The whole genomic sequence of E. histolytica clonal strain HM-1: IMSS cl6 was applied to tRNAscan-SE (Lowe and Eddy, 1997; Lowe and Chan, 2016) to predict all tRNA sequences. All predicted tRNA sequences were counted and grouped according to corresponding codons.

BLASTp search was performed to identify HSPC117/RtcB orthologs from a broad range of taxa by using Homo sapiens HSPC117 as a query. Obtained sequences were aligned by MAFFT v7.475 (Katoh and Standley, 2013) and trimmed by TrimAl v1.4 (Capella-Gutierrez et al., 2009), and phylogenetic reconstruction was conducted by the maximum likelihood method in FastTree 2.1.10 with default parameters (Price et al., 2010) with HSPC117/RtcB orthologs got from BLASTp searches.

Total RNA of E. histolytica trophozoites was extracted, and mRNA was purified. According to the manufacturer’s protocol, cDNA was synthesized by SuperScript III First-Strand Synthesis System (Invitrogen, Waltham, MA, USA). The protein-coding region of EhRtcB1 (EHI_169260) and EhRtcB2 (EHI_184560) were amplified by PCR from E. histolytica cDNA using appropriate oligonucleotides summarized in Supplementary Table 1 . To express EhRtcB1 and EhRtcB2 fused with the hemagglutinin (HA) tag at the amino terminus in E. histolytica trophozoites, PCR fragments amplified from cDNA using appropriate primers were digested with StuI and SalI, purified, and ligated to SmaI- and XhoI-digested pEhExHA (Nakada-Tsukui et al., 2009) in blunt-end and compatible cohesive ends manner to produce pEhEx-HA-EhRtcB1 and pEhEx-HA-EhRtcB2. To express EhRtcB1 and EhRtcB2 fused with green fluorescence protein (GFP) at the amino terminus in E. histolytica trophozoites, PCR fragments amplified from cDNA using appropriate primers were digested with StuI and SalI, purified, and ligated to SmaI- and XhoI-digested pEhExGFP (Somlata et al., 2017) in blunt-end and compatible cohesive ends manner to produce pEhEx-GFP-EhRtcB1 and pEhEx-GFP-EhRtcB2.

For antisense small RNA-mediated gene silencing in E. histolytica, the fragments corresponding to the full-length protein of EhRtcB1 and EhRtcB2 were PCR-amplified from cDNA by using appropriate primers, digested by StuI and SacI, and cloned into StuI- and SacI-digested psAP2-Gunma (Mi-Ichi et al., 2011) to produce psAP2-Gunma-Eh-RtcB1-Full and psAP2-Gunma-Eh-RtcB1-Full. Subsequently, plasmids for gene silencing, which contained only a 264 bp or 299 bp fragment corresponding to the amino-terminal portion of EhRtcB1 and EhRtcB2, respectively, were also constructed by PCR amplification using psAP-2-Gunma-Eh-RcB1-Full or psAP-2-Gunma-Eh-RcB2-Full and appropriate primers, and self-ligated to produce psAP-2-Gunma-Eh-RcB1-Short and psAP-2-Gunma-Eh-RcB2-Short.

To express bacterial recombinant proteins of EhRtcB1 and EhRtcB2 fused with the histidine tag at the amino terminus, the protein-coding region of EhRtcB1 and EhRtcB2 was PCR-amplified using pEhEx-HA-EhRtcB1 or pEhEx-HA-EhRtcB2 as a template and appropriate primers. The fragments were cloned into NdeI- and XhoI-double digest pET28 a (+) using Infusion cloning kit (Clontech) to produce pET28a-N-His-EhRtcB1 and pET28a-N-His-EhRtcB1. For recombinant Archease, the protein-coding region of E. histolytica Archease gene (EHI_028560) was amplified using cDNA as template and appropriate primers, digested with NdeI and BamHI, and ligated to NdeI- and BamHI-digested pET15b to produce pET15b-EhArchease. Plasmid for expressing E. coli RtcB was a gift from Takashi Yokogawa, Gifu University.

