In vitro analysis of the self-cleaving satellite RNA of barley yellow dwarf virus

Silver, Stanley
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The goals of this research are to begin to define key biological elements involved in the propagation of the satellite RNA associated with barley yellow dwarf virus, serotype RPV (sBYDV) and to design target-specific ribozymes against BYDV genomic RNA. sBYDV RNA is 322 nucleotides long, contains no significant open reading frames, requires BYDV for replication, encapsidation and spread and self-cleaves in the presence of Mg[superscript]2+ and neutral pH. Sequence comparison of several self-cleaving RNAs revealed the presence of a hammerhead-shaped structure spanning the cleavage site. The hammerhead is composed of 11 highly conserved, predominately single-stranded nucleotides and 3 non-conserved helical domains. Both the (+) and (-) strands of the sBYDV RNA have the hammerhead structure; however, the (+) strand hammerhead has several novel features. First, nucleotides within the sBYDV RNA hammerhead have the potential to basepair in a manner not observed in the consensus hammerhead. Mutants constructed that disrupted the putative basepairing resulted in a nearly 400-fold increase in cleavage rate; while the compensatory mutants cleaved at a rate similar to that observed with the wildtype hammerhead. A second feature of the sBYDV hammerhead, observed only in one other self-cleaving RNA (carnation stunt associated viroid), is the presence of two unpaired bases at the top of stem II. Deletion of either base alone had little effect on cleavage; whereas, deletion of both bases resulted in a 50-fold increase in the cleavage rate. To gain insight into the biology of satellite RNAs, I constructed a full-length sBYDV clone that when transcribed produces (+) sense sBYDV RNA. Northern blot analysis revealed that the small molecular weight RNA associated with BYDV-RPV has the ability to replicate in oat protoplasts only in the presence of the helper virus, thus fulfilling the classical definition of a satellite RNA. Finally, using a portion of the hammerhead structure as a catalytic domain I tested three different ribozymes targeted against different sequences within BYDV-PAV genomic RNA. Two of the ribozymes cleaved genomic RNA poorly and the third ribozyme construct cleaved BYDV-PAV RNA efficiently in vitro to produce fragments of the correct size.

Plant diseases, Molecular, cellular, and developmental biology