Nuage condensates: accelerators or circuit breakers for sRNA silencing pathways?

Nuage are RNA-rich condensates that assemble around the nuclei of developing germ cells. Many proteins required for the biogenesis and function of silencing small RNAs (sRNAs) enrich in nuage, and it is often assumed that nuage is the cellular site where sRNAs are synthesized and encounter target transcripts for silencing. Using C. elegans as a model, we examine the complex multicondensate architecture of nuage and review evidence for compartmentalization of silencing pathways. We consider the possibility that nuage condensates balance the activity of competing sRNA pathways and serve to limit, rather than enhance, sRNA amplification to protect transcripts from dangerous runaway silencing.

Protein-based condensation mechanisms drive the assembly of RNA-rich P granules.

Germ granules are protein-RNA condensates that segregate with the embryonic germline. In Caenorhabditis elegans embryos, germ (P) granule assembly requires MEG-3, an intrinsically disordered protein that forms RNA-rich condensates on the surface of PGL condensates at the core of P granules. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). We find that MEG-3 is a modular protein that uses its IDR to bind RNA and its C-terminus to drive condensation. The HMGL motif mediates binding to PGL-3 and is required for co-assembly of MEG-3 and PGL-3 condensates in vivo. Mutations in HMGL cause MEG-3 and PGL-3 to form separate condensates that no longer co-segregate to the germline or recruit RNA. Our findings highlight the importance of protein-based condensation mechanisms and condensate-condensate interactions in the assembly of RNA-rich germ granules.

Comprehensive annotation and characterization of planarian tRNA and tRNA-derived fragments (tRFs).

tRNA-derived fragments (tRFs) have recently gained a lot of scientific interest due to their diverse regulatory roles in several cellular processes. However, their function in dynamic biological processes such as development and regeneration remains unexplored. Here, we show that tRFs are dynamically expressed during planarian regeneration, suggesting a possible role for these small RNAs in the regulation of regeneration. In order to characterize planarian tRFs, we first annotated 457 tRNAs in S. mediterranea combining two tRNA prediction algorithms. Annotation of tRNAs facilitated the identification of three main species of tRFs in planarians-the shorter tRF-5s and itRFs, and the abundantly expressed 5'-tsRNAs. Spatial profiling of tRFs in sequential transverse sections of planarians revealed diverse expression patterns of these small RNAs, including those that are enriched in the head and pharyngeal regions. Expression analysis of these tRF species revealed dynamic expression of these small RNAs over the course of regeneration suggesting an important role in planarian anterior and posterior regeneration. Finally, we show that 5'-tsRNA in planaria interact with all three SMEDWI proteins and an involvement of AGO1 in the processing of itRFs. In summary, our findings implicate a novel role for tRFs in planarian regeneration, highlighting their importance in regulating complex systemic processes. Our study adds to the catalog of posttranscriptional regulatory systems in planaria, providing valuable insights on the biogenesis and the function of tRFs in neoblasts and planarian regeneration.

Similarities and differences in the biotransformation and transcriptomic responses of Caenorhabditis elegans and Haemonchus contortus to five different benzimidazole drugs.

We have undertaken a detailed analysis of the biotransformation of five of the most therapeutically important benzimidazole anthelmintics - albendazole (ABZ), mebendazole (MBZ), thiabendazole (TBZ), oxfendazole (OxBZ) and fenbendazole (FBZ) - in Caenorhabditis elegans and the ruminant parasite Haemonchus contortus. Drug metabolites were detected by LC-MS/MS analysis in supernatants of C. elegans cultures with a hexose conjugate, most likely glucose, dominating for all five drugs. This work adds to a growing body of evidence that glucose conjugation is a major pathway of xenobiotic metabolism in nematodes and may be a target for enhancement of anthelmintic potency. Consistent with this, we found that biotransformation of albendazole by C. elegans reduced drug potency. Glucose metabolite production by C. elegans was reduced in the presence of the pharmacological inhibitor chrysin suggesting that UDP-glucuronosyl/glucosyl transferase (UGT) enzymes may catalyze benzimidazole glucosidation. Similar glucoside metabolites were detected following ex vivo culture of adult Haemonchus contortus. As a step towards identifying nematode enzymes potentially responsible for benzimidazole biotransformation, we characterised the transcriptomic response to each of the benzimidazole drugs using the C. elegans resistant strain CB3474 ben-1(e1880)III. In the case of albendazole, mebendazole, thiabendazole, and oxfendazole the shared transcriptomic response was dominated by the up-regulation of classical xenobiotic response genes including a shared group of UGT enzymes (ugt-14/25/33/34/37/41/8/9). In the case of fenbendazole, a much greater number of genes were up-regulated, as well as developmental and brood size effects suggesting the presence of secondary drug targets in addition to BEN-1. The transcriptional xenobiotic response of a multiply resistant H. contortus strain UGA/2004 was essentially undetectable in the adult stage but present in the L3 infective stage, albeit more muted than C. elegans. This suggests that xenobiotic responses may be less efficient in stages of parasitic nematodes that reside in the host compared with the free-living stages.

