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The structural domains (PAZ and PIWI) of three EhAgo proteins in E. histolytica. The length of proteins is drawn to scale, with the length of PAZ and PIWI based on the annotation of AmoebaDB. We generated EhAgo polyclonal antibodies in rabbits using peptide from the N-terminal end of sequence as indicated by Y* (see also Materials and Methods). The PAZ Superfam domain (SSF101690) is shown in orange, and the PAZ Pfam (PF02170) domain of EhAgo2-1 and EhAgo2-2 is shown in green. The PIWI Pfam (PF02171) domain is in blue. Specific PAZ/PIWI mutations generated in this study are shown. Both EhAgo2-1 and EhAgo2-2 have 200 aa after the PIWI domain, with an RGG/RG motif (red) and repetitive DR-rich region (gray) identified in EhAgo2-2.
Source publication
The protozoan parasite Entamoeba histolytica , which causes amebiasis and affects over 50 million people worldwide, contains an important RNAi pathway for gene silencing. Gene silencing via the RNAi pathway is mediated by the Argonaute (Ago) proteins. However, we lack knowledge on Ago function(s) in this nonmodel system. In this paper, we discovere...
Contexts in source publication
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... Ago proteins are generally conserved for four structural domains (the N terminus, PAZ, middle, and PIWI domains) (20). The E. histolytica genome contains genes encoding three Ago family proteins, EhAgo2-1 (EHI_186850), EhAgo2-2 (EHI_125650), and EhAgo2-3 (EHI_177170) (21), with all three Ago proteins showing the conserved PAZ and PIWI domains (Fig. 1). Although the PAZ Superfam domain is annotated for all three EhAgos, the PAZ Pfam PF02170 domain is not identified for EhAgo2-3, probably due to its sequence divergence. Our phylogenetic analysis using the current genomic data set of AmoebaDB for species including Entamoeba moshkovskii, Entamoeba dispar, Entamoeba nuttalli, and ...
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... data set of AmoebaDB for species including Entamoeba moshkovskii, Entamoeba dispar, Entamoeba nuttalli, and Entamoeba invadens indicates that all three EhAgos are conserved among these amebic species, and each EhAgo forms its own cluster. E. moshkovskii and E. invadens are more divergent than the other three species within each cluster (see Fig. S1 in the supplemental material). Evolutionary loss of RNAi can occur in some eukaryote taxa, such as yeast Saccharomyces castellii (Ago and RNAi positive) versus S. cerevisiae (Ago and RNAi negative) (7) and T. brucei (Ago and RNAi positive) versus T. cruzi (Ago and RNAi negative) (22). Our analysis of current genomes of ameba species ...
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... three EhAgo proteins have distinct subcellular localizations. Previously, we have reported that EhAgo2-2 is localized in the nucleus (23). To characterize EhAgo2-1 and EhAgo2-3, we generated custom-made polyclonal antibodies using selected N-terminal peptide sequences (Fig. 1). The antibodies were first tested by Western blot analysis using total cell lysates from wild-type and Myc-tagged overexpressing cell lines. The expected band size of each Ago was detected ( Fig. 2A). However, due to low endogenous expression of both EhAgo2-1 and EhAgo2-3 (based on three published data sets using Affymetrix microarray ...
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... not for EhAgo2-3, for which the residues are W-K-Y (of note, two FF residues are right next to K, which may indicate that EhAgo2-3 could have W-F-Y residues). In addition, the mutation of the two tyrosine residues in PAZ was shown to abolish sRNA binding in C. elegans (40). We therefore selected these two residues for mutagenesis as indicated in Fig. ...
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... PIWI domain sequences appear to be very divergent, and the alignment of EhAgo PIWI domain with model systems shows that none have a complete D-D-X catalytic triad (Fig. S7), suggesting that all three EhAgos are likely non-Slicer Agos. The only partially aligned triad position for EhAgo2-2 is D-G-N, which we analyzed by mutagenesis as indicated in Fig. ...
Citations
... In the E. histolytica genome, three Ago proteins have been identified (EHI_125650, EHI_186850, and EHI_177170); EhAgo2-2 (EHI_125650) is the most highly expressed Ago protein in E. histolytica. We previously demonstrated that EhAgo2-2 binds to populations of small RNA, 27 nt or 31 nt, and mediates transcriptional gene silencing (25,26). ...
... tRNA-derived fragments are associated with EhAgo proteins. We previously demonstrated that two small-RNA populations, 27 and 31 nt, associate with E. histolytica Argonaute (EhAgo) proteins (25,26,28). Since Ago proteins have been reported to bind tRNA fragments in other systems (23,37), we analyzed our sequencing data from size-fractionated total RNAs isolated from immunoprecipitations (IPs) of each of the 3 EhAgos: Ago2-1, Ago2-2 and Ago2-3. ...
