Search

To counteract host immunity, Cryptosporidium parvum has evolved multiple strategies to suppress host antimicrobial defense. One such strategy is to reduce the production of the antimicrobial peptide beta-defensin 1 (DEFB1) by host epithelial cells but the underlying mechanisms remain unclear. Recent studies demonstrate that a panel of parasite RNA transcripts of low protein-coding potential are delivered into infected host cells and may modulate host gene transcription. Using in vitro models of intestinal cryptosporidiosis, in this study, we analyzed the expression profile of host beta-defensin genes in host cells following infection. We found that C. parvum infection caused a significant downregulation of the DEFB1 gene. Interestingly, downregulation of DEFB1 gene was associated with host delivery of Cdg7_FLc_1000 RNA transcript, a C. parvum RNA that has previously demonstrated to be delivered into the nuclei of infected host cells. Knockdown of Cdg7_FLc_1000 in host cells could attenuate the trans-suppression of host DEFB1 gene and decreased the parasite burden. Therefore, our data suggest that trans-suppression of DEFB1 gene in intestinal epithelial cells following C. parvum infection involves host delivery of parasite Cdg7_FLc_1000 RNA, a process that may be relevant to the epithelial defense evasion by C. parvum at the early stage of infection.

BACKGROUND Eukaryotic ribonucleoprotein (RNP) granules are important for the regulation of RNA fate. RNP granules exist in trypanosomatids; however, their roles in controlling gene expression are still not understood. XRNA is a component of granules in Trypanosoma brucei but has not been investigated in Trypanosoma cruzi. OBJECTIVES This study aimed to investigate the TcXRNA dynamic assembly and its interaction with RNP components under conditions that affect the mRNA availability. METHODS We used in vitro metacyclogenesis of T. cruzi to observe changes in RNP granules during the differentiation process. TcXRNA expression was analysed by Western blot and immunofluorescence. Colocalisation assays were performed to investigate the interaction of TcXRNA with other RNP components. FINDINGS TcXRNA is constantly present during metacyclogenesis and is localised in cytoplasmic granules. TcXRNA does not colocalise with TcDHH1 and TcCAF1 granules in the cytoplasm. However, TcXRNA granules colocalise with mRNP granules at the nuclear periphery when mRNA processing is inhibited. MAIN CONCLUSIONS TcXRNA plays a role in mRNA metabolism as a component of mRNP granules whose assembly is dependent on mRNA availability. TcXRNA granules colocalise with distinct RNP granules at the nuclear periphery, suggesting that the perinuclear region is a regulatory compartment in T. cruzi mRNA metabolism.

Dicer-dependent small noncoding RNAs play important roles in gene regulation in a wide variety of organisms. Endogenous small interfering RNAs (siRNAs) are part of an ancient pathway of transposon control in plants and animals. The ciliate, Oxytricha trifallax, has approximately 16,000 gene-sized chromosomes in its somatic nucleus. Long noncoding RNAs establish high ploidy levels at the onset of sexual development, but the factors that regulate chromosome copy numbers during cell division and growth have been a mystery. We report a novel function of a class of Dicer (Dcl-1)- and RNA-dependent RNA polymerase (RdRP)-dependent endogenous small RNAs in regulating chromosome copy number and gene dosage in O. trifallax Asexually growing populations express an abundant class of 21-nt sRNAs that map to both coding and noncoding regions of most chromosomes. These sRNAs are bound to chromatin and their levels surprisingly do not correlate with mRNA levels. Instead, the levels of these small RNAs correlate with genomic DNA copy number. Reduced sRNA levels in dcl-1 or rdrp mutants lead to concomitant reduction in chromosome copy number. Furthermore, these cells show no signs of transposon activation, but instead display irregular nuclear architecture and signs of replication stress. In conclusion, Oxytricha Dcl-1 and RdRP-dependent small RNAs that derive from the somatic nucleus contribute to the maintenance of gene dosage, possibly via a role in DNA replication, offering a novel role for these small RNAs in eukaryotes., (© 2018 Khurana et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Telomerase is a ribonucleoprotein (RNP) enzyme that maintains the ends of linear eukaryotic chromosomes and whose activation is a hallmark of 90% of all cancers. This RNP minimally contains a reverse transcriptase protein subunit (TERT) that catalyzes telomeric DNA synthesis and an RNA subunit (TER) that has templating, architectural and protein-scaffolding roles. Telomerase is unique among polymerases in that it synthesizes multiple copies of the template on the 3' end of a primer following a single binding event, a process known as repeat addition processivity (RAP). Using biochemical assays and single-molecule Förster resonance energy transfer (smFRET) experiments on Tetrahymena thermophila telomerase, we now directly demonstrate that TER contributes to template positioning within the active site and to the template translocation required for RAP. We propose that the single-stranded RNA elements flanking the template act as a molecular accordion, undergoing reciprocal extension and compaction during telomerase translocation.

