Concordance between genetic relatedness and phenotypic similarities of Trichomonas vaginalis strains

BACKGROUND: Despite the medical importance of trichomoniasis, little is known about the genetic relatedness of Trichomonas vaginalis strains with similar biological characteristics. Furthermore, the distribution of endobionts such as mycoplasmas or Trichomonas vaginalis virus (TVV) in the T. vaginalis metapopulation is poorly characterised. RESULTS: We assayed the relationship between 20 strains of T. vaginalis from 8 countries using the Random Amplified Polymorphic DNA (RAPD) analysis with 27 random primers. The genealogical tree was constructed and its bootstrap values were computed using the program FreeTree. Using the permutation tail probability tests we found that the topology of the tree reflected both the pattern of resistance to metronidazole (the major anti-trichomonal drug) (p < 0.01) and the pattern of infection of strains by mycoplasmas (p < 0.05). However, the tree did not reflect pattern of virulence, geographic origin or infection by TVV. Despite low bootstrap support for many branches, the significant clustering of strains with similar drug susceptibility suggests that the tree approaches the true genealogy of strains. The clustering of mycoplasma positive strains may be an experimental artifact, caused by shared RAPD characters which are dependent on the presence of mycoplasma DNA. CONCLUSIONS: Our results confirmed both the suitability of the RAPD technique for genealogical studies in T. vaginalis and previous conclusions on the relatedness of metronidazol resistant strains. However, our studies indicate that testing analysed strains for the presence of endobionts and assessment of the robustness of tree topologies by bootstrap analysis seem to be obligatory steps in such analyses

ISG20L2: an RNA nuclease regulating T cell activation.

ISG20L2, a 3' to 5' exoribonuclease previously associated with ribosome biogenesis, is identified here in activated T cells as an enzyme with a preferential affinity for uridylated miRNA substrates. This enzyme is upregulated in T lymphocytes upon TCR and IFN type I stimulation and appears to be involved in regulating T cell function. ISG20L2 silencing leads to an increased basal expression of CD69 and induces greater IL2 secretion. However, ISG20L2 absence impairs CD25 upregulation, CD3 synaptic accumulation and MTOC translocation towards the antigen-presenting cell during immune synapsis. Remarkably, ISG20L2 controls the expression of immunoregulatory molecules, such as AHR, NKG2D, CTLA-4, CD137, TIM-3, PD-L1 or PD-1, which show increased levels in ISG20L2 knockout T cells. The dysregulation observed in these key molecules for T cell responses support a role for this exonuclease as a novel RNA-based regulator of T cell function.This study was supported by grant P2022/BMD7209- INTEGRAMUNE from the Comunidad de Madrid, a grant from “La Caixa” Banking Foundation (HR17-00016) to FS-M; the Spanish Ministerio de Ciencia e Innovación (PDC2021-121719-I00 and PID2020-120412RB-I00 to FS-M), grant from AECC, CIBER Cardiovascular (CB16/11/00272, Fondo de Investigación Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015- 0505). Vaňáčová’s laboratory is supported by the Czech Science Foundation (20-19617S and 23-07372S to S.V.) and the institutional support CEITEC 2020 (LQ1601). ARG and SGD are supported by a grant from the Spanish Ministry of Universities. Funding agencies do not have intervened in the design of the studies, with no copyright over the study.S

A New Yeast Poly(A) Polymerase Complex Involved in RNA Quality Control

Eukaryotic cells contain several unconventional poly(A) polymerases in addition to the canonical enzymes responsible for the synthesis of poly(A) tails of nuclear messenger RNA precursors. The yeast protein Trf4p has been implicated in a quality control pathway that leads to the polyadenylation and subsequent exosome-mediated degradation of hypomethylated initiator tRNA(Met) (tRNA(i) (Met)). Here we show that Trf4p is the catalytic subunit of a new poly(A) polymerase complex that contains Air1p or Air2p as potential RNA-binding subunits, as well as the putative RNA helicase Mtr4p. Comparison of native tRNA(i) (Met) with its in vitro transcribed unmodified counterpart revealed that the unmodified RNA was preferentially polyadenylated by affinity-purified Trf4 complex from yeast, as well as by complexes reconstituted from recombinant components. These results and additional experiments with other tRNA substrates suggested that the Trf4 complex can discriminate between native tRNAs and molecules that are incorrectly folded. Moreover, the polyadenylation activity of the Trf4 complex stimulated the degradation of unmodified tRNA(i) (Met) by nuclear exosome fractions in vitro. Degradation was most efficient when coupled to the polyadenylation activity of the Trf4 complex, indicating that the poly(A) tails serve as signals for the recruitment of the exosome. This polyadenylation-mediated RNA surveillance resembles the role of polyadenylation in bacterial RNA turnover

