Satellite cereal yellow dwarf virus-RPV (satRPV) RNA requires a douXble hammerhead for self-cleavage and an alternative structure for replication.
The 110 nt hammerhead ribozyme in the satellite RNA of cereal yellow dwarf virus-RPV (satRPV RNA) folds into an alternative conformation that inhibits self-cleavage. This alternative structure comprises a pseudoknot with base-pairing between loop (L1) and a single-stranded bulge (L2a), which are located in hammerhead stems I and II, respectively. Mutations that disrupt this base-pairing, or otherwise cause the ribozyme to more closely resemble a canonical hammerhead, greatly increase self-cleavage. In a more natural multimeric sequence context containing the full-length satRPV RNA and two copies of the hammerhead, wild-type RNA cleaves much more efficiently than in the 110 nt context. Mutations in the upstream hammerhead, including a knock-out in the catalytic core, affect cleavage at the downstream cleavage site, indicating that multimers of satRPV RNA cleave via a double hammerhead. The double hammerhead includes base-pairing between two copies of the L1 sequence which extends stem I. Disruption of L1-L1 base-pairing slows cleavage of the multimer. L1-L2a base-pairing is required for efficient replication of satRPV RNA in oat protoplasts. Mutations that affect self-cleavage of the multimer do not correlate with replication efficiency, indicating that the ability to self-cleave is not a primary determinant of replication. We present a replication model in which multimeric satRPV RNA folds into alternative conformations that cannot form in the monomer. One potential metastable intermediate conformation involves L1-L2a base-pairing that may facilitate formation of the double hammerhead. However, we conclude that L1-L2a also performs some other essential function in the satRPV RNA replication cycle, because the L1-L2a base-pairing is more important than efficient self-cleavage for replication.
Song
S I
SI
Plant Pathology Department, Iowa State University, 351 Bessey Hall, Ames, IA 50011, USA.
Silver
S L
SL
Aulik
M A
MA
Rasochova
L
L
Mohan
B R
BR
Miller
W A
WA
D018554:Avena / Q000166:cytology / Q000821:virology; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D002384:Catalysis; D006207:Half-Life; D007700:Kinetics; D017793:Luteovirus / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation / Q000235:genetics; D016337:RNA, Catalytic / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019244:RNA, Satellite / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D016337:RNA, Catalytic; D012333:RNA, Messenger; D019244:RNA, Satellite; D012367:RNA, Viral