My First Year Research Experience

Hello, my name is Itzel Guillen. I am a graduate of Rio Grande High School, and a freshman at UNM. I am a participant in the FYRE (First Year Research Experience) program. FYRE gave me the opportunity to work more on a project that I participated in while I was in high school: RAPS. My research interest is the response of adults who were part of the project. As a freshman, finding a mentor can be hard. Many students need more experience, but there may not be many open opportunities. For my mentor, I contacted Courtney FitzGerald at the UNM PRC. I met Courtney through the RAPS project while I was in high school. For FYRE, I wanted to work with RAPS project data. I was able to do this with Courtney as my mentor.https://digitalrepository.unm.edu/prc-posters-presentations/1000/thumbnail.jp

Entamoeba histolytica Up-Regulates MicroRNA-643 to Promote Apoptosis by Targeting XIAP in Human Epithelial Colon Cells

MicroRNAs (miRNAs) are small non-coding RNAs that function as negative regulators of gene expression. Recent evidences suggested that host cells miRNAs are involved in the progression of infectious diseases, but its role in amoebiasis remains largely unknown. Here, we reported an unexplored role for miRNAs of human epithelial colon cells during the apoptosis induced by Entamoeba histolytica. We demonstrated for the first time that SW-480 colon cells change their miRNAs profile in response to parasite exposure. Our data showed that virulent E. histolytica trophozoites induced apoptosis of SW-480 colon cells after 45 min interaction, which was associated to caspases-3 and -9 activation. Comprehensive profiling of 667 miRNAs using Taqman Low-Density Arrays showed that 6 and 15 miRNAs were significantly (FC > 1.5; p < 0.05) modulated in SW-480 cells after 45 and 75 min interaction with parasites, respectively. Remarkably, no significant regulation of the 6-miRNAs signature (miR-526b-5p, miR-150, miR-643, miR-615-5p, miR-525, and miR-409-3p) was found when SW-480 cells were exposed to non-virulent Entamoeba dispar. Moreover, we confirmed that miR-150, miR-643, miR-615-5p, and miR-525 exhibited similar regulation in SW-480 and Caco2 colon cells after 45 min interaction with trophozoites. Exhaustive bioinformatic analysis of the six-miRNAs signature revealed intricate miRNAs-mRNAs co-regulation networks in which the anti-apoptotic XIAP, API5, BCL2, and AKT1 genes were the major targets of the set of six-miRNAs. Of these, we focused in the study of functional relationships between miR-643, upregulated at 45 min interaction, and its predicted target X-linked inhibitor of apoptosis protein (XIAP). Interestingly, interplay of amoeba with SW-480 cells resulted in downregulation of XIAP consistent with apoptosis activation. More importantly, loss of function studies using antagomiRs showed that forced inhibition of miR-643 leads to restoration of XIAP levels and suppression of both apoptosis and caspases-3 and -9 activation. Congruently, mechanistic studies using luciferase reporter assays confirmed that miR-643 exerts a postranscripcional negative regulation of XIAP by targeting its 3′-UTR indicating that it's a downstream effector. In summary, we provide novel lines of evidence suggesting that early-branched eukaryote E. histolytica may promote apoptosis of human colon cells by modulating, in part, the host microRNome which highlight an unexpected role for miRNA-643/XIAP axis in the host cellular response to parasites infection

mRNA Decay Proteins Are Targeted to poly(A)⁺ RNA and dsRNA-Containing Cytoplasmic Foci That Resemble P-Bodies in <em>Entamoeba histolytica</em>

