Screening by deep sequencing reveals mediators of microRNA tailing in C. elegans.

microRNAs are frequently modified by addition of untemplated nucleotides to the 3' end, but the role of this tailing is often unclear. Here we characterize the prevalence and functional consequences of microRNA tailing in vivo, using Caenorhabditis elegans. MicroRNA tailing in C. elegans consists mostly of mono-uridylation of mature microRNA species, with rarer mono-adenylation which is likely added to microRNA precursors. Through a targeted RNAi screen, we discover that the TUT4/TUT7 gene family member CID-1/CDE-1/PUP-1 is required for uridylation, whereas the GLD2 gene family member F31C3.2-here named GLD-2-related 2 (GLDR-2)-is required for adenylation. Thus, the TUT4/TUT7 and GLD2 gene families have broadly conserved roles in miRNA modification. We specifically examine the role of tailing in microRNA turnover. We determine half-lives of microRNAs after acute inactivation of microRNA biogenesis, revealing that half-lives are generally long (median = 20.7 h), as observed in other systems. Although we observe that the proportion of tailed species increases over time after biogenesis, disrupting tailing does not alter microRNA decay. Thus, tailing is not a global regulator of decay in C. elegans. Nonetheless, by identifying the responsible enzymes, this study lays the groundwork to explore whether tailing plays more specialized context- or miRNA-specific regulatory roles.

RNA editing restricts hyperactive ciliary kinases.

Protein kinase activity must be precisely regulated, but how a cell governs hyperactive kinases remains unclear. In this study, we generated a constitutively active mitogen-activated protein kinase DYF-5 (DYF-5CA) in Caenorhabditis elegans that disrupted sensory cilia. Genetic suppressor screens identified that mutations of ADR-2, an RNA adenosine deaminase, rescued ciliary phenotypes of dyf-5CA We found that dyf-5CA animals abnormally transcribed antisense RNAs that pair with dyf-5CA messenger RNA (mRNA) to form double-stranded RNA, recruiting ADR-2 to edit the region ectopically. RNA editing impaired dyf-5CA mRNA splicing, and the resultant intron retentions blocked DYF-5CA protein translation and activated nonsense-mediated dyf-5CA mRNA decay. The kinase RNA editing requires kinase hyperactivity. The similar RNA editing-dependent feedback regulation restricted the other ciliary kinases NEKL-4/NEK10 and DYF-18/CCRK, which suggests a widespread mechanism that underlies kinase regulation.

CDE-1 suppresses the production of risiRNA by coupling polyuridylation and degradation of rRNA.

BACKGROUND: Modification of RNAs, particularly at the terminals, is critical for various essential cell processes; for example, uridylation is implicated in tumorigenesis, proliferation, stem cell maintenance, and immune defense against viruses and retrotransposons. Ribosomal RNAs can be regulated by antisense ribosomal siRNAs (risiRNAs), which downregulate pre-rRNAs through the nuclear RNAi pathway in Caenorhabditis elegans. However, the biogenesis and regulation of risiRNAs remain obscure. Previously, we showed that 26S rRNAs are uridylated at the 3'-ends by an unknown terminal polyuridylation polymerase before the rRNAs are degraded by a 3' to 5' exoribonuclease SUSI-1(ceDIS3L2). RESULTS: Here, we found that CDE-1, one of the three C.elegans polyuridylation polymerases (PUPs), is specifically involved in suppressing risiRNA production. CDE-1 localizes to perinuclear granules in the germline and uridylates Argonaute-associated 22G-RNAs, 26S, and 5.8S rRNAs at the 3'-ends. Immunoprecipitation followed by mass spectrometry (IP-MS) revealed that CDE-1 interacts with SUSI-1(ceDIS3L2). Consistent with these results, both CDE-1 and SUSI-1(ceDIS3L2) are required for the inheritance of RNAi. CONCLUSIONS: This work identified a rRNA surveillance machinery of rRNAs that couples terminal polyuridylation and degradation.

Characterization of exosome-like vesicles derived from Taenia pisiformis cysticercus and their immunoregulatory role on macrophages.