Trophozoites of the E. histolytica strain HM-1:IMSS cl-6 (Diamond et al., 1978) and G3 (Bracha et al., 2006) were maintained axenically in Diamond’s BI-S-33 medium (Diamond et al., 1978) at 35.5°C. The plasmids for expression of HA-tagged and GFP-fused EhRtcBs, generated as above, were introduced into HM-1 trophozoites by lipofection as previously established (Nozaki et al., 1999). For HA-tagged or GFP-fused EhRtcBs expressing strains, 5 or 20 µg/ml of G418 (Gibco/Life Technologies, Waltham, MA, USA) was used to achieve appropriate expression levels, respectively. The plasmids for gene silencing were introduced into G3 strain, and transfectants were maintained with 10 µg/ml of G418.

cDNAs from EhRtcB1-gs and EhRtcB2-gs strains were synthesized using 2 μg of total RNA (DNase I treated) and SuperScript III First-Strand Synthesis System (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol.

cDNAs of tRNA^Ile(UAU), tRNA^Tyr(GUA), and tRNA^His(GUG) were reverse transcribed using 2 μg of total RNA [DNase I (Invitrogen) treated] and ReverTra Ace qPCR RT kit (TOYOBO, Japan) by tRNA^Ile(UAU) common reverse primer, tRNA^Tyr(GUA) common reverse primer, and tRNA^His(GUG) reverse primer, respectively, according to the manufacturer’s protocol.

cDNAs of tRNA^Ile(UAU), tRNA^Tyr(GUA), and tRNA^His(GUG) were used as template for traditional PCR by using Pfu-X DNA polymerase (Jena Bioscience, Germany). tRNA^Ile(UAU) and tRNA^Tyr(GUA) common forward and reverse primers were used to synthesize both unspliced and spliced form of tRNA^Ile(UAU) and tRNA^Tyr(GUA), respectively, followed with 30 amplification cycles. tRNA^Ile(UAU) common forward primer and spliced form-specific reverse primer were used to target the spliced tRNA^Ile(UAU) specifically followed with 25 amplification cycles. tRNA^His(GUG) is synthesized by designed primer followed with 30 amplification cycles. All the PCR products were run on 10% polyacrylamide gel, stained by 0.5 µg/ml EtBr.

PCR products targeting tRNA^Ile(UAU) transcripts were amplified using cDNA from psAP-mock strain by a common forward primer containing BamHI site and a common reverse primer containing XhoI site. Amplified PCR products were digested by BamHI and XhoI and ligated to a derivative of pET151/D-TOPO vector in which BamHI and XhoI sites had been engineered. Ligated plasmids were introduced into E. coli, and three to four colonies each for precursor (unprocessed) and mature (processed) tRNA^Ile(UAU) transcripts were selected and sequenced by Sanger sequencing.

The mRNA levels of EhRtcB1 and EhRtcB2 were measured by qRT-PCR, with RNA polymerase II gene (EHI_056690) as an internal control. Respective primers were used to target a 193 bp segment of EhRtcB1, a 226 bp segment of EhRtcB2, and a 204 bp segment of RNApolII. The amounts of transcripts of spliced tRNA^Ile(UAU) were also quantitated by qRT-PCR. tRNA^His(GUG) was used as a reference, which does not contain an intron in the E. histolytica genome based on the prediction by tRNAscan-SE. Respective primers were used to target a 52 bp segment of mature tRNA^Ile(UAU) and a 70 bp segment of tRNA^His(GUG). Twenty-fold diluted cDNAs from each strain were used as a template to interact with Fast SYBR Green Master Mix (Applied Biosystem, Foster City, CA, USA) according to the manufacturer’s protocol. qRT-PCR was conducted using StepOne Plus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The data were analyzed by DataAssist software (ThermoFisher).