<div><p>In higher eukaryotes, mRNA degradation and RNA-based gene silencing occur in cytoplasmic foci referred to as processing bodies (P-bodies). In protozoan parasites, the presence of P-bodies and their putative role in mRNA decay have yet to be comprehensively addressed. Identification of P-bodies might provide information on how mRNA degradation machineries evolved in lower eukaryotes. Here, we used immunofluorescence and confocal microscopy assays to investigate the cellular localization of mRNA degradation proteins in the human intestinal parasite <em>Entamoeba histolytica</em> and found evidence of the existence of P-bodies. Two mRNA decay factors, namely the <em>Eh</em>XRN2 exoribonuclease and the <em>Eh</em>DCP2 decapping enzyme, were localized in cytoplasmic foci in a pattern resembling P-body organization. Given that amoebic foci appear to be smaller and less rounded than those described in higher eukaryotes, we have named them “P-body-like structures”. These foci contain additional mRNA degradation factors, including the <em>Eh</em>CAF1 deadenylase and the <em>Eh</em>AGO2-2 protein involved in RNA interference. Biochemical analysis revealed that <em>Eh</em>CAF1 co-immunoprecipitated with <em>Eh</em>XRN2 but not with <em>Eh</em>DCP2 or <em>Eh</em>AGO2-2, thus linking deadenylation to 5′-to-3′ mRNA decay. The number of <em>Eh</em>CAF1-containing foci significantly decreased after inhibition of transcription and translation with actinomycin D and cycloheximide, respectively. Furthermore, results of RNA-FISH assays showed that (i) <em>Eh</em>CAF1 colocalized with poly(A)⁺ RNA and (ii) during silencing of the <em>Ehpc4</em> gene by RNA interference, <em>Eh</em>AGO2-2 colocalized with small interfering RNAs in cytoplasmic foci. Our observation of decapping, deadenylation and RNA interference proteins within P-body-like foci suggests that these structures have been conserved after originating in the early evolution of eukaryotic lineages. To the best of our knowledge, this is the first study to report on the localization of mRNA decay proteins within P-body-like structures in <em>E. histolytica</em>. Our findings should open up opportunities for deciphering the mechanisms of mRNA degradation and RNA-based gene silencing in this deep-branching eukaryote.</p> </div

mRNA expression profiles of genes for <i>E. histolytica</i> mRNA degradation proteins.

<p>Quantitative real-time PCR assays were performed to analyze the relative expression of representative mRNA degradation genes after heat shock, UV-C induced DNA damage and sodium nitroprusside treatments. For each triplicate experiment, the mean of the relative concentrations obtained for the tested mRNA were divided by the mean of the corresponding values obtained for endogenous ribosomal <i>L31</i> amplification. Each PCR experiment was carried out three times and three independent biological samples were analyzed. The relative expression (fold change) of mRNA degradation genes in the different treatments was calculated by the 2(−ΔΔCt method) using as reference the Ct data for the untretated condition. Bars indicate the mean ± SD.</p

Colocalization of mRNA decay factors with <i>Eh</i>CAF1 in cytoplasmic foci.

<p>(A–L) Confocal immunofluorescence microscopy assays. Trophozoites were incubated with anti-<i>Eh</i>XRN2 (B), anti-<i>Eh</i>DCP2 (F), anti-<i>Eh</i>AGO2-2 (J) and anti-<i>Eh</i>CAF1 (C, G, K) primary antibodies (1∶100). Secondary antibodies included TRITC-conjugated anti-mouse IgG (1∶200) and FITC-conjugated anti-rabbit IgG (1∶200). (A, E and I): light microscopy; (B, F and J): the red channel; (C, G and K): the green channel; (D, H and L): merged images. Arrowheads mark typical, colocalized signals in cytoplasmic foci. (M) Immunoprecipitation assays using anti-<i>Eh</i>CAF1 and anti-<i>Eh</i>XRN2 antibodies conjugated to protein G beads with cytoplasmic lysates of <i>E. histolytica</i> trophozoites. Immunoprecipitates were separated with SDS-PAGE and analyzed by Western blotting with the antibodies indicated at the left side of each panel. Lanes 1 and 3: input (10% of the total lysate used for immunoprecipitation); lanes 2 and 4: immunoprecipitated proteins.</p

Comparison of XRN2 and DCP2 from eukaryotic organisms and <i>E. histolytica</i>.

