The RNA binding activity of the first identified trypanosome protein with Z-DNA-binding domains

RNA-binding proteins play a particularly important role in regulating gene expression in trypanosomes. A map of the network of protein complexes in Trypanosoma brucei uncovered an essential protein (Tb927.10.7910) that is postulated to be an RNA-binding protein implicated in the regulation of the mitochondrial post-transcriptional gene regulatory network by its association with proteins that participate in a multi-protein RNA editing complex. However, the mechanism by which this protein interacts with its multiple target transcripts remained unknown. Using sensitive database searches and experimental data, we identify Z-DNA-binding domains in T. brucei in the N- and C-terminal regions of Tb927.10.7910. RNA-binding studies of the wild-type protein, now referred to as RBP7910 (RNA binding protein 7910), and site-directed mutagenesis of residues important for the Z-DNA binding domains show that it preferentially interacts with RNA molecules containing poly(U) and poly(AU)-rich sequences. The interaction of RBP7910 with these regions may be involved in regulation of RNA editing of mitochondrial transcripts.


Results
Identification of potential Z-DNA-binding domains in RBP7910. The conventional sequence search methods BLAST 32 and PSI-BLAST 33 were used to interrogate potential biological functions of RBP7910 based on homologous proteins identified in a sequence search. The similarity search tools based only on sequence returned no relationships to proteins with known function. In an alternative strategy, we used HHpred, a highly sensitive method for searching for more remotely homologous relationships 34 . Using an improved version of profile-sequence comparison, Profile Hidden Markov Models (HMMs), HHpred predicted two potential Z-DNA-binding domains in the N-and C-terminal regions of RBP7910. The N-or C-terminal sequences were input into HHpred or I-TASSER server instead of the complete sequence 35,36 and dramatically improved the accuracy of the predicted function and secondary structure of each domain. The secondary structure prediction showed three-helix bundles and three β-sheets with an αβααββ topology for both domains. Similar α/β HTH architecture, consisting of three α-helices and three β-strands, has been observed in Z-DNA-binding proteins (ZBPs) 30,37,38 . Multiple sequence alignment of N-and C-terminal domains of RBP7910 with some of its orthologs and corresponding domains of known ZBPs is shown in Fig. 1. To date, four protein families with one or two tandem Z-DNA-binding domains have been identified: ADAR1, DLM-1 or ZBP1, a protein kinase from fish containing a Z-DNA-binding domain (PKZ) and the viral protein E3L [38][39][40][41] . ADAR1, DLM-1, and PKZ contain two Z-DNA-binding domains (Zα and Zβ, respectively), whereas E3L has one Zα domain. The nucleic acid binding activity of the Zα and Zβ domains of different ZBPs has been widely studied. The Zα domain exhibits a higher level of sequence conservation than the Zβ domain. Crystallographic data for ZBPs showed that residues from α3 and the β2/β3 wing region serve as the nucleic acid binding interfaces 31,38,42 (Fig. 1). The N-terminal domain of RBP7910 also shows a greater sequence conservation than the C-terminal sequence, particularly at the nucleic acid-contacting interfaces.
RBP7910 has higher affinity for gRNAs than mRNAs. We determined the binding affinity of recombinant RBP7910 for radiolabeled gRNAs and pre-edited and edited mRNAs using electrophoretic mobility shift assay (EMSA) to assess the RNA-binding ability of predicted Z-DNA-binding domains of RBP7910. RNA substrates including (gA6 [14]) 43 , A6U5 pre-mRNA 6 , edited A6U5 (deletion of 3Us), CYb gRNA (gCYb [558] USD-2A-42nt) 44 , native CYb gRNA (gCYb [558]) 45 , CYb pre-mRNA 46 , and CYb edited-mRNA 47 were in vitro transcribed and labeled with [α-32 P] either during transcription or after transcription at the 3′ end of the mRNA. Despite the detection of a protein-RNA complex between recombinant RBP7910 and gA6 [14] and pre-and edited CYb mRNAs, we did not detect binding between this protein and the A6 pre-mRNA or any CYb gRNA variant (data not shown).
