Repetitive prime-and-realign mechanism converts short capped RNA leaders into longer ones that may be more suitable for elongation during rice stripe virus transcription initiation
Min Yao1,†, Tianqi Zhang1,†, Tong Zhou2, Yijun Zhou2, Xueping Zhou3 and Xiaorong Tao1
Cucumber mosaic virus (CMV) RNAs were found to serve as cap donors for rice stripe virus (RSV) transcription initiation during their co-infection of Nicotiana benthamiana. The 5′ end of CMV RNAs was cleaved preferentially at residues that had multiple-base complementarity to the 3′ end of the RSV template. The length requirement for CMV capped primers to be suitable for elongation varied between 12 and 20 nt, and those of 12–16 nt were optimal for elongation and generated more CMV–RSV chimeric mRNA transcripts. The original cap donors that were cleaved from CMV RNAs were predominantly short (10–13 nt). However, the CMV capped RNA leaders that underwent long-distance elongation were found to contain up to five repetitions of additional AC dinucleotides. Sequence analysis revealed that these AC dinucleotides were used to increase the size of short cap donors in multiple prime-and-realign cycles. Each prime-and-realign cycle added an AC dinucleotide onto the capped RNA leaders; thus, the original cap donors were gradually converted to longer capped RNA leaders (of 12–20 nt). Interestingly, the original 10 nt (or 11 nt) cap donor cleaved from CMV RNA1/2 did not undergo direct extension; only capped RNA leaders that had been increased to ≥12 nt were used for direct elongation. These findings suggest that this repetitive priming and realignment may serve to convert short capped CMV RNA leaders into longer, more suitable sizes to render a more stabilized transcription complex for elongation during RSV transcription initiation.