The Eukaryotic RNA Exosome: Methods and Protocols

This volume provides a cross-section of RNA exosome research protocols, applied to a diversity of model organisms. Chapters guide readers through methods that e.g. delineate eukaryotic exosomes’ origins in prokaryotes, probe its RNA substrates, adapter complexes, and macromolecular interaction of networks, and establish critical structure-function relationships. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, The Eukaryotic RNA Exosome: Methods and Protocols aims to ensure successful results in the further study of this vital field

In Vitro Reconstitution of Trf4 Complex from Recombinant Proteins

<p>Polyadenylation assays were performed with radiolabeled unmodified (A and C) and native (B and D) tRNA_i^Met substrate incubated with 25, 50, or 100 ng of Trf4-TAP complex (Trf4-TAP; A and B), or with 5, 10, or 20 ng of recombinant Trf4 protein expressed in the baculovirus system (Trf4-bac; A and B), or with 5, 10, or 20 ng of mutant Trf4-bac (DADA-bac; C and D), or with equal amounts and dilutions of control eluates (CTRL-bac; proteins from cell lysates that unspecifically bound to the Ni²⁺-NTA matrix; C and D). In reconstitution experiments 20 ng of Trf4-bac, or DADA-bac, or control eluates were mixed with 0.5, 3, or 15 ng of recombinant Air1p and/or recombinant Air2p in the combinations indicated. The proteins were pre-incubated for 30 min on ice to allow for binding. Reactions were incubated for 50 min at 30 °C. Control reactions contained no protein (lane 1 in each gel).</p

Trf4p Is the Catalytic Subunit of a New Poly(A) Polymerase

<div><p>(A) The Trf4 complex has poly(A) polymerase activity. The 5′-end-labeled oligo(A)₁₅ was incubated 30 min with 5, 10, or 20 ng of affinity-purified fractions of the wild-type TAP-tagged Trf4p (Trf4-TAP) or mutant Trf4p with the aspartic acid residues 236 and 238 changed to alanines (DADA-TAP). Protein was omitted in lane 1. Recombinant yeast poly(A) polymerase (Pap1), 1, 2, and 4 ng, was used as a positive control. The migration position of oligo(A)₁₅ is indicated by an arrow.</p> <p>(B) The Trf4p activity is specific for the addition of adenosine monophosphate. Polyadenylation assays with 20 ng of Trf4-TAP in the presence of different ribonucleoside triphosphates. Recombinant yeast Pap1p, 5 ng, was used as a control. All samples were separated on 15% denaturing gels.</p></div

The Polyadenylation Activity of the Trf4 Complex Stimulates the Degradation of Unmodified tRNA_i^Met by the Nuclear Exosome

<div><p>(A) The PAP activity of Trf4 complex is required to stimulate the exosome activity. In a coupled exosome/polyadenylation assay, 5′-end-labeled unmodified tRNA_i^Met was incubated with 50 ng of affinity-purified Rrp6-TAP eluate for 30 min as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030189#s4" target="_blank">Materials and Methods</a> (lane 2), followed by addition of 50 ng of wild-type (Trf4-TAP), mutant complex (DADA-TAP), or buffer A (buffer). Reactions were stopped after 10 (lanes 3, 7, and 11), 30 (lanes 4, 8, and 12), 60 (lanes 5, 9, and 13), or 90 min (lanes 6, 10, and 14) and separated on a 15% gel. Arrows indicate the position of the input tRNA. Protein was omitted in lane 1 of each gel. The migration positions of the degradation products (dp) are indicated by a bracket.</p> <p>(B) Coupled polyadenylation/exosome assay. The 5′-end-labeled unmodified tRNA_i^Met was pre-adenylated with 50 ng of affinity-purified Trf4-TAP complex for 30 min (lane 2). Then 50 ng of exosome complex (Rrp6-TAP) or buffer A (buffer) was added, and the reactions were continued as in (A).</p> <p>(C) Depletion of Mtr4p results in incomplete degradation. Coupled-assay, 5′-end-labeled unmodified tRNA_i^Met was pre-incubated for 30 min with Trf4p-TAP lacking Mtr4p (Trf4-TAP w/o Mtr4), followed by the addition of 50 ng of Rrp6-TAP complex or buffer A, and the incubation was continued as in (A).</p></div

Schematic Alignment of the Domain Organization of Trf4p and Trf5p with Other Members of the Pol-β-like Nucleotidyltransferase Family

<p>The proteins are represented as lines, with conserved regions shown as boxes (CAT, catalytic domain; CD, central domain; RBD, RNA-binding domain; sizes in amino acids are indicated to the right). The green boxes in the sequence alignment mark regions with 50% similarity, light blue boxes indicate 100% similarity, and dark blue and orange boxes are regions with 100% identity. The three conserved catalytic aspartates at positions 236, 238, and 294 are in orange and marked by triangles. Regions with four additional amino acids present in Cid1 and GLD-2 are marked with a orange “4.”</p