Plasmids for expressing recombinant EhRtcB1, EhRtcB2, E. coli RtcB and EhArchease, constructed as described above, were introduced into E. coli BL21 (DE3) cells by heat shock at 42°C for 1 min. Transformed E. coli cells were grown in 100 ml of Luria Bertani medium in the presence of 30 μg/ml kanamycin at 37°C. The overnight culture was used to inoculate 500 ml of fresh medium, and the culture was further continued at 37°C with shaking at 200 rpm. When A600 reached 0.4, cultures were chilled by ice, 2% of ethanol and 1 mM of isopropyl β-d-thiogalactopyranoside was added, and cultivation was continued for another 16 h at 18°C. E. coli cells from the induced culture were harvested by centrifugation at 8,000 g for 10 min at 4°C. The cell pellet was washed with Tris-saline (10 mM Tris pH 7.6 and 150 mM NaCl), resuspended in 40 ml of the lysis buffer (50 mM Tris–HCl, pH 7.6, 250 mM NaCl, and 10% sucrose, w/v) with 100 μg/ml lysozyme, 0.5 mg/ml E64, 1× Complete Mini protease inhibitor cocktail (Roche, Mannheim, Germany), and 1 mM phenylmethyl sulfonyl fluoride, and incubated at room temperature for 20 min, French press, added 0.1% Triton X-100 (v/v), and centrifuged at 25,000 g for 30 min at 4°C. The supernatant was mixed with 1 ml of 50% Ni²⁺-NTA His-bind slurry, incubated for 1 h at 4°C with mild rotating. The resin in a column was washed three times with Wash Buffer (50 mM Tris-HCl, pH 7.6, 2M KCl). Bound proteins were eluted with Elution Buffer [50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 10% glycerol (v/v)] containing 100–500 mM imidazole to obtain recombinant EhRtcB1, EhRtcB2, E. coli RtcB, and EhArchease. After the integrity and the purity of recombinants, proteins were confirmed with 10% SDS-PAGE analysis, followed by Coomassie Brilliant Blue staining, they were extensively dialyzed twice against the 300-fold volume of 50 mM Tris-HCl, 150 mM NaCl, pH 7.6 containing 10% glycerol (v/v) for 18 h at 4°C. The dialyzed proteins were stored at –80°C with 10% glycerol in small aliquots until further use. Protein concentrations were spectrophotometrically determined by the Bradford method using bovine serum albumin as a standard as previously described.

Approximately 3×10⁴ exponentially growing trophozoites of E. histolytica G3 strain transformed with psAP2-Gunma-Eh-RtcB1-Full, psAP2-Gunma-Eh-RcB1-Short, psAP2-Gunma-Eh-RtcB2-Full, psAP2-Gunma-Eh-RcB2-Short, and psAP2G (control) were inoculated into 6 ml of fresh BI-S-33 medium containing 10 μg/ml geneticin, and the parasites were counted every 24 h on a hemocytometer.

A synthetic RNA (5’-GUCGGUUCGUGCGAUGGUUGUAGC-3’) with 5’- and 3’ hydroxyl termini was labeled with 5’-[³²P]-pCp by using T4 RNA ligase 1. The labeled RNA was purified through 7M urea-PAGE. RNA self-ligation assay mixture (total 10 μl) consisted of 2 μl of 5x RtcB assay Buffer [100 mM Tris-HCl (pH 7.6), 25 mM MgCl2, 5 mM MnCl2, 10 mM ATP, 5 mM GTP, 10mM DTT], 1 μl of RNase-OUT, 1 μl of RtcB solution containing various amount of the protein, 1 μl of EhArchease solution containing various amount of the protein, and the labeled substrate RNA with 100,000 cpm radioactivity. The assay mixture was incubated for 1 h at 37°C, and the reaction was stopped by adding 10 μl of FDE [90% (v/v) formamide, 20 mM EDTA, 0.02% (w/v) bromophenol blue, and 0.02% (w/v) xylene cyanol]. Approximately 5 μl of the assay mixture was electrophoresed by 7 M urea-PAGE. The radioactivity in gel was visualized by Phosphor Imager (Cytiva).

