Monomeric linear RNA of citrus exocortis viroid resulting from processing in vivo has 5'-phosphomonoester and 3'-hydroxyl termini: implications for the RNase and RNA ligase involved in replication

Abstract

Members of the family Pospiviroidae, like Citrus exocortis viroid (CEVd), replicate through an RNA-based asymmetric rolling-circle mechanism in which oligomeric plus-strand [(+)] RNA intermediates are cleaved to monomeric linear (ml) RNA and then circularized. Here we show, by rapid amplification of 5' and 3' cDNA ends and in vitro ligation assays, that ml CEVd (+) RNA resulting from cleavage of a dimeric transcript transgenically expressed in Arabidopsis thaliana contains 5'-phosphomonoester and 3'-hydroxyl termini. The nature of these termini and the double-stranded structure previously proposed as the substrate for cleavage in vivo suggest that a type III RNase catalyzes cleavage and an RNA ligase distinct from tRNA ligase promotes circularization.

FIG. 1.

(A) Asymmetric rolling-circle replication cycle followed by members of the family Pospiviroidae that includes the kissing-loop interaction between two contiguous hairpin I motifs and the subsequent formation of a double-stranded structure proposed to be the substrate for cleavage (14). (B) Hairpin I and double-stranded structure corresponding to CEVd (+) RNA. Black arrowheads indicate the processing sites located 2 nt apart in each strand. Nucleotide numbering refers to CEVd sequence variant (with GenBank sequence number M34917) with a deleted G between positions 70 and 74.

FIG. 2.

Analysis of the 5′ and 3′ termini of the ml CEVd (+) RNA purified from transgenic A. thaliana expressing a dimeric CEVd (+) transcript. (A) Scheme depicting 5′- and 3′-RACE procedures. (B and C) RNA aliquots not treated or subjected to different pretreatments were subjected to 5′-RACE (B) and 3′-RACE (C), with the resulting products being separated by PAGE. Lane 0, control without RNA; lane 1, untreated RNA (Ø); lanes 2 and 3, RNA treated with alkaline phosphatase (AP) and polynucleotide kinase (PNK), respectively; lane M, DNA markers (M) with sizes (in base pairs) indicated to the right of the gels.

FIG. 3.

(A) Diagram depicting results of ligation by T4 RNA ligase 1 and A. thaliana tRNA ligase of two RNAs with different termini. (B and C) Ligation in vitro of ml CEVd (+) RNA purified from infected gynura and transgenic A. thaliana expressing a dimeric CEVd (+) transcript. Products generated by T4 RNA ligase 1 (B) and A. thaliana tRNA ligase (C) were separated by denaturing PAGE and analyzed by Northern blot hybridization. Prior to ligation, aliquots of ml CEVd (+) RNA from infected gynura (lanes 2 to 4) or transgenic A. thaliana (lanes 5 to 7) and ml CEVd (+) RNA synthesized in vitro with 5′-P and 3′-OH termini (lanes 8 to 10) were not treated (−) (lanes 2, 5, and 8) or pretreated (+) with alkaline phosphatase (AP) (lanes 3, 6, and 9) or pretreated (+) with alkaline phosphatase and polynucleotide kinase (PNK) (lanes 4, 7, and 10). Lane 1, RNA from infected gynura containing mc and ml CEVd (+) RNAs with their positions indicated to the left of the gels. The product with the lowest mobility in lanes 5 and 8 most likely corresponds to the dimeric linear CEVd RNA.