Structural basis of gRNA stabilization and mRNA recognition in trypanosomal RNA editing.

In Trypanosoma brucei, the editosome, composed of RNA-editing substrate-binding complex (RESC) and RNA-editing catalytic complex (RECC), orchestrates guide RNA (gRNA)-programmed editing to recode cryptic mitochondrial transcripts into messenger RNAs (mRNAs). The mechanism of information transfer from gRNA to mRNA is unclear owing to a lack of high-resolution structures for these complexes. With cryo-electron microscopy and functional studies, we have captured gRNA-stabilizing RESC-A and gRNA-mRNA-binding RESC-B and RESC-C particles. RESC-A sequesters gRNA termini, thus promoting hairpin formation and blocking mRNA access. The conversion of RESC-A into RESC-B or -C unfolds gRNA and allows mRNA selection. The ensuing gRNA-mRNA duplex protrudes from RESC-B, likely exposing editing sites to RECC-catalyzed cleavage, uridine insertion or deletion, and ligation. Our work reveals a remodeling event facilitating gRNA-mRNA hybridization and assembly of a macromolecular substrate for the editosome's catalytic modality.

Targeting RNA Structure to Inhibit Editing in Trypanosomes.

Mitochondrial RNA editing in trypanosomes represents an attractive target for developing safer and more efficient drugs for treating infections with trypanosomes because this RNA editing pathway is not found in humans. Other workers have targeted several enzymes in this editing system, but not the RNA. Here, we target a universal domain of the RNA editing substrate, which is the U-helix formed between the oligo-U tail of the guide RNA and the target mRNA. We selected a part of the U-helix that is rich in G-U wobble base pairs as the target site for the virtual screening of 262,000 compounds. After chemoinformatic filtering of the top 5000 leads, we subjected 50 representative complexes to 50 nanoseconds of molecular dynamics simulations. We identified 15 compounds that retained stable interactions in the deep groove of the U-helix. The microscale thermophoresis binding experiments on these five compounds show low-micromolar to nanomolar binding affinities. The UV melting studies show an increase in the melting temperatures of the U-helix upon binding by each compound. These five compounds can serve as leads for drug development and as research tools to probe the role of the RNA structure in trypanosomal RNA editing.

Structure of LARP7 Protein p65-telomerase RNA Complex in Telomerase Revealed by Cryo-EM and NMR.

La-related protein 7 (LARP7) are a family of RNA chaperones that protect the 3'-end of RNA and are components of specific ribonucleoprotein complexes (RNP). In Tetrahymena thermophila telomerase, LARP7 protein p65 together with telomerase reverse transcriptase (TERT) and telomerase RNA (TER) form the core RNP. p65 has four known domains-N-terminal domain (NTD), La motif (LaM), RNA recognition motif 1 (RRM1), and C-terminal xRRM2. To date, only the xRRM2 and LaM and their interactions with TER have been structurally characterized. Conformational dynamics leading to low resolution in cryo-EM density maps have limited our understanding of how full-length p65 specifically recognizes and remodels TER for telomerase assembly. Here, we combined focused classification of Tetrahymena telomerase cryo-EM maps with NMR spectroscopy to determine the structure of p65-TER. Three previously unknown helices are identified, one in the otherwise intrinsically disordered NTD that binds the La module, one that extends RRM1, and another preceding xRRM2, that stabilize p65-TER interactions. The extended La module (αN, LaM and RRM1) interacts with the four 3' terminal U nucleotides, while LaM and αN additionally interact with TER pseudoknot, and LaM with stem 1 and 5' end. Our results reveal the extensive p65-TER interactions that promote TER 3'-end protection, TER folding, and core RNP assembly and stabilization. The structure of full-length p65 with TER also sheds light on the biological roles of genuine La and LARP7 proteins as RNA chaperones and core RNP components.

Structural basis for guide RNA selection by the RESC1-RESC2 complex.

