Nuclear ligation of RNA polynucleotide kinase products

✍ Scribed by Ilga Winicov

• Publisher: John Wiley and Sons
• Year: 1982
• Tongue: English
• Weight: 1005 KB
• Volume: 113
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041130118

✦ Synopsis

Abstract

RNA polynucleotide kinase has been shown to transfer [γ^32^P] from ATP to 5‐OH termini of endogenous nuclear RNA. The products of this reaction have been isolated in RNA larger than 125 after in vitro incubation of mouse L cell nuclei. About 20%–30% of these 5′‐OH kinase products are polyadenylated. A sizeable fraction of the [γ^32^P] label from ATP is also found in internal phosphodiester bonds after 30‐minute nuclear incubation in vitro. The possibility of substantial [^32^P] recycling via the α position of nucleoside triphosphate was ruled out because: (1) 2mM nucleoside triphosphates in the incubation medium, (2) limited nearestneighbor distribution 3′ and 5′ to the phosphodiester bond compared with that from [α^32^P] UTP, (3) different nearest‐neighbor distribution for RNA molecules > 12S and 12‐3S, (4) relative insensitivity of the [γ^32^P] incorporation to α‐amanitin as compared with total RNA synthesis, (5) internal [^32^P] appearance in RNA > 12S in less than five minutes of incubation, and (6) < 0.03% to 0.6% of the total [^32^P] in the α position of nucleoside triphosphates after 30 minutes of incubation. The [γ^32^P] incorporation was dependent on high ATP concentration and was insensitive to competition by inorganic phosphate. These results are consistent with the levels of 5′ RNA polynucleotide kinase activity in L cell nuclei and suggest the presence of an RNA ligase that can utilize the termini generated by the 5′‐OH RNA kinase in a ligation reaction.