Reduction of functional N‐methyl‐d‐aspartate receptors in neurons by RNase P‐mediated cleavage of the NR1 mRNA

Abstract

One approach to studying the functional role of individual NMDA receptor subunits involves the reduction in the abundance of the protein subunit in neurons. We have pursued a strategy to achieve this goal that involves the use of a small guide RNA which can lead to the destruction of the mRNA for a speci®c receptor subunit. We designed a small RNA molecule, termed`external guide sequence' (EGS), which binds to the NR1 mRNA and directs the endonuclease RNase P to cleave the target message. This EGS has exquisite speci®city and directed the RNase P-dependent cleavage at the targeted location within the NR1 mRNA. To improve the ef®ciency of this EGS, an in vitro evolution strategy was employed which led to a second generation EGS that was 10 times more potent than the parent molecule. We constructed an expression cassette by¯anking the EGS with self-cleaving ribozymes and this permitted generation of the speci®ed EGS RNA sequence from any promoter. Using a recombinant Herpes simplex virus (HSV), we expressed the EGS in neurons and showed the potency of the EGS to reduce NR1 protein within neurons. In an excitotoxicity assay, we showed that expression of the EGS in cortical neurons is neuroprotective. Our results demonstrate the utility of EGSs to reduce the expression of any gene (and potentially any splice variant) in neurons.

RNase P: Variations and Uses

“…Kalb and his collaborators (66) have also shown that a recombinant HSV that contains an EGS directed to the mRNA of an N-methyl-D-aspartate receptor can infect primary cultures of neuronal cells and reverse some aspects of the cytotoxic effect of Nmethyl-D-aspartate receptor ligands.…”

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

“…Indeed, our results confirm the utility of this promoter for the expression of the EGS genes in maize BMS cells. U3 snRNA is transcribed by pol III (RNA polymerase III) in plants, and its promoter was chosen for the expression of EGSs rather than a pol II promoter for several reasons [11][12][13][21][22][23]. First, the regulatory elements in this pol III promoter reside entirely upstream of the coding regions of the gene.…”

“…Although the EGS method might perform better in planta than one might anticipate from our transient assay results, it is important to appreciate that even a decrease of 70 % protein levels is adequate to begin establishing gene-function relationships or for use in metabolic engineering applications. One study that employed RNAi for disrupting expression of a transcription factor [29] and another that employed the EGS approach for downregulating the mRNA for a neuronal receptor [13] were both able to elicit the expected phenotypic alterations even when gene expression was reduced by only 50-60 %. Moreover, it has been documented that metabolic flux alterations can be accomplished even with an approx.…”

“…The observation that RNase P can cleave a bipartite RNA complex that structurally resembles a ptRNA led to the idea that any target mRNA that can form a ptRNA-like structure and a sequence-specific complex with an EGS (external guide sequence) RNA can potentially become a non-natural substrate for RNase P cleavage ( Figure 1C; [5,6,10]). This idea has been demonstrated successfully in mammalian cell cultures [11][12][13]. As part of our studies to test the viability of this approach in plants, we reported previously that partially purified rice and maize (Zea mays) RNase P, like their mammalian counterparts, are capable of cleaving a non-natural bipartite substrate in vitro [14].…”

A peptide-morpholino oligomer conjugate targeting Staphylococcus aureus gyrA mRNA improves healing in an infected mouse cutaneous wound model

“…This conserved mechanism can be directed against specific mRNA by providing a complementary external guide sequence (EGS), which binds to the target mRNA and directs RNase P cleavage (Li et al, 1992). When given the proper EGS, RNase P is able to catalytically break down bacterial, viral, and eukaryotic mRNAs (Guerrier-Takada et al, 1995; Guerrier-Takada et al, 1997; Jiang et al, 2012; Trang et al, 2001; Yen et al, 2001). Currently, EGSs can be created using phosphorodiamidate morpholino oligonucleotides (MOs), conjugated with a cell-penetrating peptide (CPP) to improve intracellular transport, and then used to alter and kill both Gram positive and negative bacteria (Shen et al, 2009; Wesolowski et al, 2011).…”