BACKGROUND: Taenia pisiformis is one of the most common intestinal parasites in canines, and leads to serious economic losses in the rabbit breeding industry. Exosome-like vesicles from parasites play crucial roles in host-parasite interactions by transferring cargo from parasites to host cells and by modulating host immunological response through inducing production of host-derived cytokines. Nevertheless, the mechanism by which exosome-like vesicles from T. pisiformis cysticercus regulate the macrophage immune response remains unknown. METHODS: Using ultracentrifugation, we isolated exosome-like vesicles from excretory/secretory products (ESP) of T. pisiformis cysticercus. The morphology and size of purified vesicles were confirmed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The components of proteins and miRNAs within these vesicles were identified by proteomic analysis and high-throughput small RNA sequencing. The biological function of targets of exosomal miRNAs was predicted by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Moreover, the expression of Th1- and Th2-type immune response associated cytokines in RAW264.7 macrophages were evaluated by qPCR and ELISA. We found that exosome-like vesicles were typical cup-shaped vesicles with diameters from 30 to 150 nm. A total of 87 proteins were identified by proteomic analysis, including proteins prominently associated with exosome-like vesicles biogenesis and vesicle trafficking. 41 known miRNAs and 18 novel miRNAs were identified in the exosome-like vesicles. Eleven selected miRNAs, including 7 known miRNAs (miR-71-5p, miR-10a-5p, miR-let-7-5p, miR-745-3p, miR-219-5p, miR-124-3p and miR-4989-3p) and 4 novel miRNAs (novel-mir-3, novel-mir-7, novel-mir-8 and novel-mir-11) were validated to exist in metacestiodes and exosome-like vesicles of T. pisiformis cysticercus by qPCR. The functions of most targets of exosomal miRNAs were mainly associated with signal transduction and the immune system. Additionally, T. pisiformis cysticercus-derived vesicles induced the production of IL-4, IL-6, IL-10, IL-13 and Arg-1, but downregulated the expression of IL-12, IFN-γ and iNOS in RAW264.7 macrophages. CONCLUSIONS: We demonstrated that proteins and miRNAs enclosed within exosome-like vesicles from T. pisiformis cysticercus have immunomodulatory functions. Furthermore, exosome-like vesicles were shown to induce the macrophage Th2-type immune response in vitro. Our study suggests that exosome-like vesicles play an important role in the interaction between cysticerci and their hosts.

Epigenetic and parasitological parameters are modulated in EBi3-/- mice infected with Schistosoma mansoni.

Schistosoma mansoni adaptive success is related to regulation of replication, transcription and translation inside and outside the intermediate and definitive host. We hypothesize that S. mansoni alters its epigenetic state in response to the mammalian host immune system, reprogramming gene expression and altering the number of eggs. In response, a change in the DNA methylation profile of hepatocytes could occurs, modulating the extent of hepatic granuloma. To investigate this hypothesis, we used the EBi3-/- murine (Mus musculus) model of S. mansoni infection and evaluated changes in new and maintenance DNA methylation profiles in the liver after 55 days of infection. We evaluated expression of epigenetic genes and genes linked to histone deubiquitination in male and female S. mansoni worms. Comparing TET expression with DNMT expression indicated that DNA demethylation exceeds methylation in knockout infected and uninfected mice and in wild-type infected and uninfected mice. S. mansoni infection provokes activation of demethylation in EBi3-/-I mice (knockout infected). EBi3-/-C (knockout uninfected) mice present intrinsically higher DNA methylation than WTC (control uninfected) mice. EBi3-/-I mice show decreased hepatic damage considering volume and reduced number of granulomas compared to WTI mice; the absence of IL27 and IL35 pathways decreases the Th1 response resulting in minor liver damage. S. mansoni males and females recovered from EBi3-/-I mice have reduced expression of a deubiquitinating enzyme gene, orthologs of which target histones and affect chromatin state. SmMBD and SmHDAC1 expression levels are downregulated in male and female parasites recovered from EBi3-/-, leading to epigenetic gene downregulation in S. mansoni. Changes to the immunological background thus induce epigenetic changes in hepatic tissues and alterations in S. mansoni gene expression, which attenuate liver symptoms in the acute phase of schistosomiasis.