Kinetoplastid parasites, such as trypanosomes or leishmania, rely on RNA-templated RNA editing to mature mitochondrial cryptic pre-mRNAs into functional protein-coding transcripts. Processive pan-editing of multiple editing blocks within a single transcript is dependent on the 20-subunit RNA editing substrate binding complex (RESC) that serves as a platform to orchestrate the interactions between pre-mRNA, guide RNAs (gRNAs), the catalytic RNA editing complex (RECC), and a set of RNA helicases. Due to the lack of molecular structures and biochemical studies with purified components, neither the spacio-temporal interplay of these factors nor the selection mechanism for the different RNA components is understood. Here we report the cryo-EM structure of Trypanosoma brucei RESC1-RESC2, a central hub module of the RESC complex. The structure reveals that RESC1 and RESC2 form an obligatory domain-swapped dimer. Although the tertiary structures of both subunits closely resemble each other, only RESC2 selectively binds 5'-triphosphate-nucleosides, a defining characteristic of gRNAs. We therefore propose RESC2 as the protective 5'-end binding site for gRNAs within the RESC complex. Overall, our structure provides a starting point for the study of the assembly and function of larger RNA-bound kinetoplast RNA editing modules and might aid in the design of anti-parasite drugs.

Temporal transcriptomic changes in long non-coding RNAs and messenger RNAs involved in the host immune and metabolic response during Toxoplasma gondii lytic cycle.

BACKGROUND: Long non-coding RNAs (lncRNAs) are important regulators of various biological and pathological processes, in particular the inflammatory response by modulating the transcriptional control of inflammatory genes. However, the role of lncRNAs in regulating the immune and inflammatory responses during infection with the protozoan parasite Toxoplasma gondii remains largely unknown. METHODS: We performed a longitudinal RNA sequencing analysis of human foreskin fibroblast (HFF) cells infected by T. gondii to identify differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs), and dysregulated pathways over the course of T. gondii lytic cycle. The transcriptome data were validated by qRT-PCR. RESULTS: RNA sequencing revealed significant transcriptional changes in the infected HFFs. A total of 697, 1234, 1499, 873, 1466, 561, 676 and 716 differentially expressed lncRNAs (DElncRNAs), and 636, 1266, 1843, 2303, 3022, 1757, 3088 and 2531 differentially expressed mRNAs (DEmRNAs) were identified at 1.5, 3, 6, 9, 12, 24, 36 and 48 h post-infection, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DElncRNAs and DEmRNAs revealed that T. gondii infection altered the expression of genes involved in the regulation of host immune response (e.g., cytokine-cytokine receptor interaction), receptor signaling (e.g., NOD-like receptor signaling pathway), disease (e.g., Alzheimer's disease), and metabolism (e.g., fatty acid degradation). CONCLUSIONS: These results provide novel information for further research on the role of lncRNAs in immune regulation of T. gondii infection.

Comparative analysis of differentially expressed genes in Acanthamoeba after ingestion of Legionella pneumophila and Escherichia coli.

Acanthamoeba spp. feeds on bacteria, fungi, and algae to obtain nutrients from the environment. However, several pathogens can survive and multiply in Acanthamoeba. Mechanisms necessary for the survival and proliferation of microorganisms in Acanthamoeba remain unclear. The object of this study was to identify effective factors for the survival of microorganisms in Acanthamoeba. Differentially expressed genes (DEGs) in A. castellanii infected by Legionella pneumophila or Escherichia coli were identified based on mRNA sequencing. A total of 2342 and 1878 DEGs were identified in Acanthamoeba with L. pneumophila and E. coli, respectively. Among these DEGs, 502 were up-regulated and 116 were down-regulated in Acanthamoeba infected by L. pneumophila compared to those in Acanthamoeba feed on E. coli. Gene ontology analysis showed that the genes encoded small GTPase-mediated signal transduction proteins in the biological process domain, intracellular proteins in the cellular component domain, and ATP binding proteins in the molecular function domain were up-regulated while integral components of membrane proteins in the cellular component domain were down-regulated in Acanthamoeba infected by Legionella compared to those in Acanthamoeba feed on E. coli. During endosymbiosis with Legionella, Acanthamoeba showed various changes in the expression of genes supposed to be involved in phagosomal maturation. Acanthamoeba infected by Legionella also showed high expression levels of aminotransferase, methyltransferase, and cysteine proteinase but low expression levels of RNA pseudouridine synthase superfamily protein and 2OG-Fe(II) oxygenase superfamily. These results provide directions for further research to understand the survival strategy of L. pneumophila in A. castellanii.