... Amoebic parasites have a functional RNAi pathway mediated through a population of 27-nt antisense RNAs (25,(58)(59)(60), including 3 Ago proteins, which have been shown to bind sRNAs (26). The possibility of tRNA-derived fragments interacting with the RNAi pathways in amoebas offers an intriguing new role for the abundant tRNA genes in the amoebic genome. ...
In the present study, we report for the first time the presence of tRNA-derived fragments in Entamoeba . tRNA-derived fragments were identified by bioinformatics analyses of small-RNA sequencing data sets from the parasites and also confirmed experimentally. We found that tRNA halves accumulated in parasites exposed to environmental stress or during the developmental process of encystation.
... It is mediated through small RNAs of 27 nt with a 5′-polyP structure (Zhang et al. 2008(Zhang et al. , 2011, and a 31 nt population that differs from the former in having 3-4 non-templated As at the 3′-end (Zhang et al. 2020). Three homologues of sRNA-binding Argonaute protein (EhAgo) have also been reported (Zhang et al. 2019). These sRNAs have been shown to mediate long-term transcriptional gene silencing. ...
Genome sequence analysis of Entamoeba species revealed various classes of transposable elements. While E. histolytica and E. dispar are rich in non-long terminal repeat (LTR) retrotransposons, E. invadens contains predominantly DNA trans-posons. Non-LTR retrotransposons of E. histolytica constitute three families of long interspersed nuclear elements (LINEs), and their short, nonautonomous partners, SINEs. They occupy ~ 11% of the genome. The EhLINE1/EhSINE1 family is the most abundant and best studied. EhLINE1 is 4.8 kb, with two ORFs that encode functions needed for retrotransposition. ORF1 codes for the nucleic acid-binding protein, and ORF2 has domains for reverse transcriptase (RT) and endonuclease (EN). Most copies of EhLINEs lack complete ORFs. ORF1p is expressed constitutively, but ORF2p is not detected. Ret-rotransposition could be demonstrated upon ectopic over expression of ORF2p, showing that retrotransposition machinery is functional. The newly retrotransposed sequences showed a high degree of recombination. In transcriptomic analysis, RNA-Seq reads were mapped to individual EhLINE1 copies. Although full-length copies were transcribed, no full-length 4.8 kb transcripts were seen. Rather, sense transcripts mapped to ORF1, RT and EN domains. Intriguingly, there was strong antisense transcription almost exclusively from the RT domain. These unique features of EhLINE1 could serve to attenuate retrotransposition in E. histolytica.
... Supporting that these small RNAs are relevant to previous approaches that exploited gene silencing in E. histolytica, in the G3 strain, 27nt small RNAs mapped to amoebapore A, and when additional genes were silenced in this background, new small RNAs mapped to the newly silenced gene [23]. There is no obvious Dicer homologue, but a potential noncanonical Dicer and Argonaute homologues have been characterized [24,25]. ...
While Entamoeba histolytica remains a globally important pathogen, it is dramatically understudied. The tractability of E . histolytica has historically been limited, which is largely due to challenging features of its genome. To enable forward genetics, we constructed and validated the first genome-wide E . histolytica RNAi knockdown mutant library. This library allows for Illumina deep sequencing analysis for quantitative identification of mutants that are enriched or depleted after selection. We developed a novel analysis pipeline to precisely define and quantify gene fragments. We used the library to perform the first RNAi screen in E . histolytica and identified slow growth (SG) mutants. Among genes targeted in SG mutants, many had annotated functions consistent with roles in cellular growth or metabolic pathways. Some targeted genes were annotated as hypothetical or lacked annotated domains, supporting the power of forward genetics in uncovering functional information that cannot be gleaned from databases. While the localization of neither of the proteins targeted in SG1 nor SG2 mutants could be predicted by sequence analysis, we showed experimentally that SG1 localized to the cytoplasm and cell surface, while SG2 localized to the cytoplasm. Overexpression of SG1 led to increased growth, while expression of a truncation mutant did not lead to increased growth, and thus aided in defining functional domains in this protein. Finally, in addition to establishing forward genetics, we uncovered new details of the unusual E . histolytica RNAi pathway. These studies dramatically improve the tractability of E . histolytica and open up the possibility of applying genetics to improve understanding of this important pathogen.
