RNase P as a tool for disruption of gene expression in maize cells

Abstract

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.

Trials, Travails and Triumphs: An Account of RNA Catalysis in RNase P

“…190 The expression of such an EGS to down-regulate target gene expression has been demonstrated in bacteria as well as plant and mammalian cell cultures (including successful inhibition of viral replication). [192][193][194][195] The second approach, termed M1GS, is based on the ribozyme M1 RNA of E. coli RNase P (Fig. 5b).…”

Current perspectives in zebrafish reverse genetics: Moving forward

“…In contrast, in the external guide sequence (EGS) technique, the antisense guide is designed to bind to the target mRNA such that together they form a complex of hairpin structures that resemble a tRNA molecule (Pei et al,2007). This structure is then recognized by the endogenous ribonuclease (RNase) P, which then cleaves the mRNA in a manner analogous to the maturation of tRNA from its precursor molecule (Liu and Altman,1996; Rangarajan et al,2004; Pei et al,2007; Fig. 2B).…”

Inactivation of expression of two genes in Saccharomyces cerevisiae with the external guide sequence methodology

“…Although the utility of EGS-directed cleavage by RNase P had been demonstrated in bacterial, animal, and plant cells (Guerrier-Takada et al 1995;Plehn-Dujowich and Altman 1998;Raj et al 2001;Dreyfus et al 2004;Rangarajan et al 2004;Pei et al 2008), similar possibilities for inhibition of gene expression in yeast cells remained unclear. We show here that M1 3/4EGS RNA alone is efficient in cleavage of yeast target RNA in vitro, and was capable of reducing the target mRNA in vivo by up to 30% as measured by Northern blots.…”

“…In several recent studies, the external guide sequence (EGS)-based cleavage by RNase P (Forster and Altman 1990) has been used successfully to inhibit gene expression in bacterial, animal, and plant cells (Guerrier-Takada et al 1997;PlehnDujowich and Altman 1998;Raj et al 2001;Dreyfus et al 2004;Rangarajan et al 2004;Pei et al 2008), while its utility in yeast remains unexplored. Therefore, we undertook this study to test the viability of this approach for disrupting expression of host genes in Saccharomyces cerevisiae.…”