In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei.

Telomerase is a unique ribonucleoprotein (RNP) reverse transcriptase that utilizes its cognate RNA molecule as a template for telomere DNA repeat synthesis. Telomerase contains the reverse transcriptase protein, TERT and the template RNA, TR, as its core components. The 5'-half of TR forms a highly conserved catalytic core comprising of the template region and adjacent domains necessary for telomere synthesis. However, how telomerase RNA folding takes place in vivo has not been fully understood due to low abundance of the native RNP. Here, using unicellular pathogen Trypanosoma brucei as a model, we reveal important regional folding information of the native telomerase RNA core domains, i.e. TR template, template boundary element, template proximal helix and Helix IV (eCR4-CR5) domain. For this purpose, we uniquely combined in-cell probing with targeted high-throughput RNA sequencing and mutational mapping under three conditions: in vivo (in WT and TERT-/- cells), in an immunopurified catalytically active telomerase RNP complex and ex vivo (deproteinized). We discover that TR forms at least two different conformers with distinct folding topologies in the insect and mammalian developmental stages of T. brucei. Also, TERT does not significantly affect the RNA folding in vivo, suggesting that the telomerase RNA in T. brucei exists in a conformationally preorganized stable structure. Our observed differences in RNA (TR) folding at two distinct developmental stages of T. brucei suggest that important conformational changes are a key component of T. brucei development.

G-quadruplex RNA motifs influence gene expression in the malaria parasite Plasmodium falciparum.

G-quadruplexes are non-helical secondary structures that can fold in vivo in both DNA and RNA. In human cells, they can influence replication, transcription and telomere maintenance in DNA, or translation, transcript processing and stability of RNA. We have previously showed that G-quadruplexes are detectable in the DNA of the malaria parasite Plasmodium falciparum, despite a very highly A/T-biased genome with unusually few guanine-rich sequences. Here, we show that RNA G-quadruplexes can also form in P. falciparum RNA, using rG4-seq for transcriptome-wide structure-specific RNA probing. Many of the motifs, detected here via the rG4seeker pipeline, have non-canonical forms and would not be predicted by standard in silico algorithms. However, in vitro biophysical assays verified formation of non-canonical motifs. The G-quadruplexes in the P. falciparum transcriptome are frequently clustered in certain genes and associated with regions encoding low-complexity peptide repeats. They are overrepresented in particular classes of genes, notably those that encode PfEMP1 virulence factors, stress response genes and DNA binding proteins. In vitro translation experiments and in vivo measures of translation efficiency showed that G-quadruplexes can influence the translation of P. falciparum mRNAs. Thus, the G-quadruplex is a novel player in post-transcriptional regulation of gene expression in this major human pathogen.

The enigmatic RNase MRP of kinetoplastids.

The ribonucleoprotein RNase MRP is responsible for the processing of ribosomal RNA precursors. It is found in virtually all eukaryotes that have been examined. In the Euglenozoa, including the genera Euglena, Diplonema and kinetoplastids, MRP RNA and protein subunits have so far escaped detection using bioinformatic methods. However, we now demonstrate that the RNA component is widespread among the Euglenozoa and that these RNAs have secondary structures that conform to the structure of all other phylogenetic groups. In Euglena, we identified the same set of P/MRP protein subunits as in many other protists. However, we failed to identify any of these proteins in the kinetoplastids. This finding poses interesting questions regarding the structure and function of RNase MRP in these species.

