RNase P, a ribonucleoprotein responsible for the 5' maturation of precursor tRNAs (ptRNAs) in all organisms, can be enticed to cleave any target mRNA that forms a ptRNA-like structure and sequence-specific complex when bound to an RNA, termed the EGS (external guide sequence). In the present study, F3H (flavanone 3-hydroxylase), a key enzyme in the flavonoid biosynthetic pathway that participates in the formation of red-coloured anthocyanins, was used as a target for RNase P-mediated gene disruption in maize cells. Transient expression of an EGS complementary to the F3H mRNA resulted in suppression of F3H to 29% of the control, as indicated by a reduced number of anthocyanin-accumulating cells. This decrease was not observed in experiments where a disabled mutant EGS was expressed. Our results demonstrate the potential of employing plant RNase P, in the presence of an appropriate gene-specific EGS, as a tool for targeted degradation of mRNAs.

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As we trek into the uncharted territories of the genomic era, there is an urgency for the development of approaches for assigning functions to the multitude of uncharacterized genes. Although currently available knock-out methodologies could be used for uncovering the function of newly discovered genes, the mixed outcomes in terms of the success of these approaches in down-regulating gene expression necessitate the development of new functional genomics tools. This chapter describes in detail the experimental method for targeted mRNA degradation inside plant cells by enticing the endogenous and ubiquitous RNase P into recognition of specific mRNAs as non-natural substrates.

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