... Our study also included two functional EhAgo2-2 mutants to pinpoint the effects of these mutants on the RISC contents. These two mutant proteins have either impaired nuclear localization (Myc-EhAgo2-2 DNLS-DR ) or impaired sRNA binding (Myc-DHFR-EhAgo2-2 PAZ-mut ) (30). Using a similar strategy of IP mass spectrometry analysis, we found 32 exclusive protein hits and one enriched hit for EhAgo2-2 DNLS-DR RISC ( Fig. S2B and Table S1A) and 98 exclusive hits and 6 enriched hits for the EhAgo2-2 PAZ-mut RISC ( Fig. S2C and Table S1B). ...
... The plasmids for overexpressing wild-type EhAgo2-2 and its two mutants are pKT3M-EhAgo2-2, pKT3M-EhAgo2-2 DNLS-DR , and pKT3M-DHFR-EhAgo2-2 PAZ-mut , respectively. These plasmids were generated in our previous studies (28,30) and retransfected for use in this study; pKT-CS-Luc overexpresses a full-length luciferase under the same CS ...
Entamoeba histolytica is a leading parasitic cause of death in developing countries, and our efforts are focused on defining the molecular basis of RNA interference (RNAi) gene regulation in this parasite. The Entamoeba RNAi pathway effectively silences a subset of endogenous genes and has also been harnessed as a gene silencing tool to study gene function in this organism.
... The RNAi pathway has been well characterized in E. histolytica. The predominant sRNAs in E. histolytica are 27 nt with a 5 ′ -polyP structure (Zhang et al., 2008(Zhang et al., , 2011, and three homologues of sRNA-binding Argonaute protein (EhAgo) have also been reported (Zhang et al., 2019). These sRNAs have been shown to mediate long-term transcriptional gene silencing. ...
... The RNAi pathway has been well characterized in E. histolytica. The predominant sRNAs in E. histolytica are 27 nt with a 5 ′ -polyP structure (Zhang et al., 2008(Zhang et al., , 2011, and three homologues of sRNA-binding Argonaute protein (EhAgo) have also been reported (Zhang et al., 2019). These sRNAs have been shown to mediate long-term transcriptional gene silencing. ...
LINEs are retrotransposable elements found in diverse organisms. Their activity is kept in check by several mechanisms, including transcriptional silencing. Here we have analyzed the transcription status of LINE1 copies in the early-branching parasitic protist Entamoeba histolytica. Full-length EhLINE1 encodes ORF1, and ORF2 with reverse transcriptase (RT) and endonuclease (EN) domains. RNA-Seq analysis of EhLINE1 copies (both truncated and full-length) showed unique features. Firstly, although 20/41 transcribed copies were full-length, we failed to detect any full-length transcripts. Rather, sense-strand transcripts mapped to the functional domains-ORF1, RT and EN. Secondly, there was strong antisense transcription specifically from RT domain. No antisense transcripts were seen from ORF1. Antisense RT transcripts did not encode known functional peptides. They could possibly be involved in attenuating translation of RT domain, as we failed to detect ORF2p, whereas ORF1p was detectable. Lack of full-length transcripts and strong antisense RT expression may serve to limit EhLINE1 retrotransposition.
... We found that E. histolytica has abundant 27 nt sRNAs with a 5′-polyP structure, a feature that is seen in the secondary sRNAs in C. elegans and nematode parasite Ascaris suum [7,24]. There are three EhAgo proteins: EhAgo2-1 (EHI_186850), EhAgo2-2 (EHI_125650), and EhAgo2-3 (EHI_177170) that have distinct subcellular locations including the nucleus (EhAgo2-2), perinuclear ring (EhAgo2-1, EhAgo2-3), and cytosol (EhAgo2-3) [25]. Our structural domain analysis showed that all three EhAgos have a conserved PAZ and PIWI domain [25]. ...
... There are three EhAgo proteins: EhAgo2-1 (EHI_186850), EhAgo2-2 (EHI_125650), and EhAgo2-3 (EHI_177170) that have distinct subcellular locations including the nucleus (EhAgo2-2), perinuclear ring (EhAgo2-1, EhAgo2-3), and cytosol (EhAgo2-3) [25]. Our structural domain analysis showed that all three EhAgos have a conserved PAZ and PIWI domain [25]. We demonstrated that EhAgo PAZ domains are essential for sRNA binding for all three EhAgos, and sRNA binding affects cellular localization of EhAgo2-1 and EhAgo2-3 but not EhAgo2-2 [25]. ...