The RNA structurome in the asexual blood stages of malaria pathogen plasmodium falciparum.

Plasmodium falciparum is a deadly human pathogen responsible for the devastating disease called malaria. In this study, we measured the differential accumulation of RNA secondary structures in coding and non-coding transcripts from the asexual developmental cycle in P. falciparum in human red blood cells. Our comprehensive analysis that combined high-throughput nuclease mapping of RNA structures by duplex RNA-seq, SHAPE-directed RNA structure validation, immunoaffinity purification and characterization of antisense RNAs collectively measured differentially base-paired RNA regions throughout the parasite's asexual RBC cycle. Our mapping data not only aligned to a diverse pool of RNAs with known structures but also enabled us to identify new structural RNA regions in the malaria genome. On average, approximately 71% of the genes with secondary structures are found to be protein coding mRNAs. The mapping pattern of these base-paired RNAs corresponded to all regions of mRNAs, including the 5' UTR, CDS and 3' UTR as well as the start and stop codons. Histone family genes which are known to form secondary structures in their mRNAs and transcripts from genes which are important for transcriptional and post-transcriptional control, such as the unique plant-like transcription factor family, ApiAP2, DNA-/RNA-binding protein, Alba3 and proteins important for RBC invasion and malaria cytoadherence also showed strong accumulation of duplex RNA reads in various asexual stages in P. falciparum. Intriguingly, our study determined stage-specific, dynamic relationships between mRNA structural contents and translation efficiency in P. falciparum asexual blood stages, suggesting an essential role of RNA structural changes in malaria gene expression programs. Abbreviations: CDS: Coding Sequence; DNA: Deoxyribonucleic Acid; dsRNA: double-stranded RNA; IDC: Intra-erythrocytic Developmental Cycle (IDC); m6A: N6-methyladenosine; mRNA: Messenger RNA; ncRNA: Non-coding RNA; RBC: Red Blood cells; RBP: RNA-Binding Protein; REC: Relative Expression Counts; RNA-seq: RNA-sequencing; RNA: Ribonucleic Acid; RNP: Ribonucleoprotein; RPKM: Reads Per Kilobase of transcript Per Million; rRNA: Ribosomal RNA 16. RUFs: RNAs of Unknown Function; SHAPE: Selective 2'-hydroxyl acylation analysed by primer extension; snoRNA: Small Nucleolar RNA; snRNA: Small Nuclear RNA; SRP-RNA: Signal Recognition Particle RNA; ssRNA: (Single-stranded RNA); TE: Translation Efficiency; tRNA: transfer RNA; UTR: Untranslated Region.

Dual RNA-Seq transcriptome analysis of chicken macrophage-like cells (HD11) infected in vitro with Eimeria tenella.

The study aimed to monitor parasite and host gene expression during the early stages of Eimeria tenella infection of chicken cells using dual RNA-Seq analysis. For this, we used chicken macrophage-like cell line HD11 cultures infected in vitro with purified E. tenella sporozoites. Cultures were harvested between 2 and 72 h post-infection and mRNA was extracted and sequenced. Dual RNA-Seq analysis showed clear patterns of altered expression for both parasite and host genes during infection. For example, genes in the chicken immune system showed upregulation early (2­4 h), a strong downregulation of genes across the immune system at 24 h and a repetition of early patterns at 72 h, indicating that invasion by a second generation of parasites was occurring. The observed downregulation may be due to immune self-regulation or to immune evasive mechanisms exerted by E. tenella. Results also suggested pathogen recognition receptors involved in E. tenella innate recognition, MRC2, TLR15 and NLRC5 and showed distinct chemokine and cytokine induction patterns. Moreover, the expression of several functional categories of Eimeria genes, such as rhoptry kinase genes and microneme genes, were also examined, showing distinctive differences which were expressed in sporozoites and merozoites.