To see the repertoire of tRNA in the genome and also validate the significance of tRNA processing in E. histolytica, a comprehensive analysis of tRNA genes was conducted using our latest updated genome of HM-1 reference strain (Kawano-Sugaya et al., 2020) by tRNAscan-SE (Lowe and Eddy, 1997; Lowe and Chan, 2016) ( Table 1 ). The genome of HM-1 apparently contains 2,670 tRNA genes. Among them, two groups of tRNA genes have a single intron: 52 tRNA^Ile(UAU) and 42 tRNA^Tyr(GUA) genes. These intron-containing genes represent approximately 3.8% of all tRNA genes. It is plausible that tRNA splicing is essential in E. histolytica because all tRNA^Ile(UAU) and tRNA^Tyr (GUA) genes contain an intron. Note that an ortholog of tRNA endonuclease (two copied genes with 100% amino acid sequence identity to each other, EHI_092410, EHI_042160) and an ortholog of archease (EHI_028560) that has been known to be required for full enzyme activity of HSPC117 were also found in the E. histolytica genome. Chromosomal distribution of tRNA^Ile(UAU), tRNA^Tyr(GUA) genes are remarkably different ( Figure 1A ). While tRNA^Ile(UAU) genes are scattered throughout the genome, tRNA^Tyr(GUA) genes are clustered on a single chromosome. In addition, all 42 tRNA^Tyr(GUA) genes are identical with one exception, whereas tRNA^Ile(UAU) genes are quite polymorphic in both exon and intron regions ( Figures 1B, C ).

To further understand the conservation of tRNA splicing in E. histolytica, we investigated conservation of two core processes of tRNA splicing: cleavage and ligation of nascent tRNA transcripts. We first searched for E. histolytica HSPC117/RtcBs orthologs by BLASTp search using human HSPC117 as a query. Two HSPC117/RtcB orthologs, EhRtcB1 (XP_651200.1/EHI_169260) and EhRtcB2 (XP_648794.1/EHI_184560), were found in the E. histolytica HM-1 reference strain. EhRtcB1 and EhRtcB2 show 67.8% mutual amino acid identity, and 48.7 and 50.0% amino acid identity to H. sapiens HSPC117, respectively. To investigate the origin of E. histolytica RtcBs, 128 HSPC117/RtcB orthologs were aligned, and phylogenetic reconstruction was conducted ( Figure S1 ). Only representative taxa from the eukaryotic supergroups were selected. Orthologs from animals other than H. sapiens were excluded because of high similarity and repetitiveness. RtcB orthologs from four Entamoeba species form two independent well-separated clades with a good bootstrap proportion, suggesting that two paralogs are conserved in four Entamoeba species. Furthermore, two Entamoeba clades, which form a monophyletic clade, are well separated from other eukaryotes, but all eukaryotes show monophyly independent orthologs of bacterial and archaeal origins. These data are consistent with the notion that Entamoeba RtcB is of eukaryotic origin and that the ancestral organism of Entamoeba possesses two isotypes of RtcB prior to the diversification of Entamoeba species, suggesting non-overlapping roles of both EhRtcBs. Similarly, Trichomonas vaginalis appears to possess four different RtcB orthologous genes, although gene multiplication is common in this organism (Alsmark et al., 2009; Aurrecoechea et al., 2009). Also, note that an ortholog of tRNA endonuclease was also found in the E. histolytica genome (two allelic genes encoding a protein of 100% amino acid identity to each other, EHI_092410, EHI_042160), but investigation on Entamoeba tRNA endonuclease is above the scope of this study.

Protein alignment of representative RtcBs (E. histolytica, Pyrococcus horikoshii, and H. sapiens) ( Figure S2 ) shows a high degree of conservation of key residues implicated in the manganese binding and the guanylylation active site for P. horikoshii RtcB, which also supports the premise that EhRtcBs resemble mammalian/archaeal tRNA ligases and both EhRtcBs are functional. In silico survey of potential organelle targeting or localization motifs such as the signal peptide, the nuclear export signal, mitochondria targeting transit peptides, membrane targeting motifs such as the transmembrane region, and GPI anchor conjugation failed to detect significant motifs or domains. NLS was predicted to exist in EhRtcB1, but not EhRtcB2, by NLS Mapper (Kosugi et al., 2009) with a score of 4.5, indicating possible nuclear localization of EhRtcB1.

