1. Volume 12, Issue 4, Pages 925-935 (October 2003)
Eukaryotic RNase P 
Hagit Mann, Yitzhak Ben-Asouli, Aleks Schein, Sana Moussa, Nayef Jarrous 
Molecular Cell 
Volume 12, Issue 4, Pages (October 2003)
DOI: /S (03)

2. Figure 1
Reconstitution of the Endonucleolytic Activity of Human RNase P
(A) The indicated recombinant Rpp polypeptides (10 pmol each) and H1 RNA (10 pmol) were used in reconstitution assays as described in Experimental Procedures. Labeled precursor tRNATyr (0.035 pmol) was used as substrate, and the cleavage reactions proceeded for 2 hr at 37°C. Cleavage products, tRNA (3′) and 5′ leader sequence (5′), were resolved in denaturing 8% polyacrylamide/7 M urea gel. Substrate (lane 1) and control assay of purified HeLa RNase P (Ctrl; lane 2) are shown. p14, p20, p30, p38, and p40 correspond to recombinant Rpp14, Rpp20, Rpp30, Rpp38, and Rpp40. Asterisk points to degraded RNA in the running front of the gel.
(B) 10 pmol of each of H1 RNA and Rpp21 were incubated with 10 pmol (lanes 5–10) or 20 pmol (lanes 11–13) of Rpp29. Cleavage reactions of pSupS1 were done in 1× (lanes 12 and 13), 2× (lanes 5–7 and 11), or 3× (lanes 8–10) RP buffer containing 1, 2, or 3 mM of 2-mercaptoethanol (2-merc). Cleavage products were analyzed as in (A). Control assay was M1 RNA tested at 30 mM MgCl2 (lane 2). H1 RNA alone (lane 3) or Rpp21 and Rpp29 only (lane 4) are shown. Asterisk points to degraded RNA in the front of the gel.
(C) Kinetic analysis of pSupS1cleavage by purified HeLa RNase P or mini-RNase P. Reconstitution of mini-RNase P was done as described in Experimental Procedures but no preincubation time was allowed. Cleavage products were analyzed as in (A), and the plots depict substrate cleavage at the indicated time points in minutes. ×, HeLa RNase P; closed box, mini-RNase P.
(D) Proposed secondary structure of the H1 RNA. The structure is based on phylogenetic comparative analysis (see Frank et al., 2000) and includes the P4 pseudoknot. Positions of nucleotides in P4 are numbered (note 5′ and 3′ orientation). The P4 sequences of H1 RNA (H1) and the mutants, H1-2, H1-7, and H1-2c are shown. H1-2c is derived from H1-2 but has compensatory mutations at positions 83–84 (AG to GU).
(E) H1 RNA, H1-2 RNA, H1-7 RNA, and H1-2c RNA were used in reconstitution assays in the absence (lanes 3, 4, 5, and 9) or presence (lanes 6, 7, 8, 10, and 11) of Rpp21 and Rpp29. Reconstitution conditions and analysis of cleavage products of pSupS1 (S; lane 1) were as in (B). Control assay of purified HeLa RNase P is shown (lane 20).
(F) Coomassie blue staining of Rpp29 (p29wt, lane 2), Rpp29Δ (lane 3), and Rpp29Δ (lane 4) purified on nickel-charged resin columns (see Experimental Procedures). Positions of size markers are shown (M; lane 1).
(G) Reconstitution assays of mini-RNase P were performed as described in (B) using 20 pmol of each of Rpp29 (lane 5), Rpp29Δ (lane 6), or Rpp29Δ (lane 7). As control, H1 RNA alone (lane 3) or Rpp21/Rpp29 only (lane 4) were used. Cleavage products, tRNA (3′) and 5′ leader sequence (5′), were resolved as in (B).
