RNase P-Mediated Sequence-Specific Cleavage of RNA by Engineered External Guide Sequences

Abstract

The RNA cleavage activity of RNase P can be employed to decrease the levels of specific RNAs and to study their function or even to eradicate pathogens. Two different technologies have been developed to use RNase P as a tool for RNA knockdown. In one of these, an external guide sequence, which mimics a tRNA precursor, a well-known natural RNase P substrate, is used to target an RNA molecule for cleavage by endogenous RNase P. Alternatively, a guide sequence can be attached to M1 RNA, the (catalytic) RNase P RNA subunit of Escherichia coli. The guide sequence is specific for an RNA target, which is subsequently cleaved by the bacterial M1 RNA moiety. These approaches are applicable in both bacteria and eukaryotes. In this review, we will discuss the two technologies in which RNase P is used to reduce RNA expression levels.

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function

“…Target IE1/2 mRNA sequences have been selected by determining accessibility for M1GS binding via dimethyl sulfate mapping [303][304][305][306]. M1GS is partially derived from the M1 RNA catalytic subunit of the E.coli RNase P ribozyme, which mediates tRNA maturation [307,308]. M1 RNA can be converted into an M1SG sequence-specific ribozyme by covalently linking it to an external guide sequence (EGS) that contains nucleotides complementary to the target mRNA sequence [307,308].…”

“…M1GS is partially derived from the M1 RNA catalytic subunit of the E.coli RNase P ribozyme, which mediates tRNA maturation [307,308]. M1 RNA can be converted into an M1SG sequence-specific ribozyme by covalently linking it to an external guide sequence (EGS) that contains nucleotides complementary to the target mRNA sequence [307,308]. The tertiary structure generated upon hybridization between the mRNA substrate and the EGS is required for recognition and cleavage by the ribozyme active site [307,308].…”

“…M1 RNA can be converted into an M1SG sequence-specific ribozyme by covalently linking it to an external guide sequence (EGS) that contains nucleotides complementary to the target mRNA sequence [307,308]. The tertiary structure generated upon hybridization between the mRNA substrate and the EGS is required for recognition and cleavage by the ribozyme active site [307,308]. The initial IE1/2-targeted study used a wild-type M1 sequence and IE1/2 exon 3 as the cleavage site.…”

In Arabidopsis thaliana distinct alleles encoding mitochondrial RNA PROCESSING FACTOR 4 support the generation of additional 5′ termini of ccmB transcripts

“…Cleavage by this endonuclease requires RNA secondary structures resembling acceptor stems of precursor tRNAs, which could be formed by intramolecular base pairing or in a bimolecular manner (Derksen et al 2015). The formation of such structure could be influenced by PPR proteins.…”

Trying on tRNA for Size: RNase P and the T-box Riboswitch as Molecular Rulers

“…The principles of pre-tRNA recognition by RNase P have been widely harnessed to selectively degrade selected cellular mRNAs that pair with an exogeneous guide RNA to form a pre-tRNA-like structure. Details of RNase P-mediated knockdown of target RNAs are summarized in an accompanying article in this Special Issue [ 65 ].…”