In vivo inhibition of Novikoff cytoplasmic messenger RNA methylation by S-tubercidinylhomocysteine

Abstract

The analogue S-tubercidinylhomocysteine (STH) has been used to study the methylation of mRNA in vivo. Partial inhibition of cytoplasmic poly(A)-RNA methylation was observed using a level of inhibitor which still permitted cell growth. Characterization of the partially methylated mRNA indicated the presence of cap structures lacking 2'-O-methylnucleosides, m7GpppN', which are normally not found in mammalian mRNA. Inhibition of additional methylated sites in mRNA at the second 2'-O-methynucleoside, and at internal N6-methyladenosine was also observed Methylation of 7-methylguanosine was not affected under the conditions used in these experiments. The methylnucleoside composition of cap structures differed in STH-inhibited and uninhibited cells. These results indicate that a completely methylated cap is not required for transport of mRNA into the cytoplasm. Furthermore, it may now be possible to assess in vivo the sequential nature of mRNA methylation and its potential role in mRNA processing.

Effect of undermethylation on mRNA cytoplasmic appearance and half-life.

“…For the duration of all experiments, media was supplemented with 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.4)-2 mM glutamine. STH was synthesized as described previously (7,19). Stock solutions of STH were prepared fresh in 10 versed-phase column (Whatman, Inc.) in a procedure similar to one previously described (1).…”

“…Although STH does inhibit the (guanine-7)-methyltransferases of Newcastle disease virus and vaccinia virus in vitro (24,25), it apparently did not inhibit m7G formation in Novikoff cells (19).…”

Nucleosides and Nucleotides

“…In addition, the 5 -α-D-glucopyranosyl derivatives of tubercidin and toyocamycin have been isolated from blue-green algae (152). The structural elucidation, physical and chemical properties, chemical syntheses, and biological activities of these nucleosides and their derivatives have been reported (1)(2)(3)(4)(153)(154)(155)(156)(157)(158)(159)(160)(161)(162)(163)(164)(165)(166)(167)(168)(169)(170)(171)(172). GTP, but not ATP, is a precursor for the biosynthesis of the pyrrolopyrimidine ring of (51-53) (161).…”

“…Compound (51) is incorporated into RNA, inhibits the processing of RNA, but does not inhibit the transcription of 45S precursor RNA to 28S and 18S RNA; DNA synthesis is inhibited. The importance of (51) as a methylase inhibitor has been studied in detail (163,164). S-Tubercidinyl homocysteine inhibits methylation (163,164).…”

“…The importance of (51) as a methylase inhibitor has been studied in detail (163,164). S-Tubercidinyl homocysteine inhibits methylation (163,164). It has been used to elucidate the requirements for the catalytic site of RNA polymerase with respect to initiation, elongation, and termination.…”