Disruption of the Caenorhabditis elegans Integrator complex triggers a non-conventional transcriptional mechanism beyond snRNA genes.

Gene expression is generally regulated by recruitment of transcription factors and RNA polymerase II (RNAP II) to specific sequences in the gene promoter region. The Integrator complex mediates processing of small nuclear RNAs (snRNAs) as well as the initiation and release of paused RNAP II at specific genes in response to growth factors. Here we show that in C. elegans, disruption of the Integrator complex leads to transcription of genes located downstream of the snRNA loci via a non-conventional transcription mechanism based on the lack of processing of the snRNAs. RNAP II read-through generates long chimeric RNAs containing snRNA, the intergenic region and the mature mRNA of the downstream gene located in sense. These chimeric sn-mRNAs remain as untranslated long non-coding RNAs, in the case of U1- and U2-derived sn-mRNAs, but can be translated to proteins in the case of SL-derived sn-mRNAs. The transcriptional effect caused by disruption of the Integrator complex is not restricted to genes located downstream of the snRNA loci but also affects key regulators of signal transduction such as kinases and phosphatases. Our findings highlight that these transcriptional alterations may be behind the correlation between mutations in the Integrator complex and tumor transformation.

The steroid derivative 6-aminocholestanol inhibits the DEAD-box helicase eIF4A (LieIF4A) from the Trypanosomatid parasite Leishmania by perturbing the RNA and ATP binding sites.

The antifungal agent 6-aminocholestanol targets the production of ergosterol, which is the principle sterol in many fungi and protozoans; ergosterol serves many of the same roles as cholesterol in animals. We found that it also is an effective inhibitor of the translation-initiation factor eIF4AI from mouse (eIF4AIMus) and the Trypanosomatid parasite Leishmania (LieIF4A). The eIF4A proteins belong to the DEAD-box family of RNA helicases, which are ATP-dependent RNA-binding proteins and RNA-dependent ATPases. DEAD-box proteins contain a commonly-shared core structure consisting of two linked domains with structural homology to that of recombinant protein A (RecA) and that contain conserved motifs that are involved in RNA and ATP binding, and in the enzymatic activity. The compound inhibits both the ATPase and helicase activities by perturbing ATP and RNA binding, and it is capable of binding other proteins containing nucleic acid-binding sites as well. We undertook kinetic analyses and found that the Leishmania LieIF4A protein binds 6-aminocholestanol with a higher apparent affinity than for ATP, although multiple binding sites were probably involved. Competition experiments with the individual RecA-like domains indicate that the primary binding sites are on RecA-like domain 1, and they include a cavity that we previously identified by molecular modeling of LieIF4A that involve conserved RNA-binding motifs. The compound affects the mammalian and Leishmania proteins differently, which indicates the binding sites and affinities are not the same. Thus, it is possible to develop drugs that target DEAD-box proteins from different organisms even when they are implicated in the same biological process.

Prospectively Isolated Tetraspanin+ Neoblasts Are Adult Pluripotent Stem Cells Underlying Planaria Regeneration.

Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) that sustain tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single-cell transplantation to address this long-standing issue. Large-scale scRNA-seq of sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein and marked by a conserved cell-surface protein-coding gene, tetraspanin 1 (tspan-1). tspan-1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody, rescued the viability of lethally irradiated animals after single-cell transplantation. The first prospective isolation of an adult PSC bridges a conceptual dichotomy between functionally and molecularly defined neoblasts, shedding light on mechanisms governing in vivo pluripotency and a source of regeneration in animals. VIDEO ABSTRACT.

FUS Regulates Activity of MicroRNA-Mediated Gene Silencing.