The incubation of a fixed amount of RBP7910 with increasing concentrations of radiolabeled gA6 [14] or pre-edited and edited CYb mRNAs resulted in the formation of a slowly migrating protein-RNA complex ( Fig. 2A). The Kd for the interaction of recombinant RBP7910 with each labeled RNA substrate was estimated from five individual experiments, and the results were analyzed using a non-linear regression model. As shown in Fig. 2A, the Kd value for the wild-type (WT) protein interacting with the U-tail-bearing A6 guide RNA substrate was determined to be 0.21 ± 0.01 nM; 95% CI:0.18, 0.27, indicating a significantly higher affinity for this target than for the edited CYb mRNA (1.57 ± 0.06 nM; 95% CI:1.25, 1.97) and pre-edited CYb mRNA (2.78 ± 0.20 nM; 95% CI:2.09, 3.66) substrates.
To support these results, we performed competition experiments using labeled gA6 [14] RNA, unlabeled CYb gRNA variants and CYb mRNAs as competitors. The natural gCYb RNA with the U-tail competed for binding between RBP7910 and gA6 [14] RNA 10 times better than the 42-mer CYb gRNA without the U-tail (Fig. 2B). The same concentrations of natural gCYb RNA and pre-CYb mRNA (10-fold molar excess of unlabeled RNAs) reduced binding of the labeled RNA by 50%. However, the edited CYb mRNA, which contains approximately double the number of Us compared to the pre-edited CYb RNA, competed for binding more efficiently by producing a 30% reduction in binding of labeled gA6 [14] RNA at a 10-fold molar excess of unlabeled edited CYb mRNA. We performed competition experiments, discussed in the next section, to examine the specificity of  gRNA and mRNA-binding specificity of RBP7910. Gel shift assays were conducted to examine the specificity of binding of each substrate using radiolabeled substrates in the presence of increasing concentrations of unlabeled RNAs, including gA6 [14] and pre-and edited CYb mRNAs. Unlabeled homologous RNAs reduced binding at the same molar ratios of labeled RNAs and eliminated RBP7910-labeled RNA interactions at 10-fold molar excess concentrations (Fig. 3A, left panel). We also examined competitive binding using a heterologous 92nt pBlueScript RNA at up to a 1000-fold excess (Fig. 3A, right panel) and observed negligible competition for binding of RBP7910 with the CYb mRNA and A6 gRNA.
We also assessed the affinity of RBP7910 for the poly (U)-tail of the gRNA by performing a competition assay using unlabeled gA6 [14] RNA lacking the U-tail with gA6 [14] RNA (Fig. 3B). While an equimolar ratio of the unlabeled gA6 [14] RNA with the U-tail completely competed for binding of labeled gA6 [14] RNA (Fig. 3A), a 100-fold molar excess of unlabeled gA6 [14] RNA lacking the U-tail only reduced complex formation by 25%. This result indicates the importance of the U-tail in the RBP7910-gRNA-binding process. We confirmed this finding using unlabeled poly U as a competitor and showed that it competed with the bound complex at an equimolar ratio of unlabeled poly U and labeled gA6 [14] RNA (Fig. 3B).
Thus, we used a uridylated non-guide RNA (49 nt) as the competitor to more comprehensively investigate the contributions of stem-loop elements (secondary structure) and the U-tail in the RBP7910-gRNA interaction (Fig. 3B). This RNA has a shorter poly U-tail (15 nt) and only one stem-loop compared to the gA6 [14] RNA. The non-guide RNA was more efficient in competing the RBP7910-gA6 [14] complex than gA6 [14] RNA lacking the U-tail, but was still 10 times less efficient than gA6 [14] RNA. This result indicates indispensable roles for the oligo U-tail and the secondary structure of the gRNA in the RBP7910-gRNA interaction, although again suggesting that the oligo U-tail is the main determinant.
In light of the high affinity of RBP7910 for gRNA, we asked if RBP7910 possesses a general gRNA stabilizing activity during the RNA editing process, similar to the gRNA-binding proteins GRBC1 and GRBC2 11 . We compared the total gRNA population between Tet-induced and uninduced cells expressing a RBP7910 knock-down RNAi construct 3 and 4 days post-induction 28 using guanylyl transferase labeling to determine the contribution of RBP7910 to the stability of the total gRNA population (Fig. 3C). No prominent changes were observed in the levels of gRNAs between induced and uninduced samples. Therefore, the major gRNA-binding activity of RBP7910 is not related to stability of gRNAs, and the gRNA-binding activity of the protein is part of the general RNA-binding activity of RBP7910. However, we cannot exclude the possibility of a transcript-specific effect of RBP7910 on gRNA stability in the absence of data on individual gRNAs.

