Characterization of exogenous and endogenous plant small RNAs: discovery of a ubiquitous terminal modification.

RNA silencing is gene regulatory process mediated by 21 to 25 nt RNA which originate from longer single- or double-stranded RNA transcripts. After synthesis, the 21 to 25 nt RNAs are incorporated into argonaute containing RNA-protein complexes acting either pre- or post-transcriptionally – silencing heterochromatin or silencing RNA transcripts, respectively. Plants can be infected with viral satellites, which are small infectious agents comprised of 200-400 nt single-stranded RNA genomes. These satellite genomes lack obvious protein coding regions and adopt characteristic secondary structures. Viral satellites depend on helper viruses for replication as the latter provide the RNA-dependent RNA polymerase. One viral satellite system is the Cucumber Mosaic Virus (CMV) and its Y-Satellite RNA (Y-Sat). This helper virus (CMV) and satellite (Y-Sat) pair can infect a range of plants including tobacco, causes particular tobacco strains to yellow. In a recent study, Wang et al. (PNAS 101:3275-3280) demonstrated that symptoms of the CMV Y-Sat were no longer evident despite an accumulation of Y-Sat RNA. This symptom suppression was due to an endogenously expressed strong RNA silencing suppressor called P1/HC-Pro. I cloned and sequenced 698 small RNA from infected and non-infected plants. Analyzing the cloning data I observed a sharp discrepancy between the size distribution of cloned small RNAs in the histogram versus the autoradiogram of 5\u27 radiolabelled RNA in P1/HC-Pro(+) CMV/Y-Sat(+) plants. Using enzymatic assays and a new band-shift assay I proved that viral small RNA in P1/HC-Pro expressing plants were not modified on the 2\u27-hydroxyl of their 3\u27 terminus, but commonly cloned endogenous micro RNAs were modified. The analysis of these results was greatly facilitated by my software called Ebbie, an automated sequencing data analysis pipeline using a dynamic web page, which assists the process of annotating cloned small RNAs. These findings imply a separation between the multiple small RNA biogenesis pathways for RNA silencing in plants via sub-cellular localization and/or recruiting distinct sets of proteins by each pathway. My research also showed that all small RNAs in plants have a 3\u27 terminal modification, a significant difference between animal and plant RNA silencing pathways