De novo genome sequencing and comparative stage-specific transcriptomic analysis of Dirofilaria repens.

The zoonotic mosquito-borne filarial nematode Dirofilaria repens causes subcutaneous and ocular infections in dogs, cats and humans. From infected vertebrate hosts, microfilariae are taken up by mosquitoes and develop into infective L3. These are transmitted to new vertebrate hosts and develop over two further moults to adult worms. The aims of the project were (i) the de novo sequencing and annotation of the D. repens genome and (ii) comparative transcriptomic analyses of the two developmental stages, mf and L3. Genomic DNA was obtained from adult male D. repens. RNA was extracted from mf from naturally infected dogs and from L3 produced in Aedes aegypti mosquitoes fed on blood spiked with mf. The 99.59 MB genome was approximately 17% larger than that of the related species Dirofilaria immitis (dog heartworm) and contained 8.9% fewer predicted genes (10,357). Approximately 1.8% of identified proteins (206/11,262) could not be mapped to D. immitis. Out of these, six (2.9%) presented an ortholog in all other considered filarial nematodes (e.g. Loa loa) and Caenorhabditis elegans. A significantly higher number of D. repens proteins, compared with D. immitis, mapped to the filarial nematode L. loa, reflecting the similarity in biology of D. repens and L. loa. A total of 876 genes were differentially expressed, of which 591 could be annotated in UniProtKB/Swiss-Prot. In particular, 155 genes with a UniProtKB/Swiss-Prot annotation to C. elegans and filarial nematodes were upregulated in the L3 and 57 in the mf stage, respectively. Fifteen Gene Ontology Biological Processes were significantly enriched for the L3 group and 12 for the mf. To our knowledge these data provide the first insight into the differential gene expression profiles of this filarial nematode and can serve future investigations of metabolic processes and stage-specific diagnostics.

Phylogeny of hymenolepidid cestodes (Cestoda: Cyclophyllidea) from mammalian hosts based on partial 28S rDNA, with focus on parasites from shrews.

The aims of the study are to enrich the partial 28S rDNA dataset for hymenolepidids by adding new sequences for species parasitic in the genera Sorex, Neomys and Crocidura (Soricidae) and to propose a new hypothesis for the relationships among mammalian hymenolepidids. New sequences were obtained for Coronacanthus integrus, C. magnihamatus, C. omissus, C. vassilevi, Ditestolepis diaphana, Lineolepis scutigera, Spasskylepis ovaluteri, Staphylocystis tiara, S. furcata, S. uncinata, Vaucherilepis trichophorus and Neoskrjabinolepis sp. The phylogenetic analysis (based on 56 taxa) confirmed the major clades identified by Haukisalmi et al. (Zool Scr 39:631-641, 2010) based on analysis of 31 species: Ditestolepis clade, Hymenolepis clade, Rodentolepis clade and Arostrilepis clade; however, the support was weak for the early divergent lineages of the tree and for the Arostrilepis clade. Novelties revealed include the molecular evidence for the monophyly of Coronacanthus, the non-monophyletic status of Staphylocystis and the polyphyly of Staphylocystoides. The analysis has confirmed the monophyly of Hymenolepis, the monophyly of hymenolepidids from glirids, the position of Pararodentolepis and Nomadolepis as sister taxa, the polyphyly of Rodentolepis, the position of Neoskrjabinolepis and Lineolepis as sister taxa, and the close relationship among the genera with the entire reduction of rostellar apparatus. Resolved monophyletic groups are supported by the structure of the rostellar apparatus. The diversification of the Ditestolepis clade is associated with soricids. The composition of the other major clades suggests multiple evolutionary events of host switching, including between different host orders. The life cycles of Coronacanthus and Vaucherilepis are recognised as secondarily aquatic as these taxa are nested in terrestrial groups.