MicroRNA-mediated gene silencing is a fundamental mechanism in the regulation of gene expression. It remains unclear how the efficiency of RNA silencing could be influenced by RNA-binding proteins associated with the microRNA-induced silencing complex (miRISC). Here we report that fused in sarcoma (FUS), an RNA-binding protein linked to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), interacts with the core miRISC component AGO2 and is required for optimal microRNA-mediated gene silencing. FUS promotes gene silencing by binding to microRNA and mRNA targets, as illustrated by its action on miR-200c and its target ZEB1. A truncated mutant form of FUS that leads its carriers to an aggressive form of ALS, R495X, impairs microRNA-mediated gene silencing. The C. elegans homolog fust-1 also shares a conserved role in regulating the microRNA pathway. Collectively, our results suggest a role for FUS in regulating the activity of microRNA-mediated silencing.

Role of Kallistatin Treatment in Aging and Cancer by Modulating miR-34a and miR-21 Expression.

Kallistatin is an endogenous protein that regulates differential signaling pathways and a wide spectrum of biological activities via its two structural elements: an active site and a heparin-binding domain. Kallistatin via its heparin-binding site inhibits vascular inflammation and oxidative stress by antagonizing TNF-α-induced NADPH oxidase activity, NF-κB activation, and inflammatory gene expression in endothelial cells. Moreover, kallistatin via its active site inhibits microRNA-34a (miR-34a) synthesis and stimulates eNOS and SIRT1 expression in endothelial progenitor cells, whereas its heparin-binding site is crucial for blocking TNF-α-induced miR-21 expression and oxidative stress, thus reducing cellular senescence. By downregulating miR-34a and miR-21 expression, kallistatin treatment attenuates oxidative damage and aortic senescence in streptozotocin-induced diabetic mice and extends Caenorhabditis elegans lifespan under stress conditions. Likewise, kallistatin through the heparin-binding site inhibits TGF-ß-induced miR-21 synthesis and oxidative stress in endothelial cells, resulting in inhibition of endothelial-mesenchymal transition, a process contributing to fibrosis and cancer. Furthermore, kallistatin's active site is essential for stimulating miR-34a and p53 expression and inhibiting the miR-21-Akt-Bcl-2 signaling pathway, thus inducing apoptosis in breast cancer cells. These findings reveal novel mechanisms of kallistatin in protection against senescence, aging, and cancer development by modulating miR-34a and miR-21 levels and inhibiting oxidative stress.

LIN41 Post-transcriptionally Silences mRNAs by Two Distinct and Position-Dependent Mechanisms.

The RNA-binding protein (RBP) LIN41, also known as LIN-41 or TRIM71, is a key regulator of animal development, but its physiological targets and molecular mechanism of action are largely elusive. Here we find that this RBP has two distinct mRNA-silencing activities. Using genome-wide ribosome profiling, RNA immunoprecipitation, and in vitro-binding experiments, we identify four mRNAs, each encoding a transcription factor or cofactor, as direct physiological targets of C. elegans LIN41. LIN41 silences three of these targets through their 3' UTRs, but it achieves isoform-specific silencing of one target, lin-29A, through its unique 5' UTR. Whereas the 3' UTR targets mab-10, mab-3, and dmd-3 undergo transcript degradation, lin-29A experiences translational repression. Through binding site transplantation experiments, we demonstrate that it is the location of the LIN41-binding site that specifies the silencing mechanism. Such position-dependent dual activity may, when studied more systematically, emerge as a feature shared by other RBPs.

Induction of hsp70, hsp90, and catalase activity in planarian Dugesia japonica exposed to cadmium.

The hsp70 and hsp90 expression patterns and catalase (CAT) activity in the freshwater planaria Dugesia japonica exposed to cadmium (Cd) under laboratory conditions were investigated. Planaria were exposed to a range of Cd concentrations (0-150 µg Cd/L) for 24 h. The expression levels of hsp70 and hsp90 were determined by relative quantitative real-time polymerase chain reaction. Within the overall dose range in the experiment, the expression level of hsp70 and the activity of CAT in D. japonica were altered significantly. Hsp70 was induced in D. japonica upon Cd exposure concentrations as low as 9.375 µg Cd/L. No significant effect on the expression level of hsp90 was observed. Our findings demonstrated that stress gene hsp70, but not hsp90, was responsive to Cd contamination in D. japonica CAT activity was significantly induced at concentrations of 18.75, 37.5, and 75 µg Cd/L after 24-h exposure. We recommend that the use of hsp70 as a biomarker should be complemented by evidence of changes in other parameters, such as CAT activity, in D. japonica.