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2
Cleavage Sites and End Group Termini of Substrate Processed by Mini-RNase P
(A) M1 RNA (lane 2), DEAE-purified HeLa RNase P (lane 3), and mini-RNase P (lane 6) were tested for processing of pSupS1. Cleavage products were then separated in 8% sequencing gel. The reconstituted mini-RNase P cleaved between positions G28 and G29, as did the authentic HeLa RNase P (lanes 3 versus 6), while M1 RNA cleaved between positions A27 and G28. Guanosine ladders (lanes 1 and 7) were obtained by partial digestion of pSupS1 by RNase T1. A size difference of 1 nucleotide exists between the 5′ leader sequences with 3′-OH and the RNA ladders with 3′-P. H1 RNA (lane 4) and Rpp21/Rpp29 (lane 5) were used as negative control.
(B) pSupS1 (S; lane 2) was processed by S. pombe nuclear RNase P (lanes 3 and 4). Cleavage products were analyzed as in (A). This authentic RNase P cleaved between positions G28 and G29. Guanosine RNA ladders were obtained by partial RNase T1 cleavage of pSupS1 (lanes 1 and 5).
(C) pSupS1 uniformly labeled with [α-32P]ATP (left column) or [α-32P]CTP (right column) was cleaved by HeLa RNase P (upper row), mini-RNase P (middle row), or M1 RNA (lower row). The tRNA products were gel extracted, digested with RNase T2, and analyzed in 2D-TLC (see Experimental Procedures). The positions of the various nucleotides are shown in the left column.
(D) Unlabeled pSupS1 was cleaved with M1 RNA, HeLa RNase P, or M1 RNA/C5. Cleavage products were subjected to 32P-pCp labeling using T4 RNA ligase, labeled 5′ leader sequences were gel extracted, and 3000 cpm of RNA was digested with RNase T2 and analyzed in 2D-TLC. Ap and Gp are shown. Arrows point to the first and second dimension of the 2D-TLC.
(E) Sequence and positions of nucleotides spanning the cleavage site of pSupS1. Positions of processing by M1 RNA versus HeLa RNase P, mini-RNase P, and M1 RNA/C5 are indicated by arrow heads.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 Ionic Requirements for the Reconstituted Mini-RNase P
(A) Reconstituted mini-RNase P was assayed for processing of precursor tRNATyr in the presence of 10–100 mM MgCl2 (lanes 5–10). Cleavage products, tRNA (3′) and 5′ leader sequence (5′), were resolved as in Figure 1A. In this particular experiment, H1 RNA alone (lane 3) and Rpp21/Rpp29 only (lane 4) were tested at 100 mM MgCl2 and showed to activity, thus excluding any possible contamination of these preparations by bacterial RNase P RNA. Arrow points to degraded RNA and asterisk indicates substrate miscleavage (lanes 2 and 5).
(B) Activity of mini-RNase P at 10–100 mM MgCl2, as measured from the determination of the optical density of the 5′ leader sequence seen in (A). Similar results have been obtained when the tRNA (3′) band was quantified (data not shown).
(C) Activity of mini-RNase P, DEAE-purified HeLa RNase P, and M1 RNA tested at various concentrations of NaCl (0.1–0.8 M). Analysis of substrate cleavage was as described in (A).
(D) Activities of mini-RNase P, DEAE-purified HeLa RNase P, and M1 RNA in 2×RP buffer adjusted by Tris-HCl to the indicated pH points. Substrate cleavage was analyzed as in (A).
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 H1 RNA Selectively Binds to Precursor tRNA
(A) H1 RNA and M1 RNA were incubated with labeled pSupS1 (lanes 1–7) or precursor tRNATyr (pTyr; lanes 8–14) in 40 μl of buffer B (see Experimental Procedures). Increasing amounts of H1 RNA and M1 RNA used for binding were 0.7 (lanes 2 and 5, respectively), 2.9 (lanes 3 and 6), and 5.8 pmol (lanes 4 and 7). C1 points to the major complex formed in this native polyacrylamide gel. Cleavage products, 5′ leader sequence (5′) and tRNA (3′), obtained in the presence of M1 RNA are indicated.
(B) Precursor tRNATyr (lane 1) was incubated with 2.6 pmol of H1 RNA (lane 1), M1 RNA (lanes 2), or MRP RNA (lane 4), and binding was performed as described in (A).
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5 Rpp29 Activates M1 RNA In Vitro
(A) A histidine-tagged Rpp29 polypeptide was purified on a nickel-charged resin column, and aliquots from eluted fractions, F2–F50, were separated in 12% SDS/PAGE before staining with Coomassie blue staining. Positions of Rpp29 (p29) and protein size markers (in kilodaltons) are shown.
