Reverse 5' caps in RNAs made in vitro by phage RNA polymerases.
We show that about one-third of the RNAs produced in vitro by viral RNA polymerases in the presence of m7GpppG dinucleotides have unusual 5' caps. In these RNAs, the initiating dinucleotide is incorporated in an orientation opposite to that expected so that the 7-methyl guanine (m7G) nucleotide is adjacent to the body of the RNA, making a "reverse" cap. The doubly methylated dinucleotide, m7GpppGm, containing a 2' O-methylated guanine (Gm) is incorporated only in the reverse orientation. Precursors of U1 snRNAs containing reverse caps are recognized by antibodies specific for the m7G cap structure. When injected into Xenopus laevis oocyte nuclei, reverse-capped pre-U1 RNAs are exported considerably more slowly than normal. Furthermore, U1 RNAs with reverse caps exhibit a striking defect in nuclear import that can be attributed to the failure of reverse caps to be hypermethylated to m2,2,7G caps. Thus, the presence of reverse-capped RNAs in RNA preparations may affect conclusions about the efficiency and extent of certain m7G cap-dependent processes.
Department of Biomolecular Chemistry, University of Wisconsin, Madison 53706-1532, USA.
A E
AE
Pasquinelli
J E
JE
Dahlberg
E
E
Lund
D000818:Animals; D000906:Antibodies; D017903:Bacteriophage T3 / Q000201:enzymology; D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000378:metabolism*; D015226:Dinucleoside Phosphates / Q000737:chemistry / Q000378:metabolism; D005260:Female; D008745:Methylation; D008845:Microinjections; D008969:Molecular Sequence Data; D009865:Oocytes / Q000502:physiology; D011233:Precipitin Tests; D012315:RNA Caps / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D012322:RNA Precursors / Q000737:chemistry / Q000276:immunology; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014982:Xenopus laevis
D000906:Antibodies; D015226:Dinucleoside Phosphates; D012315:RNA Caps; D012322:RNA Precursors; D017412:Ribonucleoprotein, U1 Small Nuclear; C062229:7-methyl-diguanosine triphosphate; D012321:DNA-Directed RNA Polymerases