To examine if both intron-containing tRNA, tRNA^Ile(UAU) and tRNA^Tyr(GUA), are spliced in vivo, the transcripts of the genes were amplified by PCR from cDNA from E. histolyitca control strain (psAP-mock transfected G3 strain), and their sizes were compared against those of the fragments amplified from the corresponding intron-containing genes, using genomic DNA as the template ( Figure 2A ). Common forward and reverse primers were designed to amplify both intron-containing and intron-spliced forms of tRNA^Ile(UAU) and tRNA^Tyr(GUA). One band corresponding to the intron-containing region of tRNA^Ile(UAU) was amplified from genomic DNA, while three bands corresponding to intron-retained transcript (62 bp), intron-spliced transcript (52 bp), and the top band (~120 bp) of unknown sequence were amplified from cDNA. These PCR products from cDNA were cloned to a pET151/D-TOPO vector and sequenced as described in Materials and Methods. Sequencing results of precursor and mature tRNA^Ile(UAU) are identical to the tRNA^Ile(UAU) sequence predicted by tRNAscan-SE ( Figure S3 ). Note that the ~120 bp band that had been amplified from cDNA and visualized in polyacrylamide gel electrophoresis was not visible in 3% agarose gel. Considering the partially self-complementary nature of tRNA, together with platform-dependent (agarose vs polyacrylamide) presence or absence of the ~120 bp band, we speculate that the ~120 bp band may be a differently annealed form or artifactual dimer of the main PCR product, probably easily denatured in the agarose gel electrophoresis, in which electrophoresis was carried out at higher temperature than polyacrylamide gel electrophoresis ( Figure S4 ). Similarly, PCR amplified one band corresponding to intron-containing tRNA^Tyr(GUA) fragment (65 bp) from genomic DNA, while two bands corresponding to intron-retained (65 bp) and intron-spliced (52 bp, faint) fragments were amplified from cDNA. However, the abundance of spliced tRNA^Tyr(GUA) was lower than that of unspliced form, suggesting that most tRNA^Tyr(GUA) remained unspliced in vivo.

To verify if both EhRtcB candidates possess RNA ligase activity, RNA self-ligation assay was performed ( Figure 2B ). Synthetic RNA possessing 5’- and 3’-hydroxyl termini were labeled with 5’-[³²P]-pCp ( Figure S5 ). EhRtcBs are expected to produce circular RNA, which migrates faster than linear RNA on urea-PAGE. As human tRNA ligase complex was previously reported to be stimulated by Archease, recombinant protein of E. histolytica Archease, whose encoding gene was found in the E. histolytica genome, was produced in E. coli and used for the RNA self-ligation assay. Circularizing activity by recombinant EhRtcB1 and EhRtcB2 was observed, although activity was weaker than that of E. coli RtcB, which was used as a positive control. The intensity of the circularized RNA was measured by densitometric scanning of the gel image ( Figure 2C ). The intensity of the bands corresponding to circularized RNA produced by EhRtcB1, EhRtcB2 only, EhRtcB1 plus EhArchease, or EhRtcB2 plus EhArchease is shown relative to that produced by E. coli RtcB. The ligase activity of EhRtcB1/2 was enhanced by EhArchease: relative EhRtcB1 and EhRtcB2 ligase activity (normalized against E. coli RtcB) were 11.4 and 48.2%, respectively, which increased by 2.89 and 12.8-folds, respectively, compared to that without EhArchease. The drastic increase in circular RNA produced by EhRtcB2 plus EhArchease was much higher than the increase found in a reaction with EhRtcB1 and EhArchease. Furthermore, archease was previously proven to interact with RtcB ligase and enhance its enzymatic activity. Archease itself failed to show RNA ligation ability, as previously shown (Desai et al., 2014). These data reinforce that both EhRtcBs possess RNA ligase activity (EhRtcB2>EhRtcB1), and their activity was enhanced by EhArchease, like human HSPC117. The slow migrating bands were visible only when the substrate was mixed with EhRtcB2, but not when mixed with both EhRtcB1/2 and EhArchease. We presume that the slow migrating band likely represents EhRtcB2/RNA complex. EhArchease presumably helped dissociation of the protein from the substrate.