RBP7910 shows distinct affinity for AU-enriched sequences. Mitochondrial mRNAs and gRNAs are
AU-rich transcripts with multiple biological functions. According to Brown and colleagues 48 , AU elements in the pre-edited CYb mRNA function as the primary assembly point for the editosome machinery. Following the binding of the gRNA to the pre-edited CYb mRNA, editing factors are transferred to the AU elements of the gRNA. Similarly, another study showed the importance of the AU sequence for formation of the pre-edited/gRNA duplex using A to C point mutations within the gRNA-binding site that interfered with the formation of the pre-edited/ gRNA duplex 49 and reduced editing by 80%.
Another AU structure in mitochondrial transcripts is the long AU-tail, a post-editing AU extension of the primary short A-tail of pre-edited transcripts. The long AU-tail is a hallmark of the translation process of fully-edited transcripts 26 . In addition to the general factors involved in synthesis of the long AU-tail, such as RET1, KPAP1, and KPAF1, other RBPs selectively affect the stability of mitochondrial mRNAs containing AU-tails and activate their translation at the insect life stage 50 .
Considering the RNA-binding activity of RBP7910, we next questioned the potential AU sequence-binding affinity of RBP7910. We labeled a poly AU sequence previously found to be enriched in 3′ untranslated region of many trypanosomatid genes 51 . Incubation of increasing concentrations of RBP7910 with a fixed amount of labeled poly AU RNA led to the formation of a RNA-protein complex. The specificity of the protein-RNA interaction was confirmed in a competition assay using the unlabeled RNA (Fig. 4A).
In light of the importance of the AU sequence during the editing process and for duplex formation of gRNA/ pre-edited mRNA 48,49 , we assayed the interaction of RBP7910 with a modified poly AU sequence containing U to C substitutions. The ability of this U to C-substituted poly AU RNA to compete for binding was largely abolished compared to the poly AU substrate (Fig. 4B).
We tested the abilities of poly U, poly A, and poly G RNAs to compete with the RBP7910 -poly AU interaction as a method to determine whether RBP7910 prefers poly AU or poly U as substrate. Poly U RNA was the most competitive substrate, as it decreased complex formation by 50% at an equimolar concentration, while poly A and poly G RNAs were similar to the U to C-substituted poly AU RNA, as shown above.
Based on these results, we conclude that RBP7910 binds to AU-containing RNAs. However, we were unable to determine whether RBP7910 binds to the internal AU sequence of the mitochondrial substrates (gRNAs and DLM-1 from Homo sapiens in hZαDLM-1 and Mus musculus, mZαDLM-1; E3L from orf virus in orfZαE3L and yabZαE3L from Yaba-like disease virus; PKZ from goldfish, caZαPKZ and drZαPKZ in zebrafish; ADAR1 from Mus musculus, mZαADAR1, and hZαADAR1 in Homo sapiens. Zβs include goldfish PKZ, caZβPKZ and zebrafish PKZ, drZβPKZ; ADAR1 in hZβADAR1 from Homo sapiens and Mus musculus, mZβADAR1; DLM-1 in Mus musculus, mZβDLM-1, and hZβDLM-1 from Homo sapiens. www.nature.com/scientificreports www.nature.com/scientificreports/ mRNAs) or the poly AU-tail of mitochondrial transcripts. Although we have shown binding of RBP7910 to the AU sequence, we could not determine if this interaction is purely sequence specific or mediated by the secondary structures of the sequence. Considering the binding preference of RBP7910 for the poly AU sequence, we propose that this protein is likely to be involved in RNA editing or translation of mRNAs containing an AU-tail.
Functional analysis of the RNA-binding activity of RBP7910 using structure-based mutagenesis. Following experimental establishment of the RNA-binding activity of RBP7910, we were interested in identifying residues that affect RNA binding based on sequence and structural alignments. Structural predictions and sequence analysis of RBP7910 identified two putative Z-DNA-binding domains in the N-and C-termini of RBP7910. The Z-DNA-binding domain family belongs to the superclass of protein with WHTH domains, and this superclass is largely present in the DNA-binding domain of prokaryotic transcription factors and some eukaryotic transcription factors 52 . This domain specifically recognizes the Z-form of DNA/RNA molecules in a conformation-specific manner. Because RBP7910 and ZBPs exhibit a similar fold, we examined whether they also shared the same nucleotide-binding interface. Zα and Zβ are structurally homologous domains with a similar  [14] to other guides and mRNAs. Competition assays were done by incubation of a fixed concentration of purified protein and labeled gA6 [14] in the absence and presence of increasing concentrations of unlabeled competitors (gCYb RNA variants, pre-edited, and edited CYb mRNAs). Asterisk indicates the input labeled RNA in the absence of the protein and the white star shows the labeled RNA with protein in the absence of the competitor RNA. Numbers above the panels indicate the fold excess of the unlabeled RNA competitors and numbers below of each panel is the shift percentage in the presence of competitor RNAs normalized to the shift in the absence of a competitor whitestar ( ) . The name of unlabeled RNA substrate used for each assay is indicated above each panel along with the complete sequence under each panel.