(B) M1 RNA (0.7 pmol) was tested for processing of pSupS1 (S) in the absence (lanes 1–8) or presence (lanes 9–16) of 2 μl of each of the indicated fractions of recombinant Rpp29 (fraction F14 has 14 pmol of protein in 2 μl). As control, M1 RNA alone was tested in the presence of 10 mM MgCl2 (lane 18) or 80 mM MgCl2 (lane 17) in 1×RP buffer. Cleavage products, tRNA (3′) and 5′ leader sequence (5′), were resolved in denaturing polyacrylamide gel. Asterisk points to degraded RNA.
(C) Western blot analysis of serial dilutions of recombinant C5 protein (14, 7, 3.5, 1.75, 0.87, 0.43, and pmol) detected by affinity-purified polyclonal rabbit anti-C5 antibodies. Positions of protein size marker are shown.
(D) 40 μl of selected fractions, F5–F50, seen in (A), were concentrated and subjected to Western blot analysis using anti-C5 antibodies as in (C).
(E) Reconstitution assays of M1 RNA (0.7 pmol) in the presence of 7, 14, 21, and 28 pmol of each of Rpp29 (lanes 6–9), Rpp29Δ (lanes 10–13), or Rpp29Δ (lanes 14–17). As controls, M1 RNA (lane 1), Rpp29 (lane 3), Rpp29Δ (lane 4), or Rpp29Δ (lane 5) alone were tested for processing of pSupS1 at 10 mM MgCl2. Active M1 RNA was assays at 80 mM MgCl2 (lane 2). Cleavage products were analyzed as in (B).
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
Kinetic Analysis of M1 RNA/Rpp29 Reaction with pSupS1 Substrate
(A) M1 RNA (0.35 pmol) was assayed in the presence or absence of Rpp29 (7 pmol) for processing of precursor tRNATyr at 3–150 mM MgCl2. Cleavage products were analyzed as described in Figure 5B. The optical density of the 5′ leader sequence was determined and plotted. Similar results have been obtained when the tRNA (3′) band was quantified (data not shown).
(B) Lineweaver-Burk plot for M1 RNA/C5 (see Experimental Procedures). Calculated Vmax (pmol/min) and Km (pmol/μl) values are shown.
(C) Lineweaver-Burk plot for M1 RNA/Rpp29 (see Experimental Procedures). Calculated Vmax (pmol/min) and Km (pmol/μl) values are shown.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7 M1 RNA and Rpp29 Form a Stable Ribonucleoprotein Complex
(A) 30 pmol of M1 RNA was preincubated with 300 pmol of Rpp29 for 20 min on ice, and the mixture was then analyzed by velocity sedimentation in 15%–30% glycerol gradient as described (Jarrous and Altman, 2001). Aliquots from fractions F3 to F15 derived from the gradient were assays for processing of precursor tRNATyr. Assays were performed in 1xRP buffer containing 10 mM MgCl2, in which M1 RNA alone was inactive (lane 2). As positive control, M1 RNA/C5 was assayed (lane 1). Cleavage products were analyzed as in Figure 5B.
(B) Fractions F6–F34 eluted from a Sephacryl S-100 HR column, in which M1 RNA and Rpp29 cofractionated were assayed for processing of pSupS1, as described in Experimental Procedures. The optical density of the 5′ leader sequence was measured and plotted as shown. Arrow points to the IgG peak (∼160 kDa; 9E10 monoclonal antibody) seen in fraction F20.
(C) Western blot analysis of fractions F8–F24 using polyclonal rabbit anti-Rpp29 antibodies (Jarrous et al., 1999). Arrowhead points to possible dimeric form of Rpp29. Positions of protein size marker are indicated.
(D) Membrane seen in (C) was analyzed by anti-mouse IgG HRP antibody. IgG (“H”) and IgG (“L”) indicate heavy (55 kDa) and light (25 kDa) chains of the monoclonal 9E10 antibody.
Molecular Cell  , DOI: ( /S (03) )