In recent years it has became evident that the transcriptome of most species has little protein-coding capacity and that the abundance of non-coding RNA was previously overlooked. Non-coding RNAs were initially thought to be transcriptional noise, however, a growing number of studies is showing that many of these RNAs have important regulatory functions. Here, we review the progress done in apicomplexan parasites in this rapidly growing field., (Copyright 2010 Elsevier B.V. All rights reserved.)

Malaria is caused by the unicellular apicomplexan parasites of the genus Plasmodium, some of which, including the major human parasite Plasmodium falciparum, have extreme genome compositions (A/T content > 80%). In this overview of RNA production, roles and degradation, we show that despite their unusual genome composition these parasites generally exhibit the standard eukaryotic features of these processes. Thus genes are monocistronic and transcribed by RNA polymerases that conform to the general categories of I, II, and III. Plasmodium spp. are unusual in that they possess structurally distinct rRNA genes that are expressed at different points in the complicated life cycle of the parasite. Transcription in blood stage asexual parasites follows a cascade consistent with a dependency upon plant-like apetala 2 (AP2) DNA-binding proteins. mRNA is transported to, translated and degraded in the cytoplasm and the transcription pattern is largely inflexible and responsive to temperature and glucose but not drugs. Furthermore, although Plasmodium spp. undertake controlled repression of mRNA species at a number of points in their life cycle only one mechanism, employed by female gametocytes (gamete precursor cells), is clear; it resembles that of metazoan female gametes, consisting of a complex of repression-associated proteins in an architecture formed with the mRNA 5' cap and dependent on U-rich untranslated region (UTR) elements. Extensive antisense transcription has been documented resulting in the production of both short and long transcripts of generally unknown functional significance. This review attempts to summarize what is currently known about the biology of Plasmodium RNA., (Copyright © 2010 John Wiley & Sons, Ltd.)

Mitochondrial dysfunction underlies a large number of acute or progressive diseases, as well as aging. However, proposed therapies for mitochondrial mutations suffer from poor transformation of mitochondria with exogenous DNA, or lack of functionality of the transferred nucleic acid within the organelle. We show that a transfer RNA import complex (RIC) from the parasitic protozoon Leishmania tropica rapidly and efficiently delivered signal-tagged antisense (STAS) RNA or DNA to mitochondria of cultured human cells. STAS-induced specific degradation of the targeted mitochondrial mRNA, with downstream effects on respiration. These results reveal the existence of a novel small RNA-mediated mRNA degradation pathway in mammalian mitochondria, and suggest that RIC-mediated delivery could be used to target therapeutic RNAs to the organelle within intact cells.

Transport mechanisms involved in pH homeostasis are relevant for the survival of Leishmania parasites. The presence of chloride conductive pathways in Leishmania has been anticipated since anion channel inhibitors limit the proton extrusion mediated by the H+ATPase, which is the major regulator of intracellular pH in amastigotes. In this study, we used Xenopus laevis oocytes as a heterologous expression system in which to study the expression of ion channels upon microinjection of polyA mRNA from Leishmania amazonensis. After injection of polyA mRNA into the oocytes, we measured three different types of currents. We discuss the possible origin of each, and propose that Type 3 currents could be the result of the heterologous expression of proteins from Leishmania since they show different pharmacological and biophysical properties as compared to endogenous oocyte currents.