Noncoding regions of C. elegans mRNA undergo selective adenosine to inosine deamination and contain a small number of editing sites per transcript.

ADARs (Adenosine deaminases that act on RNA) "edit" RNA by converting adenosines to inosines within double-stranded regions. The primary targets of ADARs are long duplexes present within noncoding regions of mRNAs, such as introns and 3' untranslated regions (UTRs). Because adenosine and inosine have different base-pairing properties, editing within these regions can alter splicing and recognition by small RNAs. However, despite numerous studies identifying multiple editing sites in these genomic regions, little is known about the extent to which editing sites co-occur on individual transcripts or the functional output of these combinatorial editing events. To begin to address these questions, we performed an ultra-deep sequencing analysis of 4 Caenorhabditis elegans 3' UTRs that are known ADAR targets. Synchronous editing events were determined for the long duplexes in vivo. Furthermore, the validity of each editing event was confirmed by sequencing the same regions of mRNA from worms that lack A-to-I editing. This analysis identified a large number of editing sites that can occur within each 3' UTR, but interestingly, each individual transcript contained only a small fraction of these A-to-I editing events. In addition, editing patterns were not random, indicating that an editing event can affect the efficiency of editing at subsequent adenosines. Furthermore, we identified specific sites that can be both positively and negatively correlated with additional sites leading to mutually exclusive editing patterns. These results suggest that editing in noncoding regions is selective and hyper-editing of cellular RNAs is rare.

microRNA profiling in the zoonotic parasite Echinococcus canadensis using a high-throughput approach.

BACKGROUND: microRNAs (miRNAs), a class of small non-coding RNAs, are key regulators of gene expression at post-transcriptional level and play essential roles in fundamental biological processes such as development and metabolism. The particular developmental and metabolic characteristics of cestode parasites highlight the importance of studying miRNA gene regulation in these organisms. Here, we perform a comprehensive analysis of miRNAs in the parasitic cestode Echinococcus canadensis G7, one of the causative agents of the neglected zoonotic disease cystic echinococcosis. METHODS: Small RNA libraries from protoscoleces and cyst walls of E. canadensis G7 and protoscoleces of E. granulosus sensu stricto G1 were sequenced using Illumina technology. For miRNA prediction, miRDeep2 core algorithm was used. The output list of candidate precursors was manually curated to generate a high confidence set of miRNAs. Differential expression analysis of miRNAs between stages or species was estimated with DESeq. Expression levels of selected miRNAs were validated using poly-A RT-qPCR. RESULTS: In this study we used a high-throughput approach and found transcriptional evidence of 37 miRNAs thus expanding the miRNA repertoire of E. canadensis G7. Differential expression analysis showed highly regulated miRNAs between life cycle stages, suggesting a role in maintaining the features of each developmental stage or in the regulation of developmental timing. In this work we characterize conserved and novel Echinococcus miRNAs which represent 30 unique miRNA families. Here we confirmed the remarkable loss of conserved miRNA families in E. canadensis, reflecting their low morphological complexity and high adaptation to parasitism. CONCLUSIONS: We performed the first in-depth study profiling of small RNAs in the zoonotic parasite E. canadensis G7. We found that miRNAs are the preponderant small RNA silencing molecules, suggesting that these small RNAs could be an essential mechanism of gene regulation in this species. We also identified both parasite specific and divergent miRNAs which are potential biomarkers of infection. This study will provide valuable information for better understanding of the complex biology of this parasite and could help to find new potential targets for therapy and/or diagnosis.

Genome-wide sequencing of small RNAs reveals a tissue-specific loss of conserved microRNA families in Echinococcus granulosus.