<p>(A) A schematic representation of XRN2 protein from <i>E. histolytica</i>, <i>H. sapiens</i> and <i>S. pombe</i>. Red box: the XRN domain; cyan box: the tower domain (TD). The numbers are relative to the initial methionine in each protein. Lower panel: multiple alignments of amino acid residues from the TD of homologous proteins. Black box: a conserved residue; grey box: a conserved substitution. Asterisks mark the conserved residues in the active site. (B, C) Ribbon diagrams of <i>Sc</i>RAT1's tertiary structure (B) and the predicted model of <i>Eh</i>XRN2 (C). Red: the N-terminal XRN_N domain; cyan: the TD. (D) A schematic representation of the DCP2 protein from <i>E. histolytica</i>, <i>H. sapiens</i>, and <i>S. cerevisiae</i>. Red box: the DCP2 domain; blue box: the nudix domain. The numbers are relative to the initial methionine in each protein. Lower panel: multiple alignments of amino acid residues from the nudix motif (PF00293) of homologous proteins. Asterisks indicate the conserved residues involved in catalytic activity. Black box: a conserved residue; grey box: a conserved substitution. (E, F) Ribbon diagrams of <i>Sc</i>DCP2's tertiary structure (E) and the predicted model of <i>Eh</i>DCP2 (F). Red: the DCP2 domain; blue: the nudix domain; yellow: the nudix motif. Three-dimensional models were displayed and refined using the RCSB PBD Protein Workshop 3.9 viewer.</p

Determination of the number of <i>Eh</i>CAF1-containing cytoplasmic foci after treatment with actinomycin D, cycloheximide or puromycin.

<p>After treatment with actinomycin D (5 µg/ml, panel A), cycloheximide (10 µg/ml, panel B) or puromycin (200 µg/ml, panel C), trophozoites were fixed, incubated with anti-<i>Eh</i>CAF1 and FITC-labeled secondary antibodies, counterstained with DAPI and analyzed under an immunofluorescence confocal microscope. Left panel: change over time in the mean number of foci per trophozoite, in response to inhibition of transcription (A) or translation (B–C). The data correspond to six randomly chosen fields. Right panel: representative images of cells treated with DMSO vehicle (control) and actinomycin D, cycloheximide or puromycin (observed in the green channel).</p

Expression and immunolocalization of <i>Eh</i>XRN2, <i>Eh</i>DCP2 and <i>Eh</i>CAF1.

<p>(A–B) Expression of 6xHis-tagged <i>Eh</i>XRN2 (A) and <i>Eh</i>DCP2 (B) proteins. Bacterial proteins were separated by 12% SDS-PAGE and the gels were stained with Coomassie blue. Lane 1: non-induced bacterial extracts; lane 2: IPTG-induced bacterial extracts. Western blot assays were performed using anti-6xHis tag antibodies and non-induced bacterial extracts (lane 3) or IPTG-induced bacterial extracts (lane 4). Recombinant <i>Eh</i>XRN2 and <i>Eh</i>DCP2 proteins were also immmunodetected with specific antibodies (lane 5). (C) Immunodetection of <i>Eh</i>XRN2 (lanes 1 and 2), <i>Eh</i>DCP2 (lanes 3 and 4) and <i>Eh</i>PC4 (lanes 5 and 6) proteins in trophozoites, using specific antibodies. Lanes 1, 3 and 5: nuclear extracts; lanes 2, 4 and 6: cytoplasmic extracts. (D–R) Confocal immunofluorescence microscopy assays for the localization of <i>Eh</i>XRN2 (E), <i>Eh</i>DCP2 (J) and <i>Eh</i>CAF1 (O) in fixed trophozoites using FITC-antibodies. Middle panels (F, K, P) show nuclear staining with DAPI and right-hand panels (G, L, Q) show overlays of the two signals. Different magnifications of single cells were used to enhance the visibility of cytoplasmic structures (H, M, R). Arrowheads indicated some typical foci.</p