In trypanosome RNA editing, uridylate (U) residues are inserted and deleted at numerous sites within mitochondrial pre-mRNAs by an approximately 20S protein complex that catalyzes cycles of cleavage, U addition/U removal, and ligation. We used RNA interference to deplete TbMP18 (band VII), the last unexamined major protein of our purified editing complex, showing it is essential. TbMP18 is critical for the U-deletional and U-insertional cleavages and for integrity of the approximately 20S editing complex, whose other major components, TbMP99, TbMP81, TbMP63, TbMP52, TbMP48, TbMP42 (bands I through VI), and TbMP57, instead sediment as approximately 10S associations. Additionally, TbMP18 augments editing substrate recognition by the TbMP57 terminal U transferase, possibly aiding the recognition component, TbMP81. The other editing activities and their coordination in precleaved editing remain active in the absence of TbMP18. These data are reminiscent of the data on editing subcomplexes reported by A. Schnaufer et al. (Mol. Cell 12:307-319, 2003) and suggest that these subcomplexes are held together in the approximately 20S complex by TbMP18, as was proposed previously. Our data additionally imply that the proteins are less long-lived in these subcomplexes than they are when held in the complete editing complex. The editing endonucleolytic cleavages being lost when the editing complex becomes fragmented, as upon TbMP18 depletion, should be advantageous to the trypanosome, minimizing broken mRNAs.

Endogenous small RNAs function in RNA interference (RNAi) pathways to guide RNA cleavage, translational repression, or methylation of DNA or chromatin. In Tetrahymena thermophila, developmentally regulated DNA elimination is governed by an RNAi mechanism involving approximately 27-30-nucleotide (nt) RNAs. Here we characterize the sequence features of the approximately 27-30-nt RNAs and a approximately 23-24-nt RNA class representing a second RNAi pathway. The approximately 23-24-nt RNAs accumulate strain-specifically manner and map to the genome in clusters that are antisense to predicted genes. These findings reveal the existence of distinct endogenous RNAi pathways in the unicellular T. thermophila, a complexity previously demonstrated only in multicellular organisms.

Approximately 6000 DNA elements, totaling nearly 15 Mb, are coordinately excised from the developing somatic genome of Tetrahymena thermophila. An RNA interference (RNAi)-related mechanism has been implicated in the targeting of these germline-limited sequences for chromatin modification and subsequent DNA rearrangement. The excision of individual DNA segments can be inhibited if the homologous sequence is placed within the parental somatic nucleus, indicating that communication occurs between the parental and developing genomes. To determine how the DNA content of one nucleus is communicated to the other, we assessed DNA rearrangement occurring in wild-type cells that were mated to cells that contained the normally germline-limited M element within their somatic nuclei. M-element rearrangement was blocked in the wild-type cell even when no genetic exchange occurred between mating partners, a finding that is inconsistent with any genetic imprinting models. This inhibition by the parental somatic nucleus was rapidly established between 5 and 6 hr of conjugation, near or shortly after the time that zygotic nuclei are formed. M-element small RNAs (sRNAs) that are believed to direct its rearrangement were found to rapidly accumulate during the first few hours of conjugation before stabilizing to a low, steady-state level. The period between 5 and 6 hr during which sRNA levels stabilize correlates with the time after which the parental genome can block DNA rearrangement. These data lead us to suggest that homologous sRNAs serve as mediators to communicate sequence-specific information between the parental and developing genomes, thereby regulating genome-wide DNA rearrangement, and that these sRNAs can be effectively compared to the somatic genome of both parents.