A New Type of Circular RNA derived from Nonconventional Introns in Nuclear Genes of Euglenids.

Nuclear protein-coding genes of euglenids (Discoba, Euglenozoa, Euglenida) contain conventional (spliceosomal) and nonconventional introns. The latter have been found only in euglenozoans. A unique feature of nonconventional introns is the ability to form a stable and slightly conserved RNA secondary structure bringing together intron ends and placing adjacent exons in proximity. To date, little is known about the mechanism of their excision (e.g. whether it involves the spliceosome or not). The tubA gene of Euglena gracilis harbors three conventional and three nonconventional introns. While the conventional introns are excised as lariats, nonconventional introns are present in the cell solely as circular RNAs with full-length ends. Based on this discovery as well as on previous observations indicating that nonconventional introns are observed frequently at unique positions of genes, we suggest that this new type of intronic circRNA might play a role in intron mobility.

Human peripheral blood mononuclear cells as an ex vivo model to study the host parasite interaction in Toxoplasma gondii.

Toxoplasma gondii is a parasite that can invade any cell in the human body. Here, we implemented and described an ex vivo model with human peripheral blood mononuclear cells (PBMCs) without using culture supplements/antibiotics and without cryopreserved cells (EXMOWS) to study the interactions between T. gondii and human cells. To establish the EXMOWS, three independent tests were carried out. Firstly, blood samples from 5 individuals were included to assess the viability and adherence of PBMCs in plate culture. In a second trial, blood samples from three seropositive and two seronegative individuals for T. gondii were used to evaluate human PBMCs cells: parasites, multiplicity of infection (MOI) 1:1, 1:3 and 1:5 at different times post infection (1 h, 6 h and 24 h). The possible immunomodulatory effect of the infection for this EXMOWS were evaluated in a third trial where HFF cells were infected with T. gondii and co-cultured with PBMCs obtained from anti-Toxoplasma IgG positive and IgG negative individuals. One hour was enough time for T. gondii infection of human PBMCs and 2 h was the minimum incubation time to guarantee adherence before carrying out any infection assay. A minimum of 1:3 MOI was necessary to guarantee efficient infection in human PBMCs with T. gondii RH-GFP. All protocols, including PBMCs isolation and stimulation, should be conducted the same day. This EXMOWS can be adapted to study the early stages of interaction with other microorganisms of human interest, without need of using cryopreservation and supplements/antibiotics.

Poly(A) binding KPAF4/5 complex stabilizes kinetoplast mRNAs in Trypanosoma brucei.

In Trypanosoma brucei, mitochondrial pre-mRNAs undergo 3'-5' exonucleolytic processing, 3' adenylation and uridylation, 5' pyrophosphate removal, and, often, U-insertion/deletion editing. The 3' modifications are modulated by pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs). We have shown that KPAF3 binding to the 3' region stabilizes properly trimmed transcripts and stimulates their A-tailing by KPAP1 poly(A) polymerase. Conversely, poly(A) binding KPAF4 shields the nascent A-tail from uridylation and decay thereby protecting pre-mRNA upon KPAF3 displacement by editing. While editing concludes in the 5' region, KPAF1/2 dimer induces A/U-tailing to activate translation. Remarkably, 5' end recognition and pyrophosphate hydrolysis by the PPsome complex also contribute to mRNA stabilization. Here, we demonstrate that KPAF4 functions as a heterodimer with KPAF5, a protein lacking discernable motifs. We show that KPAF5 stabilizes KPAF4 to enable poly(A) tail recognition, which likely leads to mRNA stabilization during the editing process and impedes spontaneous translational activation of partially-edited transcripts. Thus, KPAF4/5 represents a poly(A) binding element of the mitochondrial polyadenylation complex. We present evidence that RNA editing substrate binding complex bridges the 5' end-bound PPsome and 3' end-bound polyadenylation complexes. This interaction may enable mRNA circularization, an apparently critical element of mitochondrial mRNA stability and quality control.