www.nature.com/scientificreports www.nature.com/scientificreports/ arrangement of α helices and β sheets (αβααββ), with the exception of the presence of one extra helix (α4) in ZβADAR1, which is mostly involved in dimerization of the protein 37 .
The RNA-binding function of RBP7910 was probed by replacing candidate RNA-contact residues in the Nand C-terminal domains of RBP7910 with alanine. Sequence comparisons of different ZBPs suggested the presence of a common nucleic acid recognition core containing hydrophobic and positively charged amino acids in the α3 core and the β2/β3 wing 53 . As shown in Fig. 1, Asn173, Tyr177, and Trp195 of hzαADAR1 are the most conserved core residues in ZBPs 30,54 . These residues are also conserved in the hZβDLM-1/Z-DNA complex 53 , although with a different hydrogen bonding pattern. Furthermore, ZBPs contain one or two proline (P192-P193 of hzαADAR1) residues that contribute to the Zα DNA-binding activity via hydrophobic interactions. These proline residues are usually located adjacent to a polar residue such as Thr or Asn, which interacts with DNA through water-mediated hydrogen bonds 30,38,55 . No equivalent residue for the Pro or Thr residues of Zα are present in Zβ domains.
A few mutagenesis studies have investigated the Z-DNA/RNA-binding activities of ZBPs. For instance, alanine substitution for Asn173 and Tyr 77 in hZαADAR1 54,56 or the corresponding residues in mZαDLM-1 and mZβDLM-1 57 eliminated the DNA-binding ability of each domain without altering protein stability.
The N-terminal domain of RBP7910 showed a high level of conservation for residues in the nucleic acid recognition core of ZαZBPs (Fig. 1). Thr52 and Trp56 replace Asn173 and Tyr177 from hZαADAR1 in the third www.nature.com/scientificreports www.nature.com/scientificreports/ predicted helix, although Thr52 is conserved among Trypanosoma genera and Trp56 is conserved in kinetoplastids. Arg53 is also shared among Trypanosoma genera, Pro76 in Trypanosoma genera and C. fasciculata, and Pro77 and Trp79 are conserved in kinetoplastids. Different amino acids from the N-and C-terminal domains of RBP7910 were selected for mutagenesis studies based on (1) conservation of amino acids located in the recognition core of ZBPs, (2) previously reported point mutations affecting nucleic acid-binding activity of ZBPs, and (3) avoiding residues previously reported to be crucial for the protein stability.
A gel retardation assay was employed to assess the effects of each point mutation on the binding of 32 P-labeled gA6 RNA to RBP7910. Selected amino acids in the N-terminal domain and the third helix (α3) of RBP7910 were Thr52, Arg53, and Trp56; Pro76, Pro77, Trp79 were selected from the β2/β3 wing. Because of the lower conservation of the Zβ domain of ZBPs and kinetoplastids, only Pro164, Phe167, and Trp188 from the Zβ recognition core were chosen for mutagenesis analysis of the second predicted Z-DNA-binding domain of RBP7910.

Discussion
The data presented here identify the first Z-DNA-binding domain in a T. brucei protein with RNA-binding activity, which functions in mitochondrial RNA processing. The RNA binding activity of RBP7910 was suggested by its RNA-dependent interactions with REMC5A and TbRGG2 in the RESC 28 . This conclusion is supported by another study that detected RBP7910 through pull-down experiments of several individual members of the RESC 12 , while the RNA molecule mainly enforced these interactions. A recent study of biotinylated interacting partners of RBP7910 using a RB7910-BirA biotin ligase fusion protein 29 confirmed REMC5A and TbRGG2 as the main interacting proteins in the RESC 28 . This work also showed interactions of RBP7910 with MERS1 NUDIX (nucleoside diphosphates linked to any moiety (x)) hydrolase, which with MERS2 PPR RNA-binding factor constitutes a 5′ pyrophosphohydrolase complex termed the PPsome. Therefore, we provide new insights into the role of RBP7910 in 5′processing of pre-edited transcripts as part of the PPsome. Identification of the PPsome www.nature.com/scientificreports www.nature.com/scientificreports/ purine-rich sites in 5′pre-edited transcripts with three Us at their 3′ end as the binding site of MERS2 along with the poly U binding affinity of RBP7910 suggests a role for RBP7910 in mitochondrial editing by engaging an RNA-dependent interaction of the PPsome with the RESC.
