I.S.Q.C. · Improving seed quality in cereals by manipulating gene expression and partitioning

Final Activity Report Summary - I.S.Q.C. (Improving seed quality in cereals by manipulating gene expression and partitioning)

Reverse genetic tools relying on transient knock down are powerful approaches to fast-track gene function. A commonly approach used for silencing is RNA interference. One of the implement of RNA interference is virus-induced gene silencing (VIGS) where a virus-derived vector harbouring a portion of a gene from the host triggers a silencing response directed against the selected gene. We found that a Barley stripe mosaic virus (BSMV)-derived VIGS vector was the most efficient method to generate a silencing response in barley plants. Although the VIGS vector triggers the spreading a silencing signal throughout the whole plant, persistence of silencing signal in older plant tissue is hard to achieve. Attempts to trigger a robust silencing of gene expression in seed tissue were unsuccessful. During these studies, a novel regulatory mechanism associated to the control of gene expression, so far not described in plants, was discovered. Genes consist of coding and non-coding sequences known respectively as exons and introns. Intron needs to be spliced-out and other maturation processes has to be happen to generate a matured mRNA that will be recognised for initation of the translation process. Splicing is mediated by nucleotidic signals located on the exon-intron boundaries. RNA that is improperly matured is perceived as aberrant. Such RNA failed to be translated into protein, and engage into a degradation process. We found that a so-called 'aberrant RNA' which do not contains all necessary splicing signals can be improperly maturated and subsequently becomes a target for degradation by endogenous quality control machinery. Further a plant cell use such aberrant RNA or its degradation products to mediate recognition and degradation of other RNA particles carrying the same sequence in the process identified as RNA interference. In other words, we found a mechanism which is: (a) able to recognise aberrant RNA particle incapable to undergo splicing process; (b) inhibit maturation of such RNA; (c) initiate degradation process of recognised RNA; and (d) trigger RNA interference process against all RNA particles carrying the same sequence. In the light of these results, we propose a novel approach that can be used as a reverse genetic tool and named Aberrant RNA technology (ART). The new tool allows us to produce library of silencers made from mRNA expressed in cells / tissues / organisms in one step by simple ligation of cDNA library into designed ART-vector. Such library could be further employ for screening for gene function. In future, ART-based library of silencer seems to be very attractive tool in utilisation in mammalian science areas.

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