An element of the tertiary structure of Peach latent mosaic viroid RNA revealed by UV irradiation.
Following UV irradiation, denaturing polyacrylamide gel electrophoresis and Northern blot hybridization revealed a cross-link in Peach latent mosaic viroid (PLMVd) plus-strand RNA. Primer extension and partial alkaline hydrolysis of the UV-irradiated PLMVd plus-strand RNA resulting from the hammerhead-mediated self-cleavage mapped the cross-link at U81 and at the 3'-terminal C289 (or at a very proximal nucleotide). Supporting this notion, in vitro-synthesized PLMVd plus-strand RNAs with short insertions/deletions at their 3' termini failed to cross-link. Because U81 and C289 are conserved in PLMVd variants and because the initiation site of PLMVd minus-strand RNA maps at a short double-stranded motif containing C289, the UV-photo-cross-linkable element of tertiary structure may be functionally significant. A second cross-linked species similar in size and sequence to the monomeric circular PLMVd form, observed in some PLMVd variants, probably derives from UV-induced ligation of the two termini resulting from self-cleavage.
Hernández
Carmen
C
Instituto de Biología Molecular y Celular de Plantas (UPV-CSIC), Universidad Politécnica de Valencia, 46022 Valencia, Spain. rflores@ibmcp.upv.es
Di Serio
Francesco
F
Ambrós
Silvia
S
Daròs
José-Antonio
JA
Flores
Ricardo
R
D001483:Base Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D005821:Genetic Techniques; D014644:Genetic Variation; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009029:Mosaic Viruses / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010942:Plant Viruses / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D012367:RNA, Viral