To further examine whether both EhRtcBs function as tRNA ligases in vivo, quantitation of nascent unprocessed, cleaved, and ligated forms of tRNA^Ile(UAU) was performed by RT- and qRT-PCR using common forward primer [for non-spliced and spliced forms of tRNA^Ile(UAU))] and either common reverse [for non-spliced and spliced forms of tRNA^Ile(UAU))] or spliced form-specific reverse primer [for only the spliced form of tRNA^Ile(UAU)] ( Figure S6 ). Two primer sets were designed to specifically amplify only the spliced (cleaved and ligated) form or amplify both the spliced and unprocessed forms of tRNA^Ile(UAU).

In order to investigate if EhRtcBs are involved in tRNA splicing of tRNA^Ile(UAU), transcriptional gene silencing via antisense small RNA was exploited. Since both EhRtcB genes are highly similar at the nucleic acid level (74.06% positional identity), we attempted to repress individual genes by producing anti-sense small RNA targeting either the entire protein-coding region or variable regions in the amino-terminal portion (first 264 and 298 nucleotides for EhRtcB1 and EhRtcB2, respectively). Two N-terminal region targeting (psAP-RtcB1-N terminus and psAP-RtcB2-N terminus) and two full-length gene targeting (psAP-RtcB1-full length and psAP-RtcB2-full length) plasmids were introduced into parental G3 strain. Gene silencing was evaluated by qRT-PCR, and the transcript levels of EhRtcB1 and EhRtcB2 in the gene silenced strains are shown relative to those in the psAP mock control strain ( Figure 3A ). In the strains transformed with psAP-EhRtcB1-full length and psAP-RtcB1-N terminus, the transcript level of EhRtcB1 was decreased by 92.5 ± 1.5% and 85.3 ± 5.6%, respectively, compared to psAP-mock control strain, while that of EhRtcB2 remained unchanged. In contrast, when two plasmids designed to repress EhRtcB2, psAP-EhRtcB2-full length, and psAP-EhRtcB2-N terminus were introduced into G3, EhRtcB2 transcript was almost completely abolished (98.9 ± 1.0% and 99.6 ± 0.2% decrease). However, repression of EhRtcB1 gene expression (67 ± 25% decrease) was also observed in the strain transformed with psAP-RtcB2-full length, but not in the strain transfected with psAP-RtcB2-N terminus, due to the expected cross gene silencing.

We further investigated if EhRtcBs gene silencing affected tRNA processing ( Figures 3B, C ). In the psAP mock control strain, two bands were identified by polyacrylamide gel electrophoresis: a 62 bp fragment corresponding to the unprocessed tRNA^Ile(UAU) and a 52 bp fragment corresponding to the spliced/ligated form ( Figure 3B ). In gene silenced strains, particularly the strains transfected with psAP-EhRtcB1-full length or psAP-EhRtcB1-short length, in which EhRtcB1 gene expression was abolished or largely reduced, the amount of both the unprocessed and the spliced/ligated forms of tRNA^Ile(UAU) transcript decreased (to the greatest extent for the spliced form in the strain transfected with psAP-EhRtcB1-full length). The decreased amount in unprocessed form is most likely due to the accelerated intron-splicing process when there is a lack of spliced/ligated forms of tRNA^Ile(UAU). A slight decrease in the abundance of both the unprocessed and the spliced/ligated forms was also observed in the strain transfected with psAP-RtcB2-full length, in which EhRtcB1 transcript decreased by about 75% ( Figure 3A ). Control tRNA, tRNA^His(GUG), which is not subjected to splicing, was unchanged in all the strains. To further quantitate the amount of the spliced and functional tRNA^Ile(UAU) transcript, the transcript was selectively PCR-amplified by common forward primer and spliced form-specific primer ( Figure 3C ). qRT-PCR showed that in the strains transfected with psAP-EhRtcB1gs-full length, psAP-EhRtcB1gs-N terminus, or psAP-EhRtcB2gs-N terminus, the amount of spliced tRNA^Ile(UAU) was reduced by 94.0 ± 3.0%, 85.3 ± 5.6%, and 66.9 ± 25.3%, respectively ( Figure 3D ). In contrast, the amount of spliced tRNA^Ile(UAU) was only reduced in the strain transfected with psAP-EhRtcB2gs-N terminus decreased by 16.1 ± 9.7%. The reduction of spliced tRNA^Ile(UAU) agreed well with the reduction in the EhRtcB1 gene transcript levels.