The high affinity of RBP7910 for U-rich and AU-rich RNA is consistent with Z-like steps found in RNA as r(U/ApA) dinucleotide repeats at key locations in single-stranded RNA regions and riboswitches [58][59][60] . Based on the results of the RBP7910 binding assays, in addition to the 3′oligo (U)-tail, the secondary structure of gRNAs is also important for the gRNA-binding activity of RBP7910. The secondary structure conformation of purine-pyrimidine repeats in DNA/RNA strands is the main factor responsible for the recognition of these molecules by ZBPs 59 . By considering the importance of the secondary structure for gRNA-binding by RBP7910 and the AU sequence binding preference of this protein, we suggest a crucial role for the secondary structure of the purine-pyrimidine AU-rich sequences for RBP7910 RNA-binding. The interaction of Zα with the sugar-phosphate backbone of left-handed Z-DNA/RNA has been widely investigated 38,54 , suggesting that Zα binds to Z-DNA/RNA substrates using similar binding interfaces 31,61 . Compared to other ZBPs, the residues involved in nucleic acid recognition by RBP7910 are conserved but not identical, with few exceptions. However, alanine substitution point mutations of residues in the predicted binding interfaces only resulted in ~2-4 fold reduction in affinity of RBP7910 for RNA, lower than reported in other mutational studies of ZBPs 40,41,61 . One example of the Kd estimation is the Y177A substitution in ZαADAR1, which resulted in a 17.5-fold decrease in binding of a Z-DNA substrate compared to the WT protein 40 . It should be noted, however, that previous mutational analyses were performed based on the interaction of ZαADAR1 with DNA. We speculate that the discrepancy between the mutational effects is due to higher stability of the RNA-protein interaction. The ribose 2′-OH groups of RNA can make either direct or water-mediated hydrogen bonds with amino acids at the binding interfaces, and therefore single point mutations would not have a significant effect on RNA binding 31,62 .
In summary, mutational studies support the RNA-binding function of the recognition core in the Z-DNA-binding domains of RBP7910. Further experiments, such as the construction of RBP7910 Zα and Zβ truncations, will facilitate studies of the contribution of each domain to the RNA-binding activity of the protein.
The nucleic acid binding activities of winged HTH domain-containing proteins have different biological implications in cells, such as the regulation of transcription, RNA biogenesis, translation, and immune responses. Similarly, the elucidation of the mode of RNA-binding activity in RBP7910 will be an interesting topic for future research to characterize possible regulatory roles of RBP7910 in mitochondrial RNA processing in T. brucei.

Materials and Methods
Database searches and sequence alignment. The RBP7910 sequence was analyzed for the presence of recognizable domains using HHpred 63

Purification of the recombinant protein. The pET30-a expression vector was transformed into the T7
Express lysy/I q competent E. coli strain (New England Biolabs SITE), which was grown to a density of 0.6 OD before induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Bacterial cultures were grown after induction for either 5 h at 30 °C or 8 h at 16 °C, and then collected by centrifugation at 8,000 x g for 15 min at 4 °C. The cell pellet from 1 L of induced culture was resuspended in 50 ml cold PBS (pH 7.2), 10% glycerol, and 1X protease inhibitor mixture (Roche Applied Science), and cells were lysed by sonication on ice for 5 min, followed by centrifugation at 16,000 x g for 15 min at 4 °C. The cleared lysate was applied to a column with 2 mL IMAC Nickel charged resin (Bio-Rad). Proteins were eluted with an increasing gradient of imidazole from 10 mM to 320 mM, prepared in cold PBS containing 10% glycerol. Eluted fractions were dialyzed against two changes of buffer (PBS with 10% glycerol). The dialyzed recombinant proteins were applied to an Amicon centrifugal filter device (Millipore) and concentrated to 1/5 of the starting volume.
The relative sizes of the recombinant proteins were examined using SDS-PAGE (Fig. 5B) using an anti-6x His tag antibody (631212, Clontech) and visualized using a VersaDoc instrument (Bio-Rad) while the concentrations were measured using Quantity One software (Bio-Rad).
