Giant heterogeneous polyadenylic acid on vesicular stomatitis virus mRNA synthesized in vitro in the presence of S-adenosylhomocysteine

Abstract

An in vitro transcription system in which vesicular stomatitis virus (VSV) mRNA species have been synthesized is described. In addition to purified VSV virions, which contain an RNA-dependent RNA polymerase, this system contained a cytoplasmic cell extract that enhanced correct transcription. Gel electrophoretic analysis of the methylated polyadenylic acid [poly(A)]-containing VSV mRNA produced in this system in the presence of S-adenosylmethionine showed the discrete VSV mRNA species. However, when unmethylated mRNA was synthesized in the presence of S-adenosylhomocysteine, the poly(A)-containing transcripts were large and heterogeneous in molecular weight and did not contain discrete VSV mRNA species. Two-dimensional fingerprint analysis of the methylated and unmethylated products suggested that identical nucleotide sequences were present in the RNAs. Further analysis showed the presence of very large heterogeneous poly(A), 200 to 2,000 nucleotides in length, in the unmethylated transcript. Proof that this large poly(A) was covalently linked to the correct VSV mRNA transcripts was obtained by removal of the poly(A) by hybridization with oligodeoxythymidylic acid and digestion with RNase H. This digestion produced unmethylated VSV mRNA transcripts with the same discrete sizes as the deadenylated RNAs produced from VSV mRNA initially isolated from VSV-infected cells. The results suggest that there is a relationship between methylation at the 5'-end and polyadenylation at the 3'-end of VSV mRNA's. Furthermore, addition of the very large poly(A) does not affect the normal process of sequential transcription of the VSV genome, suggesting that this poly(A) addition is occurring independently of further transcription. Infection by vesicular stomatitis virus (VSV) quences sufficient to encode the poly(A) (22). involves intracellular synthesis of five polyade-Viral transcription may provide a relatively nylic acid [poly(A)I-containing mRNA species simple model system for studying the mechathat are complementary to the single strand of nism of poly(A) addition. RNA found within VSV virions, and these While we were studying the synthesis and RNAs encode the five viral proteins (10, 17, 20, methylation of VSV mRNA in vitro, we found 23, 27). Purified VSV virions contain an RNA that the presence of S-adenosylhomocysteine polymerase activity (4) that will transcribe the (S-Ado-Hcy), which is required to prevent entire VSV genome under appropriate condimethylation in our in vitro system, resulted in tions (8). Virions also contain enzymes that will the synthesis of large heterogeneous tranadd a sequence of about 200 nucleotides of scripts that contained normal VSV mRNA sepoly(A) to VSV mRNA's (5, 6, 33) and also quences. We show here that these transcripts generate the methylated 5'-terminal structure were generated by the addition of very large m7G5' ppp5' AmpAp on the mRNA's (1). poly(A) to normally sized, but unmethylated, Little is known about the mechanism of VSV mRNA's. These results suggest a relationpoly(A) addition t...

cis-Acting signals involved in termination of vesicular stomatitis virus mRNA synthesis include the conserved AUAC and the U7 signal for polyadenylation

“…Since these templates did not direct detectable termination of upstream mRNA synthesis (Fig. 3), this result agrees with previous findings that polymerase slippage is not in itself sufficient to cause transcriptional termination (12)(13)(14)23). Our findings suggest that the C residue of the AUAC tetranucleotide is not important in causing polymerase slippage but that it may play an important role in allowing this slippage to result in productive termination.…”

“…However, the onset of reiterative transcription does not seem to guarantee that termina-tion of mRNA synthesis will occur. The appearance of poly(A) sequences in bicistronic readthrough RNAs (12)(13)(14)23) indicates that polymerase slippage and the consequent polyadenylation of upstream mRNAs is not the only factor involved in the termination process. It has been suggested that the mechanism of mRNA termination may be closely linked to, and depend on, the initial events of downstream mRNA synthesis (23,27).…”

Transcript Initiation and 5′-End Modifications Are Separable Events during Vesicular Stomatitis Virus Transcription

“…In vitro transcription in the absence of the methyl donor SAM occurs efficiently, producing capped yet unmethylated transcripts (1,16,19,35,51); therefore methylation of the nascent transcript is not a prerequisite for transcription. Interestingly, during in vitro transcription in the presence of the methylation inhibitor S-adenosylhomocystine (SAH), nonmethylated transcripts that contain large heterogeneous poly(A) tails are generated, indicating that there may be some connection between modification at the 5Ј end of the transcript and polyadenylation (20,21,41).…”

Nucleotide sequences of the mRNA's encoding the vesicular stomatitis virus G and M proteins determined from cDNA clones containing the complete coding regions

“…RNA synthesis. VSV mRNA was synthesized in vitro in a 10-ml reaction exactly as described previously (26), with the following modification. After 4 h the reaction was stopped by addition of sodium dodecyl sulfate and sodium acetate to final concentrations of 1% and 0.5 M, respectively.…”