Eukaryotic RNase P RNA mediates cleavage in the absence of protein

Abstract

The universally conserved ribonucleoprotein RNase P is involved in the processing of tRNA precursor transcripts. RNase P consists of one RNA and, depending on its origin, a variable number of protein subunits. Catalytic activity of the RNA moiety so far has been demonstrated only for bacterial and some archaeal RNase P RNAs but not for their eukaryotic counterparts. Here, we show that RNase P RNAs from humans and the lower eukaryote Giardia lamblia mediate cleavage of four tRNA precursors and a model RNA hairpin loop substrate in the absence of protein. Compared with bacterial RNase P RNA, the rate of cleavage (k(obs)) was five to six orders of magnitude lower, whereas the affinity for the substrate (appK(d)) was reduced approximately 20- to 50-fold. We conclude that the RNA-based catalytic activity of RNase P has been preserved during evolution. This finding opens previously undescribed ways to study the role of the different proteins subunits of eukaryotic RNase P.

Fig. 1.

Secondary structures of pATSerUG and the precursor to tRNA^Ser, pSu1. The canonical RNase P cleavage sites are indicated with arrows, and numbering is in accordance with standard numbering of tRNA. For the secondary structures of the well characterized tRNA precursors pSu3 and pHis[UAG], see SI Fig. 6 and ref. , whereas characterization of pTS-L(-1U) as substrate for E. coli RNase P RNA is unpublished (but see ref. , where the secondary structure and construction of the original pTS-L are outlined).

Fig. 2.

Illustration of the predicted secondary structures of the different RNase P RNAs in the present study: E. coli RNase P RNA (M1 RNA) and a variant lacking the P15-17 region (indicated area in M1 RNA; M1_ΔP15–17 RNA); human RNase P RNA (H1 RNA) variants with changes at the indicated positions: H1_Δ298C325 RNA, H1_Δ298U325 RNA, H1_C298C325 RNA, H1_C298U325 RNA, and H1_{Δ80–82Δ298} RNA; and the lower eukaryote G. lamblia RNase P RNA, G1 RNA, and G1_ΔJ5/6 RNA. For references, see refs. , , , and . These RNase P RNAs were generated as described in Materials and Methods.

Fig. 3.

Cleavage of pATSerUG and pSu1 with different RNase P RNAs and TLC analysis of cleavage products. (A) Cleavage of 5′ end-labeled pATSerUG and pSu1 with different RNase P RNAs as indicated. Time of incubation is as indicated, and the position of the 5′ cleavage fragments is marked with arrows. Neg ctrl, substrates incubated for 23 h in the reaction buffer without RNase P RNA. The different positions of the 5′ cleavage fragments in G1 RNA vs. M1 RNA- and H1 RNA-mediated cleavage were due to the fact that the G1 RNA and M1 RNA/H1 RNA experiments were run on different polyacrylamide gels. (B) Two-dimensional TLC demonstrating the presence of pGp (the hallmark in RNase P RNA-mediated cleavage) at the 5′ end of the 5′ matured cleavage product after cleavage of [α-³²P]GTP internally labeled pATSerUG (specific activity ≥5 Ci/mmol) with wild-type M1 RNA, M1_ΔP15–17 RNA, H1 RNA, and no RNase P RNA added as indicated.

Fig. 4.

RNase P RNA-mediated cleavage of pATSerUG expressed as a percentage of cleavage per minute as a function of [Mg²⁺]. The different RNase P RNAs used are indicated, and the concentrations were 3.2 μM for M1 RNA, M1_ΔP15–17 RNA, and H1 RNA; 6.4 μM for G1 RNA; and 0.02 μM for the substrate. The reactions were performed in buffer C at 37°C (see Materials and Methods). Time of incubation was adjusted to ensure that they were in the linear part of the curve of kinetics. For the calculations, we used the 5′ cleavage fragments. The given data are the average of two independent experiments, and error bars indicate the experimental range. The data for wild-type M1 RNA are based on three independent experiments and taken from ref. .

Fig. 5.

Cleavage of various internally ³²P-labeled full-size tRNA precursors [pSu3, pHis[UAG], and pTS-L(-1U)] by wild-type M1 RNA and H1 RNA. For experimental details, see Materials and Methods. Lanes: 1, wild-type M1 RNA (10-sec reaction time); 2, H1 RNA (23-h reaction time); 3, negative control (23-h reaction time). pretRNA^His, precursor to tRNA^His; pSu3, precursor to tRNA^TyrSu3; pTS-L, chimeric tRNA precursor generated by replacement of the pSu3 acceptor stem with the acceptor stem of tRNA^SerSu1 (see also ref. 15). Specific activity for all substrates was ≥5 Ci/mmol.