The RNase (P) for everyone

Ribonuclease P, or RNase P for short, is a catalytic RNA found in nearly every organism on earth (Fig 1). Discovered in 1978 by Stark, Kole, Bowman, and Altman, RNase P was the second type of ribozyme, an RNA that does the work of a protein, discovered, and the first to act exclusively on sequences not part of the same molecule as itself (1). While it was initially thought that both the RNA (called P RNA) and associated protein were necessary, further studies showed that the RNA alone has catalytic activity, making huge waves in the enzyme world (2).

Figure 1. RNAase P RNA structure across all domains of life and hypothesized "RNA world" with associated proteins. Adapted from Walker et al. 2008

P RNA folds into multiple helices and loops (Fig 2A) that connect with each other through base pairing (A-U and C-G) like in DNA and base stacking where the rings in each base stack and stick on top of each other to form a very stable structure (Fig 2B). Across all domains of life a few regions of the RNA are very conserved, most of which make up the sites where the RNA RNase P will modify is recognized and the part of RNase P able to cut the RNA (Fig 2C). The major difference between different domains' P RNA is the length and stability - archaea and eukaryote P RNA is shorter, making it less stable. To help this problem, archaea and eukaryotes have more proteins that stabilize RNase P (Fig. 1) (4).

Figure 2. Structure of P RNA. A) Predicted structure of Bacillus subtilis P RNA with conserved regions (CR) highlighted. adapted from Evans et al. 2006. B) Crystal structure of P RNA with CRI shown in red, CRII in orange, CRIII in yellow, and CRV in cyan. C) Comparison of predicted structures from bacteria, archaea, and eukarya with a consensus sequence shown in the center. Adapted from Evans et al. 2006.

RNase P works in the cell by removing the 5' end of pre-transfer RNA (tRNA), resulting in mature tRNA that's able to work with the ribosome (4). tRNA forms a 4 clover structure that resembles more of a L shape in 3D space (Fig 3). RNase P recognizes pre-tRNA primarily through its structure, binding to the T loop, acceptor stem, and a conserved CCA sequence (Fig 4A), which are found on all tRNAs and are exclusively found on tRNA. RNase P recognizes the T loop through base stacking, where amino acids from RNase P and tRNA stack on top of each other, leading to a stable interaction. The acceptor stem of tRNA fits into a tunnel made in RNase P (Fig 4B). The tunnel binds to the CCA sequence on tRNA, both helping RNase P recognize the tRNA and allowing the end containing the CCA sequence to detach from the 5' end RNase P will cleave (5).

Figure 3. tRNA structure. A) 4 leaf clover structure of tRNA with the RNase P cleavage site marked by an orange arrow. B) Crystal structure of tRNA with the 5' end shown in red, D loop in yellow, anticodon loop in green, T loop in cyan, and acceptor (3') stem in blue. 

Figure 4. Major interactions between RNase P and tRNA. A) overview of RNase P and tRNA interaction, with P RNA in black, tRNA in medium grey with same coloring as in fig. 3, and the bacterial RNase P protein in light grey. B) Base stacking between P RNA and the T loop (cyan), most noticably the last T loop on the right. C) base pairing between P RNA and  the acceptor stem (blue).

RNase P's specificity for RNA structure rather than sequence can be used to our advantage. New therapies are being developed using RNA called External Guide Sequences (EGS) that mimic the T and acceptor stem structures (Fig 5). This allows the EGS to be recognized by the RNase P already in the cell. EGSs are also able to bind to mRNA through complimentary base pairing, most often being designed to target mRNA from viruses or bacteria such as influenza or to mRNA of inflammatory signaling proteins such as interleukin-4 (IL-4). The target mRNA fits into the active site of the RNase P found in the host cell and is cleaved, leaving the EGS free to bind and guide another mRNA to RNase P (6). While EGS's have been found to be successful in vitro, it might be a while until you're getting EGS therapy for your flu.

Figure 5. Overview of EGS mechanism. mRNA is targeted by EGS through sequence complementarity, and is recognized by RNase P through CCA base pairing and TψC loop base stacking. The target mRNA is cleaved and then further degraded, leading to a decrease in viral/bacterial growth or prevention of inflammation signaling depending on the target mRNA. Figure adapted from Dreyfus et al. 2007.

References

1. Stark, B.C., Kole, R., Bowman, E.J., and Altman, S. (1978) Ribonuclease P: an enxyme with an essential RNA component. Proc. Natl. Avad. Sci. 75 (8), 3717-3721

2. Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N., and Altman, S. (1983) The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35, 849-857

3. Walker, S.C. and Engelke, D.R. (2008) A Protein-only RNase P in human mitochondria. Cell 135, 412-414

4. Evans, D., Marquez, S.M., and Pace, N.R. (2006) RNase P: interface of the RNA and protein worlds. Trends in Biochemical Sciences 31 (6)

5. Reiter, N.J., Osterman, A., Torres-Larios, A., Swinger, K.K., Pan, T., and Mondragón, A. (2010) Structure of a bacterial ribonuclease P holoenzyme in a complex with tRNA. Nature 468, 784-789

6. Dreyfus, D.H., Tompkins, S.M., Fuleihan, R., and Ghoda, L.Y. (2007) Gene silencing in the therapy of influenza and other respiratory diseases: targeting to RNase P by use of external guide sequences (EGS) Biologics: Targets & Therapy 1 (4), 425-432