Small RNAs produced by an RNAi-related mechanism are involved in DNA elimination during development of the somatic macronucleus from the germline micronucleus in Tetrahymena. The properties of these small RNAs can explain how the primary sequence of the parental macronucleus epigenetically controls genome rearrangement in the new macronucleus and provide the first demonstration of an RNAi-mediated process that directly alters DNA sequence organization. Methylation of histone H3 on lysine 9 and accumulation of chromodomain proteins, hallmarks of heterochromatin, also occur specifically on sequences undergoing elimination and are dependent on the small RNAs. These findings contribute to a new paradigm of chromatin biology: regulation of heterochromatin formation by RNAi-related mechanisms in eukaryotes.

Differentiation of kinetoplastid protozoa during their complex life cycles is accompanied by stepwise changes in mitochondrial functions. Recent studies have begun to reveal multilevel post-transcriptional regulatory mechanisms by which the expression of the nuclear and mitochondrially encoded components of respiratory enzymes is coordinated, as well as the identities of some general and gene-specific factors controlling mitochondrial differentiation.

During development of the somatic macronucleus from the germline micronucleus in ciliates, chromosome rearrangements occur in which specific regions of DNA are eliminated and flanking regions are healed, either by religation or construction of telomeres. We identified a gene, TWI1, in Tetrahymena thermophila that is homologous to piwi and is required for DNA elimination. We also found that small RNAs were specifically expressed prior to chromosome rearrangement during conjugation. These RNAs were not observed in TWI1 knockout cells and required PDD1, another gene required for rearrangement, for expression. We propose that these small RNAs function to specify sequences to be eliminated by a mechanism similar to RNA-mediated gene silencing.

Most mitochondrial mRNAs are edited in Trypano soma brucei by a series of steps that are catalyzed by a multienzyme complex that is in its initial stages of characterization. RNA interference (RNAi)-mediated repression of the expression of TbMP81, a zinc finger protein component of the complex, inhibited growth of bloodstream and insect forms, and blocked in vivo RNA editing. This repression preferentially inhibited insertion editing compared with deletion editing in vitro. It resulted in reduced specific endoribonucleolytic cleavage and a greater reduction of U addition and associated RNA ligation activities than U removal and associated RNA ligation activities. The repressed cells retained 20S editing complexes with several demonstrable proteins and adenylatable TbMP52 RNA ligase, but adenlyatable TbMP48 was not detected. Elimination of TbMP48 by RNAi repression did not inhibit cell growth or in vivo editing in either bloodstream or procyclic forms. These results indicate that TbMP81 is required for RNA editing and suggest that the editing complex is functionally partitioned.

This review focuses on the spliced leader (SL) RNA and uridylic acid-rich small nuclear RNAs (U-snRNAs) involved in pre-mRNA processing in trypanosomatid protozoa, with particular emphasis on the mechanism of transcription and cap formation. The SL RNA plays a central role in mRNA biogenesis by providing the unique cap 4 structure to the 5' end of all mRNAs by trans-splicing. The trimethylguanosine capped U-snRNAs, on the other hand, represent an unusual example among eukaryotic snRNAs in that they are transcribed by RNA polymerase III. This implies the existence of a distinctive mechanism for capping enzyme selection by the transcriptional machinery. Furthermore, the transcription units of U-snRNA genes offer yet another example of the variety of choices that have been established during eukaryotic evolution, namely that an upstream tRNA gene or tRNA-like gene provides extragenic promoter elements for a downstream small RNA gene.

The human malaria parasite, Plasmodium falciparum, maintains at least two distinct types, A and S, of developmentally controlled ribosomal RNAs. To investigate specific functions associated with these rRNAs, we replaced the Saccharomyces cerevisiae GTPase domain of the 25S rRNA with GTPase domains corresponding to the Plasmodium A- and S-type 28S rRNAs. The A-type rRNA differs in a single nonconserved base pair from the yeast GTPase domain. The S-type rRNA GTPase domain has three additional changes in highly conserved residues, making it unique among all known rRNA sequences. The expression of either A- or S-type chimeric rRNA in yeast increased translational accuracy. Yeast containing only A-type chimeric rRNA and no wild-type yeast rRNA grew at the wild-type level. In contrast, S-type chimeric rRNA severely inhibited growth in the presence of wild-type yeast rRNA, and caused lethality in the absence of the wild-type yeast rRNA. We show what before could only be hypothesized, that the changes in the GTPase center of ribosomes present during different developmental stages of Plasmodium species can result in fundamental changes in the biology of the organism.