... Our structural domain analysis showed that all three EhAgos have a conserved PAZ and PIWI domain [25]. We demonstrated that EhAgo PAZ domains are essential for sRNA binding for all three EhAgos, and sRNA binding affects cellular localization of EhAgo2-1 and EhAgo2-3 but not EhAgo2-2 [25]. To better understand the RNAi mechanism(s) in this parasite, we ask three questions (i) what are the full spectrum of sRNA species in this parasite? ...
Background
The RNA interference (RNAi) pathway is a gene regulation mechanism that utilizes small RNA (sRNA) and Argonaute (Ago) proteins to silence target genes. Our previous work identified a functional RNAi pathway in the protozoan parasite Entamoeba histolytica, including abundant 27 nt antisense sRNA populations which associate with EhAgo2–2 protein. However, there is lack of understanding about the sRNAs that are bound to two other EhAgos (EhAgo2–1 and 2–3), and the mechanism of sRNA regulation itself is unclear in this parasite. Therefore, identification of the entire pool of sRNA species and their sub-populations that associate with each individual EhAgo protein would be a major step forward.
Results
In the present study, we sequenced sRNA libraries from both total RNAs and EhAgo bound RNAs. We identified a new population of 31 nt sRNAs that results from the addition of a non-templated 3–4 adenosine nucleotides at the 3′-end of the 27 nt sRNAs, indicating a non-templated RNA-tailing event in the parasite. The relative abundance of these two sRNA populations is linked to the efficacy of gene silencing for the target gene when parasites are transfected with an RNAi-trigger construct, indicating that non-templated sRNA-tailing likely play a role in sRNA regulation in this parasite. We found that both sRNA populations (27 nt and 31 nt) are present in the related parasite Entamoeba invadens, and are unchanged during the development. In sequencing the sRNAs associating with the three EhAgo proteins, we observed that despite distinct cellular localization, all three EhAgo sRNA libraries contain 27 nt sRNAs with 5′-polyphosphate (5′-polyP) structure and share a largely overlapping sRNA repertoire. In addition, our data showed that a fraction of 31 nt sRNAs associate with EhAgo2–2 but not with its mutant protein (C-terminal deletion), nor other two EhAgos, indicating a specific EhAgo site may be required for sRNA modification process in the parasite.
Conclusion
We identified a new population of sRNA with non-templated oligo-adenylation modification, which is the first such observation amongst single celled protozoan parasites. Our sRNA sequencing libraries provide the first comprehensive sRNA dataset for all three Entamoeba Ago proteins, which can serve as a useful database for the amoeba community.
... We found that E. histolytica has abundant 27nt sRNAs with 5′-polyP termini, a feature that is seen in the secondary siRNAs in C. elegans and other nematode parasites [24,25]. There are three EhAgo proteins: EhAgo2-1 (EHI_186850), EhAgo2-2 (EHI_125650), and EhAgo2-3 (EHI_177170), that have distinct subcellular locations including the nucleus (EhAgo2-2), perinuclear ring (EhAgo2-1, EhAgo2-3), and cytosol (EhAgo2-3) [26]. We have previously demonstrated that PAZ domains are essential for sRNA binding for all three EhAgos, and that sRNA binding affects cellular localization of EhAgo2-1 and EhAgo2-3 but not EhAgo2-2 [26]. ...
... There are three EhAgo proteins: EhAgo2-1 (EHI_186850), EhAgo2-2 (EHI_125650), and EhAgo2-3 (EHI_177170), that have distinct subcellular locations including the nucleus (EhAgo2-2), perinuclear ring (EhAgo2-1, EhAgo2-3), and cytosol (EhAgo2-3) [26]. We have previously demonstrated that PAZ domains are essential for sRNA binding for all three EhAgos, and that sRNA binding affects cellular localization of EhAgo2-1 and EhAgo2-3 but not EhAgo2-2 [26]. To better understand the RNAi mechanism(s) in this non-model system, we now want to ask three questions (i) do the three EhAgos bind different subpopulations of sRNAs? ...
... As indicated by Argonaute PAZ crystal structure, PAZ domain shows a poor binding ability to the 3'-adenylated RNAs [39]. We have shown that EhAgo2-2 binds abundant 27nt sRNA populations and that mutating its PAZ domain abolishes sRNA binding [26]. With modi cation with oligo-As, 31nt sRNAs presumably are in a process of disassociation with EhAgo2-2. ...
Background: The RNA interference (RNAi) pathway is a gene regulation mechanism that uitilizes small RNA (sRNA) and Argonaute (Ago) proteins to silence target genes. Our previous work identified a functional RNAi pathway in the protozoan parasite Entamoeba histolytica, including abundant 27nt antisense sRNA populations derived from the secondary RNAi pathway which associate with EhAgo2-2 protein. However, there is lack of understanding about sRNAs that are bound to two other EhAgos (EhAgo2-1 and 2-3), and the mechanism of sRNA regulation itself is unclear in this parasite.
