A Deletion Site Editing Endonuclease in Trypanosoma brucei James Raffaello Trotter, Nancy Lewis Ernst, Jason Carnes, Brian Panicucci, Kenneth Stuart  Molecular Cell  Volume 20, Issue 3, Pages 403-412 (November 2005) DOI: 10.1016/j.molcel.2005.09.016 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Effects of KREN1 and KREPB2 Repression on Growth Growth of KREN1 (A) or KREPB2 (B) PF RNAi cell lines in the absence (closed diamonds) or presence (open diamonds) of RNAi induction with tet is indicated. Growth of BF KREN1 RKO cells that contain a tet-inducible KREN1 ectopic allele in the 18S rDNA locus but lack both endogenous alleles (C) are shown. Cells were grown in the presence of tet, allowing KREN1 expression (closed diamonds), or absence of tet for KREN1 repression (open diamonds). KREN1 expression was reactivated after three days of repression (arrow) by the reintroduction of tet (closed triangles). Molecular Cell 2005 20, 403-412DOI: (10.1016/j.molcel.2005.09.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Effects of KREN1 and KREPB2 Repression on RNA Editing In Vivo Real-time PCR analysis of RNA from PF cells in which KREN1 (A) or KREPB2 (B) expression was repressed for four days by RNAi and from BF cells in which KREN1 expression was conditionally repressed for three days (C) are shown. The abundance of the preedited (pre, white bars) and edited (edit, dark gray bars) mitochondrial RNAs from repressed cells is shown relative to that from cells in which KREN1 and KREPB2 are expressed. The relative abundance of the KREN1 or KREPB2 mRNAs (black bars) and ND4 mRNA (light gray bars), which does not get edited, is also shown. The RNA levels are normalized to β-tubulin mRNA (left bars) and 18S rRNA (right bars). The thick black line at 1 indicates no relative change in mRNA level, with anything above or below this line representing an increase or decrease in mRNA levels, respectively. Error bars represent the standard deviation from at least three replicates. Molecular Cell 2005 20, 403-412DOI: (10.1016/j.molcel.2005.09.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Editosome Sedimentation after KREN1 or KREPB2 Repression Western blotting analysis of fractions of glycerol-gradient-purified mitochondrial lysates from PF RNAi cells in which KREN1 (A) or KREPB2 (B) was expressed and repressed for four days or whole-cell lysates of BF KREN1 RKO cells in which KREN1 was expressed or repressed for three days (C) is shown. Western blotting analysis used a mixture of four Mabs specific for the editosome proteins indicated. A fraction that contains ∼20S editosomes was used as a positive control (+), and blots were reprobed with hsp70 in (C) to ensure equal loading. Molecular Cell 2005 20, 403-412DOI: (10.1016/j.molcel.2005.09.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Repression of KREN1 Expression Inhibits Deletion Cleavage in Both PFs and BFs Full-round and pre-cleaved editing assays using peak ∼20S glycerol gradient fractions from mitochondrial lysates from PF KREN1 (N1) and KREPB2 (B2) RNAi lines (day 4) or whole-cell lysates from BF KREN1 RKO cells (day 3) in which KREN1 or KREPB2 was expressed (E) or repressed (R) are shown. Assays were of A6 full-round deletion (A), A6 precleaved deletion (B), A6 full-round insertion (C), A6 precleaved insertion (D), and CYb insertion endonuclease assays (E). The 3′ cleavage products are indicated by arrows, and controls in which gRNA is omitted (−g) show their gRNA dependence. The radiolabeled input RNAs (I), products from which four Us are removed (−4U) or in which two Us are added (+2U), ligated products of unprocessed 5′ and 3′ input RNAs (Lig), and edited (Ed) products are indicated. Positive control reactions (+) were performed using a fraction that contains peak editing activity, which was omitted in negative control reactions (−). T1-digested input RNA (T1) was used as a size marker. Molecular Cell 2005 20, 403-412DOI: (10.1016/j.molcel.2005.09.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Mutations within the Conserved RNase III Signature Motif of KREN1 Are Lethal to BFs Multiple sequence alignment of RNase III catalytic domains (A) is shown. Representative sequences are shown for KREN1, KREPB2, and KREN2 editosome proteins as well as Escherichia coli RNase III (Ec-RNase III, SWISS-PROT database number P05797), Aquifex aeolicus RNase III (Aa-RNase III, SWISS-PROT database number O67082), Drosophila melanogaster Drosha (Dm-Drosha, SWISS-PROT database number Q9XYN5), and Homo sapiens Dicer (Hs-Dicer, GenBank number AB028449). Both Dm-Drosha and Hs-Dicer have two RNase III domains. The alignment is numbered according to Aa-RNase III. Boldface characters within the alignment indicate highly conserved residues involved in catalysis (D44 and E110) or possibly substrate binding or dimerization (E37 and E64). The corresponding conserved residues that were mutated in KREN1 (E271V or D278A) are indicated above E37 and D44. The RNase III signature motif is underlined. Growth of the BF KREN1 WT (closed, open diamonds), E271V (closed, open triangles), and the D278A (closed, open squares) cells with KREN1 Reg expressed or repressed, respectively (B). Western blotting analysis of fractions of glycerol gradient purified whole-cell lysates of the BF KREN1 WT cells when KREN1 Reg was expressed or repressed for three days, as described in Figure 3 (C), is shown. Molecular Cell 2005 20, 403-412DOI: (10.1016/j.molcel.2005.09.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Mutations within the Conserved RNase III Signature Motif of KREN1 Block RNA Editing In Vivo Real-time PCR analysis of RNA from BF KREN1 WT cells at day 3 (A), E271V cells at day 3 (B), or D278A cells at day 2 (C) after repression of KREN1 Reg allele expression. The abundance of mRNAs from KREN1 Reg (black bars); constitutively expressed KREN1 WT, E271V, and D278A alleles (black bars with gray outlines); preedited (pre, white bars); edited (edit, dark gray bars); and ND4 mRNA that does not get edited (light gray bars) is shown relative to when KREN1 Reg is expressed. Normalization was carried out as described in Figure 2. Error bars represent the standard deviation from at least three replicates. Molecular Cell 2005 20, 403-412DOI: (10.1016/j.molcel.2005.09.016) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Mutations within the Conserved RNase III Signature Motif of KREN1 Abolish Deletion Cleavage Full-round and precleaved editing assays using peak ∼20S glycerol-gradient fractions from whole-cell lysates of the BF KREN1 WT (day 3), E271V (day 3), and D278A (day 2) cells when the KREN1 Reg allele was expressed (E) or repressed (R) are shown. In all cases, KREN1 is abbreviated to N1. A6 full-round deletion assays (A), A6 precleaved deletion assays (B), A6 full-round insertion assays (C), and A6 precleaved insertion assays (D) are depicted. Labels are as described in Figure 4. The endonuclease doublet products in (A) reflect incomplete isolation of single-length substrate RNA after labeling. This doublet is also seen in the T1 digest lane. Molecular Cell 2005 20, 403-412DOI: (10.1016/j.molcel.2005.09.016) Copyright © 2005 Elsevier Inc. Terms and Conditions