Polysome Profiling and Metabolic Labeling Methods to Measure Translation in Trypanosoma brucei.

The amount of a protein that is made in a cell is determined not only by the corresponding mRNA level but also by the efficiency with which the mRNA is translated. Very powerful transcriptome-wide methods are available to analyze both the density of ribosomes on each mRNA and the rate at which polypeptides are elongated. However, for many research questions, simpler, less expensive methods are more suitable. Here we describe two methods to assess the general translation status of cells: polysome profiling by sucrose density gradient centrifugation and metabolic labeling using radioactive amino acids. Both methods can also be used to examine translation of individual mRNAs.

Analysis of the Physiological and Metabolic State of Leishmania Using Heavy Water Labeling.

This protocol describes the use of heavy water (2H2O) labeling to determine the growth rate and metabolic state of Leishmania parasites in culture and in infected animals. In vitro labeling studies are undertaken by cultivating defined parasite developmental stages in standard medium supplemented with 5% 2H2O, resulting in the incorporation of deuterium (2H) into a range of metabolic precursors used in macromolecule (DNA, RNA, protein, lipid, and glycan) synthesis. The rate of turnover of different parasite macromolecules can subsequently be determined by analysis of deuterium enrichment in the different constituents of these macromolecules by gas chromatography-mass spectrometry (GC-MS). To measure the growth rate and physiological state of parasite stages in lesion tissue, infected mice were provided with 9% 2H2O in their drinking water for various periods of time and 2H-enrichment in the macromolecular constituents of isolated lesion-derived parasite stages determined by GC-MS. This protocol provides quantitative information on key cellular processes, such as replication (DNA turnover), transcription (RNA turnover), translation (protein turnover), membrane biogenesis (lipid turnover), and central carbon metabolism (glycan turnover) that define the growth state and phenome of different parasite stages in vitro and in vivo. This approach can be used to assess the impact of host immune responses on parasite growth and physiology (using different Leishmania strains/species, mouse lines), characterize different parasite populations during chronic and acute infections, and assess parasite responses to drug treatments. It is also broadly applicable to other microbial pathogens.

Proteins involved in the biosynthesis of lipophosphoglycan in Leishmania: a comparative genomic and evolutionary analysis.

BACKGROUND: Leishmania spp. are digenetic parasites capable of infecting humans and causing a range of diseases collectively known as leishmaniasis. The main mechanisms involved in the development and permanence of this pathology are linked to evasion of the immune response. Crosstalk between the immune system and particularities of each pathogenic species is associated with diverse disease manifestations. Lipophosphoglycan (LPG), one of the most important molecules present on the surface of Leishmania parasites, is divided into four regions with high molecular variability. Although LPG plays an important role in host-pathogen and vector-parasite interactions, the distribution and phylogenetic relatedness of the genes responsible for its synthesis remain poorly explored. The recent availability of full genomes and transcriptomes of Leishmania parasites offers an opportunity to leverage insight on how LPG-related genes are distributed and expressed by these pathogens. RESULTS: Using a phylogenomics-based framework, we identified a catalog of genes involved in LPG biosynthesis across 22 species of Leishmania from the subgenera Viannia and Leishmania, as well as 5 non-Leishmania trypanosomatids. The evolutionary relationships of these genes across species were also evaluated. Nine genes related to the production of the glycosylphosphatidylinositol (GPI)-anchor were highly conserved among compared species, whereas 22 genes related to the synthesis of the repeat unit presented variable conservation. Extensive gain/loss events were verified, particularly in genes SCG1-4 and SCA1-2. These genes act, respectively, on the synthesis of the side chain attached to phosphoglycans and in the transfer of arabinose residues. Phylogenetic analyses disclosed evolutionary patterns reflective of differences in host specialization, geographic origin and disease manifestation. CONCLUSIONS: The multiple gene gain/loss events identified by genomic data mining help to explain some of the observed intra- and interspecies variation in LPG structure. Collectively, our results provide a comprehensive catalog that details how LPG-related genes evolved in the Leishmania parasite specialization process.