From a random amplified polymorphism cDNA study using vegetative, precystic and mature resting cysts of the ciliate Colpoda inflata, a stage-specific fragment was isolated from the resting cyst cDNA population. This fragment was used as an encystment probe to study the corresponding mRNA expression during this eukaryotic cell differentiation process. This transcript is accumulated in the resting cyst of this ciliate, and it is the first time that accumulation of specific mRNA molecules associated with the ciliate cryptobiosis process is reported. The meaning of this stored mRNA in microbial cryptobiotic stages is discussed.

Thermotolerance is an inducible state that endows cells with an enhanced resistance to thermal killing. Heat shock proteins are believed, and in a few instances have been shown, to be the agents conferring this resistance. The role of a small cytoplasmic RNA (G8 RNA) in developing thermotolerance in Tetrahymena thermophila was investigated by creating a strain devoid of all functional G8 genes. These G8 null cells mounted an apparently normal heat shock response, but they were unable to establish thermotolerance.

Background: Catalytic RNAs, or ribozymes, possessing both a genotype and a phenotype, are ideal molecules for evolution experiments in vitro. A large, heterogeneous pool of RNAs can be subjected to multiple rounds of selection, amplification and mutation, leading to the development of variants that have some desired phenotype. Such experiments allow the investigator to correlate specific genetic changes with quantifiable alterations of the catalytic properties of the RNA. In addition, patterns of evolutionary change can be discerned through a detailed examination of the genotypic composition of the evolving RNA population., Results: Beginning with a pool of 10(13) variants of the Tetrahymena ribozyme, we carried out in vitro evolution experiments that led to the generation of ribozymes with the ability to cleave an RNA substrate in the presence of Ca2+ ions, an activity that does not exist for the wild-type molecule. Over the course of 12 generations, a seven-error variant emerged that has substantial Ca(2+)-dependent RNA-cleavage activity. Advantageous mutations increased in frequency in the population according to three distinct dynamics--logarithmic, linear and transient. Through a comparative analysis of 31 individual variants, we infer how certain mutations influence the catalytic properties of the ribozyme., Conclusions: In vitro evolution experiments make it possible to elucidate important aspects of both evolutionary biology and structural biochemistry on a reasonable short time scale.

This is a review based upon a lecture given at the Developmental Biology School near Moscow in November 1991. Cases of successive and simultaneous nuclear differentiation are defined, and examples from various groups of protozoa are considered. Three cases of simultaneous nuclear differentiation, leading to the nuclear dualism phenomenon (heteromorphism) are analysed in detail. In heterokaryotic agamonts of some Foraminifera, nuclear differentiation occurs at the diploid level, proved to be irreversible and caused by either deletion or stable repression of some genes. In the Karyorelictid ciliates, the somatic nuclei (macronuclei) are paradiploid, metabolically active but unable to divide. They are irreversibly differentiated due to deletion of part of the initial (micronuclear) genome. Differentiation occurs in every cell cycle from the generative nuclei (micronuclei), which retain omnipotency and reproduce by mitosis. In most (higher) ciliates the differentiation of macronuclei occurs at an early stage of replication and involves a more or less drastic reorganization of the micronuclear genome, including both deletions and transpositions. Thereafter, the reorganized (and generally reduced) genome is strongly amplified to provide a high dose of active genes. The chromosomes of the majority of ciliates are fragmented in their macronuclei into either subchromosome-sized or gene-sized molecules, both being acentric. Systems regulating the differential replication of these molecules are likely to exist in macronuclei to keep the respective gene doses within certain limits.