BACKGROUND: MicroRNAs (miRNAs) are important post-transcriptional regulators which control growth and development in eukaryotes. The cestode Echinococcus granulosus has a complex life-cycle involving different development stages but the mechanisms underpinning this development, including the involvement of miRNAs, remain unknown. RESULTS: Using Illumina next generation sequencing technology, we sequenced at the genome-wide level three small RNA populations from the adult, protoscolex and cyst membrane of E. granulosus. A total of 94 pre-miRNA candidates (coding 91 mature miRNAs and 39 miRNA stars) were in silico predicted. Through comparison of expression profiles, we found 42 mature miRNAs and 23 miRNA stars expressed with different patterns in the three life stages examined. Furthermore, considering both the previously reported and newly predicted miRNAs, 25 conserved miRNAs families were identified in the E. granulosus genome. Comparing the presence or absence of these miRNA families with the free-living Schmidtea mediterranea, we found 13 conserved miRNAs are lost in E. granulosus, most of which are tissue-specific and involved in the development of ciliated cells, the gut and sensory organs. Finally, GO enrichment analysis of the differentially expressed miRNAs and their potential targets indicated that they may be involved in bi-directional development, nutrient metabolism and nervous system development in E. granulosus. CONCLUSIONS: This study has, for the first time, provided a comprehensive description of the different expression patterns of miRNAs in three distinct life cycle stages of E. granulosus. The analysis supports earlier suggestions that the loss of miRNAs in the Platyhelminths might be related to morphological simplification. These results may help in the exploration of the mechanism of interaction between this parasitic worm and its definitive and intermediate hosts, providing information that can be used to develop new interventions and therapeutics for the control of cystic echinococcosis.

Roles of microRNAs in the Caenorhabditis elegans nervous system.

The first microRNA was discovered in Caenorhabditis elegans in 1993, and since then, thousands of microRNAs have been identified from almost all eukaryotic organisms examined. MicroRNAs function in many biological events such as cell fate determination, metabolism, apoptosis, and carcinogenesis. So far, more than 250 microRNAs have been identified in C. elegans; however, functions for most of these microRNAs are still unknown. A small number of C. elegans microRNAs are associated with known physiological roles such as developmental timing, cell differentiation, stress response, and longevity. In this review, we summarize known roles of microRNAs in neuronal differentiation and function of C. elegans, and discuss interesting perspectives for future studies.

Inducing effect of PGRs on small regulatory si/miRNA in resistance to sugar beet cyst nematode.

Sugar beet cyst nematode Heterodera schachtii Schmidt is an economically important plant parasite of sugar beet in Ukraine. The pest control options are limited. Sugar beet cyst nematode resistant varieties are not available on the market. Carbamate and organophosphate pesticides have been banned due to the high toxicity. The problem is aggravated by continuously increasing of oilseed rape (which is suitable host for H. schachtii) growing area due to biofuel demands. Several studies' results indicate that PGRs have role in management of plant parasitic nematodes but for sugar beet it is not studied well. We had an objective- studying of the role of four compositional PGRs created based of avermectin in suppression of sugar beet cyst nematode population on sugar beet and oilseed rape caused by enhancing of endogenous si/miRNA complementary to H. schachtii mRNA. Laboratory study was conducted in 2011 with using method DOT-blot hybridization si/miRNA with mRNA and by testing inhibitory activity in cell free system protein biosynthesis. That was shown that application of the PGRs enhances sugar beet and oilseeds rape plant immune-protective properties and resistance against plant-parasitic nematode Heterodera schochtii through enhancement of synthesis of small regulatory si/miRNA related (complementary) to an mRNA structure of the parasitic organisms. As a result, translation of mRNA of the nematode is blocked and causes the mortality of plant parasite juveniles.

RNA processing in C. elegans.