Mapping the RNA Chaperone Activity of the T. brucei Editosome Using SHAPE Chemical Probing.

Mitochondrial pre-mRNAs in African trypanosomes adopt intricately folded, highly stable 2D and 3D structures. The RNA molecules are substrates of a U-nucleotide-specific insertion/deletion-type RNA editing reaction, which is catalyzed by a 0.8 MDa protein complex known as the editosome. RNA binding to the editosome is followed by a chaperone-mediated RNA remodeling reaction. The reaction increases the dynamic of specifically U-nucleotides to lower their base-pairing probability and as a consequence generates a simplified RNA folding landscape that is critical for the progression of the editing reaction cycle. Here we describe a chemical mapping method to quantitatively monitor the chaperone-driven structural changes of pre-edited mRNAs upon editosome binding. The method is known as selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). SHAPE is based on the differential electrophilic modification of ribose 2'-hydroxyl groups in structurally constraint (double-stranded) versus structurally unconstrained (single-stranded) nucleotides. Electrophilic anhydrides such as 1-methyl-7-nitroisatoic anhydride are used as probing reagents, and the ribose 2'-modified nucleotides are mapped as abortive cDNA synthesis products. As a result, SHAPE allows the identification of all single-stranded and base-paired regions in a given RNA, and the data are used to compute experimentally derived RNA 2D structures. A side-by-side comparison of the RNA 2D folds in the pre- and post-chaperone states finally maps the chaperone-induced dynamic of the different pre-mRNAs with single-nucleotide resolution.

Antisense probing of dynamic RNA structures.

RNA regulation is influenced by the dynamic changes in conformational accessibility on the transcript. Here we discuss the initial validation of a cell-free antisense probing method for structured RNAs, using the Tetrahymena group I intron as a control target. We observe changes in signal that qualitatively match prior traditional DMS footprinting experiments. Importantly, we have shown that application of this technique can elucidate new RNA information given its sensitivity for detecting rare intermediates that are not as readily observed by single-hit kinetics chemical probing techniques. Observing changes in RNA accessibility has broad applications in determining the effect that regulatory elements have on regional structures. We speculate that this method could be useful in quickly observing those interactions, along with other phenomena that influence RNA accessibility including RNA-RNA interactions and small molecules.

mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites.

In humans, mRNA polyadenylation involves the participation of about 20 factors in four main complexes that recognize specific RNA sequences. Notably, CFIm25, CPSF73, and PAP have essential roles for poly(A) site selection, mRNA cleavage, and adenosine residues polymerization. Besides the relevance of polyadenylation for gene expression, information is scarce in intestinal protozoan parasites that threaten human health. To better understand polyadenylation in Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum, which represent leading causes of diarrhea worldwide, genomes were screened for orthologs of human factors. Results showed that Entamoeba histolytica and C. parvum have 16 and 12 proteins out of the 19 human proteins used as queries, respectively, while G. lamblia seems to have the smallest polyadenylation machinery with only six factors. Remarkably, CPSF30, CPSF73, CstF77, PABP2, and PAP, which were found in all parasites, could represent the core polyadenylation machinery. Multiple genes were detected for several proteins in Entamoeba, while gene redundancy is lower in Giardia and Cryptosporidium. Congruently with their relevance in the polyadenylation process, CPSF73 and PAP are present in all parasites, and CFIm25 is only missing in Giardia. They conserve the functional domains and predicted folding of human proteins, suggesting they may have the same roles in polyadenylation.