The Integrator complex regulates differential snRNA processing and fate of adult stem cells in the highly regenerative planarian Schmidtea mediterranea.

In multicellular organisms, cell type diversity and fate depend on specific sets of transcript isoforms generated by post-transcriptional RNA processing. Here, we used Schmidtea mediterranea, a flatworm with extraordinary regenerative abilities and a large pool of adult stem cells, as an in vivo model to study the role of Uridyl-rich small nuclear RNAs (UsnRNAs), which participate in multiple RNA processing reactions including splicing, in stem cell regulation. We characterized the planarian UsnRNA repertoire, identified stem cell-enriched variants and obtained strong evidence for an increased rate of UsnRNA 3'-processing in stem cells compared to their differentiated counterparts. Consistently, components of the Integrator complex showed stem cell-enriched expression and their depletion by RNAi disrupted UsnRNA processing resulting in global changes of splicing patterns and reduced processing of histone mRNAs. Interestingly, loss of Integrator complex function disrupted both stem cell maintenance and regeneration of tissues. Our data show that the function of the Integrator complex in UsnRNA 3'-processing is conserved in planarians and essential for maintaining their stem cell pool. We propose that cell type-specific modulation of UsnRNA composition and maturation contributes to in vivo cell fate choices, such as stem cell self-renewal in planarians.

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.

Identification and characterization of microRNAs in a cestode Hydatigera taeniaeformis using deep sequencing approach.

Hydatigera taeniaeformis (formerly known as Taenia taeniaeformis) is a parasitic tapeworm that has a worldwide distribution. H. taeniaeformis is naturally transmitted between mice and cats and threatens to human health, especially those who are in close contact with pets. MicroRNAs (miRNAs) are a class of small regulatory non-coding RNAs involved in the regulation of parasite growth and development, parasite infection and immunology, and host-pathogen interactions. The miRNA profile of H. taeniaeformis remains to be elucidated. Herein, 47 conserved miRNAs (grouped into 34 miRNA families) and 4 novel miRNAs were identified in H. taeniaeformis metacestodes using deep sequencing approach. Among them, hta-miR-71, -let-7, and-miR-87 was absolutely predominant in H. taeniaeformis metacestodes. Moreover, comparative analysis revealed the presence of miR-71/2 and miR-4989/277 clusters in H. taeniaeformis. Nucleotide bias analysis of identified miRNAs showed that the adenine (A) was the dominant nucleotide at the beginning of the miRNAs, particularly at the positions of third and 7th nucleotides. The study provides rich data for further understandings of H. taeniaeformis biology.

Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses.

Enrichment methodologies enable the analysis of minor members in multi-species transcriptomic data. We compared the standard enrichment of bacterial and eukaryotic mRNA to a targeted enrichment using an Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with their unenriched counterparts (r2 = 0.559-0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r2 = 0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote enrichment from a single RNA sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase in multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries compared to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using AgSS targeted enrichment on poly(A)-enriched libraries for eukaryotic captures, and total RNA libraries for prokaryotic captures, to increase the robustness of multi-species transcriptomic studies.

Identification of long noncoding RNAs in Schistosoma mansoni and Schistosoma japonicum.

Schistosomiasis is a major parasitic disease caused by 3 principal species of schistosome. Studies of schistosome transcriptomes have focused on protein-coding transcripts and although miRNAs are attracting increased attention, few reports have concerned the long noncoding RNAs (lncRNAs). These have been shown to play key roles in the regulation of gene expression through interactions with mRNAs, proteins and miRNAs. In this study, we first identified lncRNAs from RNA-seq data in Schistosoma mansoni and Schistosoma japonicum: 3247 and 3033 potential lncRNAs were found in these two species respectively. ChIP-seq analysis to determine H3K4me3 profiles along the gene regions corresponding to lncRNAs showed that in 12% of cases this mark was enriched in regions proximal to the transcription start sites, supporting their validity as actively transcribed genes. Besides, the sequence conservation of lncRNAs between schistosome species was much lower than that of mRNAs, but higher than that of the randomly selected genomic sequences, which is consistent with that in mammals. Our results demonstrate that lncRNAs form a significant part of the schistosome transcriptome and suggest that they play an important role in the biology of the parasite.