In vitro transcription and radiolabeling of RNAs. Purified PCR fragments of gA6 [14] Δ16G were amplified from the previously described plasmid encoding gA6 [14] Δ16G 6 , which specifies the first ES of the ATPase subunit 6 (A6) pre-mRNA. A Riboprobe System-T7-promega kit was used for in vitro transcription of 2 μg template DNA 22 . The CYb pre-mRNA (102 nt) 67 and edited CYb mRNA were transcribed from BamHI linearized plasmid and synthetic DNA antisense template with a T7 promoter sequence, respectively, using a RiboMAX Express-T7-promega kit. Transcripts were either labeled with [α-32 P] UTP (Perkin Elmer) during transcription or were radiolabeled after transcription with [α-32 P] pCp at the 3′ end using T4 RNA ligase (New England Biolabs).
Unlabeled RNAs used in competition assays were synthesized from the DNA oligonucleotides listed in Table 1, in combination with a T7 promoter oligonucleotide. The 90-nt pBlueScript SK + (Stratagene) RNA was generated by in vitro transcription of the NotI linearized plasmid. The pre-edited A6U5 transcript template was (2019) 9:5904 | https://doi.org/10.1038/s41598-019-42409-1 www.nature.com/scientificreports www.nature.com/scientificreports/ PCR amplified from the plasmid containing its sequence and used in the in vitro transcription reaction containing the A6U5 pre-mRNA. All RNAs were purified on 9% polyacrylamide/7 M urea gels.
Gel shift assays. The apparent equilibrium dissociation constant (Kd app) was calculated for each RNA substrate by performing EMSAs 68 . For estimating Kd, increasing concentrations of purified RBP7910 (wild-type and point mutations) proteins were incubated with fixed concentrations of the labeled RNA (gA6 [14] substrate and pre-and edited CYb mRNAs). For the gel shift assays, labeled RNAs were heated at 75 °C for 3 min followed by a slow cooling period with a rate of 1 °C/min to 23 °C, and held for 30 min at 23 °C before transferring the RNAs to the ice. Binding reactions were conducted in RBB50 buffer (20 mM Tris-HCl, pH 7.6, 50 mM KCl, 5 mM MgCl 2 , 100 mg/mL BSA, 10% glycerol, and 1 mM DTT), 100 mM KCl, and 20 units RNasin (Promega) in a 20 μl volume for 30 min at RT. Samples were mixed with gel loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, and 30% glycerol) before loading onto native 10% TBE gels that were pre-run at 110 V for 15 min in 0.5 X TBE at 4 °C. After 2 h, gels were fixed with 10% isopropanol plus 7% acetic acid for 30 min and visualized using a PhosphorImager (Bio-Rad). Free and bound RNAs were quantified using Quantity One software (Bio-Rad). The sum of the bound complexes in each lane was considered the total bound fraction. Data were analyzed with nonlinear curve fitting methods using GraphPad Prism 7 software (GraphPad Software, Inc.). The values of Kd app and active protein concentrations, Bmax, were determined as best fits to the experimental data. The obtained Kd app values were used to calculate the active protein concentration and the corrected equilibrium dissociation constant using increasing concentrations of labeled RNAs relative to a fixed concentration of protein (wild-type and point mutants). The protein concentration was equivalent to approximately two times the estimated Kd app values.
Competition experiments were performed as described above using a fixed amount of protein that resulted in approximately 30-50% bound RNA. A saturating concentration of the radiolabeled gA6 [14], CYb pre-mRNA, edited CYb mRNA, and AU target substrate was used in separate binding reactions and mixed with 1-, 10-, 100-, and 1000-fold molar excess concentrations of unlabeled competitor RNA in the RBB50 binding buffer prior to addition of the protein. Percent competition was estimated as the ratio of bound RNA in the presence of unlabeled competitor relative to RNA bound in the absence of competitor.
Guanylyl transferase assay. RNA was isolated from (−Tet) and (+Tet) PF Tb927.10.7910 RNAi cells 3 and 4 days after Tet induction, and treated with DNase as described above. Eight micrograms of DNase-treated RNA were labeled with 10 μCi [α-32 P] of GTP (3000 Ci/mmol) using a ScriptCap ™ m7G Capping System kit (CELLSCRIPT ™ ), according to the manufacturer's instructions. Reactions were extracted with phenol: chloroform twice and chloroform once and precipitated. Samples were mixed with 80% formamide loading buffer and resolved on 8% acrylamide-7 M urea gel in 1 X TBE.

Data Availability
The datasets are available from the corresponding author.