In Caenorhabditis elegans, newly transcribed RNA is processed in several novel ways. Although introns are removed by a canonical spliceosome, they have evolved several specialized features that reflect the differences in the way they are recognized and the way they are spliced. C. elegans introns are unusually short, in part because they have no specific branch-point sequences and contain minimal polypryimidine tracts. Instead, their 3' splice site is characterized by a highly conserved consensus sequence, which alone may be sufficient to position all spliceosomal elements at the 3' end of the intron. Many RNA molecules are also trans-spliced: a capped 22nt RNA leader is donated by one of a family of specialized snRNPs and spliced to an unpaired 3' splice site, usually just upstream of the start codon. The RNA upstream of this splice site, the outron, is removed during trans-splicing and presumably degraded, making the identification of the transcriptional start site problematic. Transcripts from approximately 70% of all genes are trans-spliced. Trans-splicing has enabled the evolution of operons - multigene clusters in which a single upstream promoter drives the transcription of a polycistronic pre-mRNA. The C. elegans genome contains more than 1000 such operons. The polycistronic pre-mRNA is processed into individual gene-encoding mRNAs by coordinated upstream 3' end formation and downstream trans-splicing. An intercistronic RNA sequence, the Ur element, plays a key role in specifying downstream trans-splicing.

Active turnover modulates mature microRNA activity in Caenorhabditis elegans.

MicroRNAs (miRNAs) constitute a large class of regulatory RNAs that repress target messenger RNAs to control various biological processes. Accordingly, miRNA biogenesis is highly regulated, controlled at both transcriptional and post-transcriptional levels, and overexpression and underexpression of miRNAs are linked to various human diseases, particularly cancers. As RNA concentrations are generally a function of biogenesis and turnover, active miRNA degradation might also modulate miRNA accumulation, and the plant 3'-->5' exonuclease SDN1 has been implicated in miRNA turnover. Here we report that degradation of mature miRNAs in the nematode Caenorhabditis elegans, mediated by the 5'-->3' exoribonuclease XRN-2, affects functional miRNA homeostasis in vivo. We recapitulate XRN-2-dependent miRNA turnover in larval lysates, where processing of precursor-miRNA (pre-miRNA) by Dicer, unannealing of the miRNA duplex and loading of the mature miRNA into the Argonaute protein of the miRNA-induced silencing complex (miRISC) are coupled processes that precede degradation of the mature miRNA. Although Argonaute:miRNA complexes are highly resistant to salt, larval lysate promotes efficient release of the miRNA, exposing it to degradation by XRN-2. Release and degradation can both be blocked by the addition of miRNA target RNA. Our results therefore suggest the presence of an additional layer of regulation of animal miRNA activity that might be important for rapid changes of miRNA expression profiles during developmental transitions and for the maintenance of steady-state concentrations of miRNAs. This pathway might represent a potential target for therapeutic intervention on miRNA expression.

LAB-1 antagonizes the Aurora B kinase in C. elegans.

The Shugoshin/Aurora circuitry that controls the timely release of cohesins from sister chromatids in meiosis and mitosis is widely conserved among eukaryotes, although little is known about its function in organisms whose chromosomes lack a localized centromere. Here we show that Caenorhabditis elegans chromosomes rely on an alternative mechanism to protect meiotic cohesin that is shugoshin-independent and instead involves the activity of a new chromosome-associated protein named LAB-1 (Long Arm of the Bivalent). LAB-1 preserves meiotic sister chromatid cohesion by restricting the localization of the C. elegans Aurora B kinase, AIR-2, to the interface between homologs via the activity of the PP1/Glc7 phosphatase GSP-2. The localization of LAB-1 to chromosomes of dividing embryos and the suppression of mitotic-specific defects in air-2 mutant embryos with reduced LAB-1 activity support a global role of LAB-1 in antagonizing AIR-2 in both meiosis and mitosis. Although the localization of a GFP fusion and the analysis of mutants and RNAi-mediated knockdowns downplay a role for the C. elegans shugoshin protein in cohesin protection, shugoshin nevertheless helps to ensure the high fidelity of chromosome segregation at metaphase I. We propose that, in C. elegans, a LAB-1-mediated mechanism evolved to offset the challenges of providing protection against separase activity throughout a larger chromosome area.