Small but large enough: structural properties of armless mitochondrial tRNAs from the nematode Romanomermis culicivorax.

As adapter molecules to convert the nucleic acid information into the amino acid sequence, tRNAs play a central role in protein synthesis. To fulfill this function in a reliable way, tRNAs exhibit highly conserved structural features common in all organisms and in all cellular compartments active in translation. However, in mitochondria of metazoans, certain dramatic deviations from the consensus tRNA structure are described, where some tRNAs lack the D- or T-arm without losing their function. In Enoplea, this miniaturization comes to an extreme, and functional mitochondrial tRNAs can lack both arms, leading to a considerable size reduction. Here, we investigate the secondary and tertiary structure of two such armless tRNAs from Romanomermis culicivorax. Despite their high AU content, the transcripts fold into a single and surprisingly stable hairpin structure, deviating from standard tRNAs. The three-dimensional form is boomerang-like and diverges from the standard L-shape. These results indicate that such unconventional miniaturized tRNAs can still fold into a tRNA-like shape, although their length and secondary structure are very unusual. They highlight the remarkable flexibility of the protein synthesis apparatus and suggest that the translational machinery of Enoplea mitochondria may show compensatory adaptations to accommodate these armless tRNAs for efficient translation.

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.

Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition.

RNA-binding proteins regulate all aspects of RNA metabolism. Their association with RNA is mediated by RNA-binding domains, of which many remain uncharacterized. A recently reported example is the NHL domain, found in prominent regulators of cellular plasticity like the C. elegans LIN-41. Here we employ an integrative approach to dissect the RNA specificity of LIN-41. Using computational analysis, structural biology, and in vivo studies in worms and human cells, we find that a positively charged pocket, specific to the NHL domain of LIN-41 and its homologs (collectively LIN41), recognizes a stem-loop RNA element, whose shape determines the binding specificity. Surprisingly, the mechanism of RNA recognition by LIN41 is drastically different from that of its more distant relative, the fly Brat. Our phylogenetic analysis suggests that this reflects a rapid evolution of the domain, presenting an interesting example of a conserved protein fold that acquired completely different solutions to RNA recognition.

Quantitative RNA-seq meta-analysis of alternative exon usage in C. elegans.

Almost 20 years after the completion of the C. elegans genome sequence, gene structure annotation is still an ongoing process with new evidence for gene variants still being regularly uncovered by additional in-depth transcriptome studies. While alternative splice forms can allow a single gene to encode several functional isoforms, the question of how much spurious splicing is tolerated is still heavily debated. Here we gathered a compendium of 1682 publicly available C. elegans RNA-seq data sets to increase the dynamic range of detection of RNA isoforms, and obtained robust measurements of the relative abundance of each splicing event. While most of the splicing reads come from reproducibly detected splicing events, a large fraction of purported junctions is only supported by a very low number of reads. We devised an automated curation method that takes into account the expression level of each gene to discriminate robust splicing events from potential biological noise. We found that rarely used splice sites disproportionately come from highly expressed genes and are significantly less conserved in other nematode genomes than splice sites with a higher usage frequency. Our increased detection power confirmed trans-splicing for at least 84% of C. elegans protein coding genes. The genes for which trans-splicing was not observed are overwhelmingly low expression genes, suggesting that the mechanism is pervasive but not fully captured by organism-wide RNA-seq. We generated annotated gene models including quantitative exon usage information for the entire C. elegans genome. This allows users to visualize at a glance the relative expression of each isoform for their gene of interest.

Molecular Cloning and Characterization of Ribosomal Protein RPS9 in Echinococcus granulosus.

