Efficient and accurate in vitro processing of simian virus 40-associated small RNA.
Nuclei were isolated from simian virus 40 (SV40)-infected cells with a hypotonic, detergent-free buffer and incubated in vitro in a high-ionic-strength buffer containing [alpha-32P]UTP. The labeled viral RNAs produced were analyzed by gel electrophoresis together with 3-h-labeled viral RNAs extracted from SV40-infected cells. The in vitro-synthesized RNA contained a major RNA species of 62 to 64 nucleotides that appeared on the gel at the same position as in vivo-synthesized SV40-associated small RNA (SAS-RNA). Analyses of the in vitro-synthesized 62- to 64-nucleotide RNA by hybridization to restriction fragments and by the use of an SAS-RNA deletion mutant clearly identified it as SAS-RNA. The intensity of the band of the in vitro-synthesized SAS-RNA increased with an increase in the labeling time or when a short pulse was followed by a chase. Moreover, the SAS-RNA band disappeared when ITP replaced GTP in the transcription reaction mixture. These results indicate that SAS-RNA is processed from a precursor molecule and that an RNA secondary structure could be an element recognized by the processing enzyme.
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics / Q000378:metabolism*
D012333:RNA, Messenger; D012367:RNA, Viral