Volume 1, Issue 3, Pages 401-409 (February 1998) T. brucei RNA Editing: Adenosine Nucleotides Inversely Affect U-Deletion and U- Insertion Reactions at mRNA Cleavage  Jorge Cruz-Reyes, Laura N Rusché, Kenneth J Piller, Barbara Sollner-Webb  Molecular Cell  Volume 1, Issue 3, Pages 401-409 (February 1998) DOI: 10.1016/S1097-2765(00)80040-4

Figure 1 Current Understanding of U-Deletional and U-Insertional Editing RNA editing involves gRNA-directed cleavage of the mRNA, followed by either a 3′-U exonuclease or a 3′ TUTase acting on the cleaved upstream fragment, and RNA ligase action (Blum et al. 1990; Cruz-Reyes and Sollner-Webb 1996; Kable et al. 1996; Seiwert et al. 1996). The 3′-U-removal reaction is not a reverse TUTase reaction (Cruz-Reyes and Sollner-Webb 1996; Rusché et al. 1997). The present paper demonstrates that gRNA-directed cleavages at U-deletional and U-insertional editing sites are biochemically distinct, with the former requiring an adenosine nucleotide, while the latter is inhibited by adenosine nucleotides. 90% of editing sites are U-insertional while 10% are U-deletional. Molecular Cell 1998 1, 401-409DOI: (10.1016/S1097-2765(00)80040-4)

Figure 6 The Adenosine Nucleotide Concentration Needed to Affect gRNA-Directed Cleavages (A–D) Cleavage reactions used DNA-cellulose-purified editing complex, the indicated millimolar concentrations of adenosine nucleotides, and, where indicated, PPi. (A) and (D) (lanes 1–3) used [0,4] mRNA and [2,1] gRNA; (B), (C), and (D) (lanes 4–7) used [0,4] mRNA and [2,4] gRNA (diagrammed in Figure 3G). (E) RNA ligase assay, showing ligated product RNA, was performed using the DNA-cellulose-purified editing complex, the indicated ribonucleotides at 3 mM unless stated, and 3 mM PPi where indicated, as in Rusché et al. 1997. In all reactions, most input RNA remained unligated. Molecular Cell 1998 1, 401-409DOI: (10.1016/S1097-2765(00)80040-4)

Figure 3 The Nucleotide Involvement in Cleavage at Altered Positions within Mutated A6 ES1, ES2, and ES3 Is Maintained Five mRNA:gRNA pairs with features to specify U deletion within altered sequences of ES1 (A–E) and five mRNA:gRNA pairs with features to specify U insertion within altered sequences of ES2 and ES3 (F–J) were analyzed, and all exhibited the same ATP/ADP effects as the parental sites. The examined mRNAs are: pre-edited wild-type [0,4]; ES1-edited [0,2]; ES2- and ES1-edited [2,2]; and mutants [0,5], [0,1], and [5,2]. The examined gRNAs are: wild-type [2,2] and mutants [2,1], [2,4], and [5,2]. Designations are as in Figure 2, and residues of the editing sites are in bold. Molecular Cell 1998 1, 401-409DOI: (10.1016/S1097-2765(00)80040-4)

Figure 2 The Nucleotide Involvement in A6 gRNA–Directed Cleavage Is Different at ES1 versus ES2 and ES3 (A–C) Cleavage reactions with the indicated 3′ end–labeled A6 mRNA, gRNA, and 3 mM adenosine nucleotide were performed using Q-Sepharose-purified editing complex (see Experimental Procedures). Similar adenosine nucleotide effects are observed using editing complex that had been additionally purified on DNA-cellulose. Pre-mRNA substrates are designated [x,y] where x and y are the number of U residues present at ES2 and ES1, respectively, and complementary gRNAs are designated [m,n] where m and n are the number of U residues that this gRNA directs to be at ES2 and ES1, respectively. The RNA sequence and cleavage position are indicated. Below is shown a portion of the RNA pairs that includes the anchor region and ES1 through ES3. The gRNA-directed cleavage (arrowhead enclosing the number of the cleaved editing site) occurs just 5′ of the anchor region duplex (shaded box). Solid and dotted lines indicate Watson-Crick and G:U base pairing within the anchor and potential base pairing that will direct subsequent rounds of editing; the asterisk represents the 3′-end label. RNA markers (A+U) prepared with PhyM RNase bear 5′-OH termini and migrate ∼1/2 nt slower than the extract-cleaved mRNA fragments that bear 5′-P termini (Cruz-Reyes and Sollner-Webb 1996). (D) For cleavage reactions, we used the same RNAs as in (A), 3 mM of the indicated nucleotide, and DNA-cellulose-purified editing complex. Molecular Cell 1998 1, 401-409DOI: (10.1016/S1097-2765(00)80040-4)

Figure 7 Full Round U-Deletional and U-Insertional Editings Reflect the Adenosine Nucleotide Effects of Their Component Cleavage Reactions Full round U-deletional (A) and U-insertional (B) editing reactions used the DNA-cellulose-purified editing complex, 3′ end–labeled input mRNA, and the indicated millimolar concentrations of ATP, ADP, and UTP. (A) and (B) used the [0,4] mRNA and gRNA [2,1] or [2,4], respectively. Molecular Cell 1998 1, 401-409DOI: (10.1016/S1097-2765(00)80040-4)

Figure 4 The Nucleotide Involvement at Interconverted U-Deletional and U-Insertional A6 Editing Sites Is Also Interconverted Cleavage analysis of A6 RNA pairs mutated to have features of U insertion at ES1 (A) and of U deletion at ES2 (B). Reactions and descriptions are as in Figure 2, using the Q-Sepharose fraction (A) or the DNA-cellulose fraction (B). Molecular Cell 1998 1, 401-409DOI: (10.1016/S1097-2765(00)80040-4)

Figure 5 Cleavage at Natural, Altered, and Interconverted ES1 and ES2 of CYb Exhibits the Predicted Nucleotide Specificity (A) shows cleavage of wild-type CYb mRNA at ES1 specified by gRNAs directing insertion of two U residues (the wild-type case, lanes 1–3) and one U residue (lanes 4–6). (B) and (C) show two altered CYb RNA pairs where ES1 and ES2 were converted to have features of U-deletion sites, both specifying removal of one U residue. Reactions used 3′ end–labeled mRNA substrates and DNA-cellulose-purified editing complex and are designated as in Figure 2. The G cleavage markers were prepared with nuclease P1 and have 5′-P termini, like the extract-cleaved RNAs (Cruz-Reyes and Sollner-Webb 1996; J. C.-R. et al., submitted). Molecular Cell 1998 1, 401-409DOI: (10.1016/S1097-2765(00)80040-4)