Ribosomal protein S9 (RPS9) is an essential functional gene that participates in DNA repair and developmental regulations. A sequence homolog of RPS9 has been found to be upregulated in the protoscoleces (PSCs) of Echinococcus granulosus treated with artemisinin. However, E. granulosus RPS9 (EgRPS9) has not been identified before. In the present study, the 657-base pair (bp) cDNA encoding EgRPS9 was cloned. Amino acid sequence analysis showed that EgRPS9 was similar to the RSP9 proteins from Schistosoma japonicum (SjRPS9, 86%) and Schistosoma mansoni (SmRPS9, 79%). Phylogenetic tree analysis showed that EgRPS9, SmRPS9, and SjRPS9 were clustered together. We detected the EgRPS9 gene and protein expression in PSCs exposed to artesunate (AS) which displayed a dose-dependent reduction in PSC viability for 24 hr. The results showed that the EgRPS9 ratio of the 10-µM AS-treated ( P < 0.01) and 40-µM AS-treated ( P < 0.05) groups were increased from that of the control group. In addition, the level of reactive oxygen species (ROS) in the AS-treated groups increased in a dose-dependent manner compared to the level in the control group. In conclusion, the expression of EgRPS9 could be induced by ROS and might participate in the oxidative damage-based anti-parasite mechanism of AS treatment.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project.

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Genome-wide transcriptomics of aging in the rotifer Brachionus manjavacas, an emerging model system.

BACKGROUND: Understanding gene expression changes over lifespan in diverse animal species will lead to insights to conserved processes in the biology of aging and allow development of interventions to improve health. Rotifers are small aquatic invertebrates that have been used in aging studies for nearly 100 years and are now re-emerging as a modern model system. To provide a baseline to evaluate genetic responses to interventions that change health throughout lifespan and a framework for new hypotheses about the molecular genetic mechanisms of aging, we examined the transcriptome of an asexual female lineage of the rotifer Brachionus manjavacas at five life stages: eggs, neonates, and early-, late-, and post-reproductive adults. RESULTS: There are widespread shifts in gene expression over the lifespan of B. manjavacas; the largest change occurs between neonates and early reproductive adults and is characterized by down-regulation of developmental genes and up-regulation of genes involved in reproduction. The expression profile of post-reproductive adults was distinct from that of other life stages. While few genes were significantly differentially expressed in the late- to post-reproductive transition, gene set enrichment analysis revealed multiple down-regulated pathways in metabolism, maintenance and repair, and proteostasis, united by genes involved in mitochondrial function and oxidative phosphorylation. CONCLUSIONS: This study provides the first examination of changes in gene expression over lifespan in rotifers. We detected differential expression of many genes with human orthologs that are absent in Drosophila and C. elegans, highlighting the potential of the rotifer model in aging studies. Our findings suggest that small but coordinated changes in expression of many genes in pathways that integrate diverse functions drive the aging process. The observation of simultaneous declines in expression of genes in multiple pathways may have consequences for health and longevity not detected by single- or multi-gene knockdown in otherwise healthy animals. Investigation of subtle but genome-wide change in these pathways during aging is an important area for future study.

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.

Stage-Wise Identification and Analysis of miRNA from Root-Knot Nematode Meloidogyne incognita.

In this study, we investigated global changes in miRNAs of Meloidogyne incognita throughout its life cycle. Small RNA sequencing resulted in approximately 62, 38, 38, 35, and 39 Mb reads in the egg, J2, J3, J4, and female stages, respectively. Overall, we identified 2724 known and 383 novel miRNAs (read count > 10) from all stages, of which 169 known and 13 novel miRNA were common to all the five stages. Among the stage-specific miRNAs, miR-286 was highly expressed in eggs, miR-2401 in J2, miR-8 and miR-187 in J3, miR-6736 in J4, and miR-17 in the female stages. These miRNAs are reported to be involved in embryo and neural development, muscular function, and control of apoptosis. Cluster analysis indicated the presence of 91 miRNA clusters, of which 36 clusters were novel and identified in this study. Comparison of miRNA families with other nematodes showed 17 families to be commonly absent in animal parasitic nematodes and M. incognita. Validation of 43 predicted common and stage-specific miRNA by quantitative PCR (qPCR) indicated their expression in the nematode. Stage-wise exploration of M. incognita miRNAs has not been carried out before and this work presents information on common and stage-specific miRNAs of the root-knot nematode.