј-P-RNA into a diphosphate capping substrate was recovered with immunoprecipitated cytoplasmic capping enzyme. Evidence for such an enzyme was described more than 30 years ago (23,27), but there are no contemporary reports. This was examined by incubation of the beads containing immunoprecipitated GFP, mCE, or K294A used for Fig.…”

Genome-Wide Analysis of the 5′ and 3′ Ends of Vaccinia Virus Early mRNAs Delineates Regulatory Sequences of Annotated and Anomalous Transcripts

“…These characteristics raise the question of whether such capped 5Ј ends were generated by nonstringent transcription initiation, posttranscriptional mechanisms, or imperfect CAGE methodology. With regard to posttranscriptional mechanisms, there is evidence for secondary recapping of processed RNAs in the cytoplasm (24,41,45) and a 5Ј-phosphate polynucleotide kinase was isolated from VACV cores (54). Few TSSs with relatively low read counts were mapped antisense to VACV ORFs.…”

The messenger RNA decapping and recapping pathway in Trypanosoma

“…However, the mRNAs that are decapped or cleaved by endonucleases are produced as pRNAs and thus need to be converted to diphosphorylated RNAs (ppRNAs) to be capped by a guanylyltransferase. The existence of pRNA kinase in mammalian cells has been proposed (16,17) and such an activity has been found in the cytosol, complexed with a guanylyltransferase (18). However, the responsible protein has not yet been identified.…”