Results: In the present study, we sequenced sRNA libraries from both total RNAs and EhAgo bound RNAs. We identified a new population of 31nt sRNAs that results from the addition of a non-templated 3-4 adenosine nucleotides at the 3´-end of the 27nt sRNA populations, indicating a non-templated RNA-tailing event in the parasite. We found that both sRNA populations (27nt and 31nt) are unchanged during the development of E. invadens. However, we detected an alteration in their relative abundance for the targeted gene in parasites transfected with a trigger-gene silencing construct, indicating that non-templated RNA-tailing is likely a pathway for sRNA turnover when the targeted gene is unable to be silenced in this parasite. In sequencing the sRNAs associating with the three EhAgo proteins, we observed that despite distinct cellular localization, all three EhAgo sRNA libraries contain 27nt sRNAs with 5´-polyphosphate (5´-polyP) structure and a largely overlapping sRNA repertoire, mainly targeting retrotransposons and a subset of ~226 genes that are endogenously silenced. Furthermore, our data show that 31nt sRNA populations paritally associate with wildtype EhAgo2-2 but not with its mutant protein (EhAgo2-2 C-terminal deletion), indicating an intact RISC is essential for the sRNA modification process.
Conclusion: High-throughput sequencing of sRNA in Entamoeba has identified a new population of sRNA with non-templated adenylation modification, which is the first such observation amongst single cell protozoan parasites. Our sRNA sequencing libraries provide the first comprehensive sRNA dataset for all three Entamoeba Ago proteins, which can serve as a useful database for the amoeba community.
Purpose
To describe ocular findings in infants with signs of congenital Zika virus syndrome (CZS) in Paraíba, Brazil, as well as to conduct a literature review and report correlations with published clinical cases.
Methods
In the Paraíba sample, infants with microcephaly suggestive of CZS were classified as Z (confirmed), PZ (probable), or SZ (suspected) according to serological testing and/or clinical findings of CZS. The patients underwent a clinical eye examination, and the results were correlated with published clinical cases.
Results
Ocular findings were present in 24 (42.9%) of 56 patients, consisting of gross retinal pigmentation in 11 (45.8%), macular chorioretinal atrophy in 11 (45.8%), optic nerve hypoplasia in 1 (4.2%), optic nerve pallor in 14 (58.3%), and increased cup-to-disk ratio in 2 (8.3%). The study revealed retina and optic nerve findings consistent with previous reports of ophthalmic involvement in CZS. However, external ocular changes observed in other studies were not detected.
Conclusion
Ocular findings similar and consistent with the literature on CZS were observed with considerable frequency and severity, regardless of the patients’ serological confirmation or classification. Infants with signs of CZS should undergo ocular examination.
Zika virus (ZIKV), a mosquito-borne flavivirus, can cause severe eye disease and even blindness in newborns. However, ZIKV-induced retinal lesions have not been studied in a comprehensive way, mechanisms of ZIKV-induced retinal abnormalities are unknown, and no therapeutic intervention is available to treat or minimize the degree of vision loss in patients. Here, we developed a novel mouse model of ZIKV infection to evaluate its impact on retinal structure. ZIKV (20 plaque-forming units) was inoculated into neonatal wild type C57BL/6J mice at postnatal day (P) 0 subcutaneously. Retinas of infected mice and age-matched controls were collected at various ages, and retinal structural alterations were analyzed. We found that ZIKV induced progressive neuronal and vascular damage and retinal inflammation starting from P8. ZIKV-infected retina exhibited dramatically decreased thickness with loss of neurons, initial neovascular tufts followed by vessel dilation and degeneration, increased microglia and leukocyte recruitment and activation, degeneration of astrocyte network and gliosis. The above changes may involve inflammation and endoplasmic reticulum stress-mediated cell apoptosis and necroptosis. Moreover, we evaluated the efficacy of preclinical drugs and the safety of ZIKV vaccine candidate in this mouse model. We found that ZIKV-induced retinal abnormalities could be blocked by a selective flavivirus inhibitor NITD008 and a live-attenuated ZIKV vaccine candidate could potentially induce retinal abnormalities. Overall, we established a novel mouse model and provide a direct causative link between ZIKV and retinal lesion in vivo, which warrants further investigation of the underlying mechanisms of ZIKV-induced retinopathy and the development of effective therapeutics.
Supplementary Information
The online version contains supplementary material available at 10.1186/s40478-021-01195-6.