As for tRNA^Tyr(GUA), similarly, two bands were amplified from cDNA in psAP mock control strain ( Figure S7 ). A 65 bp upper band, which apparently has the same size as a band amplified from genomic DNA, corresponds to the unprocessed form, while a 52 bp band found in psAP-mock corresponds to the spliced form of tRNA^Tyr(GUA). In psAP-EhRtcB1-full length, spliced tRNA^Tyr(GUA) was abolished, and non-spliced tRNA^Tyr(GUA) was largely diminished. Spliced form-specific amplification with various primer pairs to selectively target only the spliced form failed, which may be due to not-yet characterized base modifications or misprediction of the intron.

In order to confirm the essentiality of both EhRtcBs, we measured growth kinetics of the EhRtcB1 and EhRtcB2 gene silenced and control strains ( Figure 3E ). The strains that had been transfected with psAP-EhRtcB1gs-full length or psAP-EhRtcB1gs-N terminus, and showed EhRtcB1-specific gene repression, showed severe growth defects. In addition, the strain that had been transfected with psAP-EhRtcB2gs-full length and showed gene repression for both EhRtcB1 and EhRtcB2 also showed severe growth defects. These data indicate the essentiality of EhRtcB1 and the unique non-overlapping role of EhRtcB1. In contrast, the strain that had been transfected with psAP-EhRtcB2gs-N terminus showed EhRtcB2-specific gene silencing, presented no growth retardation, suggesting non-essentiality of the gene under normal conditions.

To examine the distribution of EhRtcB1 and EhRtcB2 in the amebae, EhRtcB1 and EhRtcB2 fused with GFP at the amino terminus were ectopically expressed in E. histolytica. The expression of the proteins was confirmed by immunoblot analysis of amebic lysates, which reacted with the anti-GFP antibody ( Figure 4A ). GFP-EhRtcB1 and GFP-EhRtcB2 were recognized as the predominant bands of approximately 84 and 86 kDa, which agrees well with the predicted molecular weight of each protein (57.6 and 59.1 kDa, respectively) plus GFP (26.9 kDa). Also, note that truncated proteins, which likely correspond to only the GFP portion, was also detected in both transformant lines; however, in GFP-EhRtcB1 and GFP-EhRtcB2 expressing strain, the proportion of truncation was 32% and 7.6% of the combined amount of the full-length GFP-EhRtcB1 or GFP-EhRtcB2 and their truncation, based on the densitometric scanning of the blot. Immunofluorescence imaging revealed that GFP-EhRtcB1 was observed predominantly in the nucleus, while GFP-EhRtcB2 was evenly distributed in the cytoplasm ( Figures 4B, C ). In contrast, GFP was distributed in the whole cell in GFP-mock control strain ( Figure 4D ). Representative movies of time-lapse imaging of GFP-EhRtcB1 and GFP-EhRtcB2 are provided ( Movie S1 ).

To exclude a possibility that the localization of GFP-fused RtcBs was influenced by GFP tagging or truncation of the fusion proteins, we also created amebae transformant strains that expressed HA-tagged EhRtcB1 and HA-EhRtcB2. The expression of HA-EhRtcB1 and HA-EhRtcB2 was validated by immunoblot analysis by anti-HA antibody ( Figure 5A ). HA-EhRtcB1 and HA-EhRtcB2 were recognized as the predominant bands of approximately 61 and 63 kDa, each corresponding to EhRtcB protein plus the HA tag (3.2 kDa). Also, note that several truncated proteins (around 28.8% of the total amount) were also detected in HA-EhRtcB2-expressing strain but not in HA-EhRtcB1-expressing strain. Immunofluorescence imaging of the fixed amebic transformants verified the disparate distribution of HA-EhRtcB1 and HA-EhRtcB2, indicated by live imaging described above ( Figures 5B, C ). The different localization of EhRtcB1 and EhRtcB2 indicates that these two proteins are likely involved in different biological functions in E. histolytica.