Detailed characterization of the posttranscriptional gene-silencing-related small RNA in a GUS gene-silenced tobacco

Posttranscriptional gene-silencing phenomena such as cosuppression and RNA interference are associated with the occurrence of small, about 21-23 nt sh ort RNA species homologous to the silenced gene. We here show that the smal l RNA present in silenced transgenic plants can easily be detected in total RNA isolated according to standard procedures. This will allow for the dev elopment of routine and early screenings for the presence of small RNA spec ies and, therefore, gene silencing in transgenic plants. We further demonst rate that the small RNA fraction can be visualized with the SYBR Green II R NA stain, isolated from a gel, labeled and used as a specific probe. Using these approaches, we have fine-mapped the sequences of the GUS gene that ar e represented in the small RNA fraction of a GUS-silenced tobacco line cont aining an inverted repeat of the GUS gene. In this tobacco line, the silenc ing-associated small RNA is a mixture of fragments that cover the 3' two-th irds of the GUS coding region. The 5' coding and the 3' noncoding ends of t he GUS mRNA are not represented in the small RNA fraction. The RNA fragment s are not likely to be a primary synthesis product of an RNA-dependent RNA polymerase, but rather degradation products from nuclease activity. Surpris ingly, RNA isolated from wild-type, untransformed plants showed the presenc e of a similar-sized small RNA pool. This might indicate the existence of s mall RNA species from putative endogenous genes that are down regulated by a similar posttranscriptional gene-silencing mechanism. The possibility of isolating and labeling the small RNA pool from wild-type plants will provid e a way to identify and study such putative genes.