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.

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.

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.

High-throughput identification of miRNAs of Taenia ovis, a cestode threatening sheep industry.

Taenia ovis is a tapeworm that is mainly transmitted between dogs and sheep or goats and has an adverse effect on sheep industry. miRNAs are short regulatory non-coding RNAs, involved in parasite development and growth as well as parasite infection. The miRNA profile of T. ovis remains to be established. Herein, 33 known miRNAs belonging to 23 different families were identified in T. ovis metacestodes using deep sequencing approach. Of them, expression of some miRNAs such as tov-miR-10 and -let-7 was absolutely predominant. Moreover, comparative analysis revealed the presence of a miR-71/2b/2c cluster in T. ovis, which was also completely conserved in other 6 cestodes. The study provides rich data for further understandings of T. ovis biology.

Genes of the bovine lungworm Dictyocaulus viviparus associated with transition from pasture to parasitism.

Genes necessary to enable nematode parasitic life after free-living larval life are of substantial interest to understand parasitism. We investigated transcriptional changes during transition to parasitism in the bovine lungworm Dictyocaulus viviparus, one of the most important parasites in cattle farming due to substantial economic losses. Upregulated transcripts in either free-living, developmentally arrested L3 or parasitic immature L5 were identified by suppression subtractive hybridization (SSH) followed by differential screening and subsequent virtual Northern blot verification. From 400 sequenced clones of parasitic L5, 372 (93.0%) upregulated high quality ESTs were obtained clustering into 30 contigs and 38 singletons. Most conceptual translated peptides were SCP/TAPS "family" members also known as pathogenesis-related protein (PRP) superfamily (28.5% of total ESTs), cysteine proteases (24.5%), and H-gal-GP orthologues (9.9%). These proteins are predicted to play key roles in fundamental biological processes such as nutrition and development but also parasite-host interactions and immune defense mechanisms. Increased energy requirement of the rapidly developing L5 lungworm stage was obvious in a proportion of 12.2% upregulated ESTs being components of the respiratory chain. From the developmentally arrested L3 stage sequencing of 200 clones resulted in 195 high quality ESTs (97.0%) clustering into 7 contigs and 3 singletons only. Besides a hypothetical protein (70.1% of total ESTs) most transcripts encoded the cleavage stimulation factor subunit 2 (17.5%), which is a component of the poly(A(+)) machinery and found to be involved in gene silencing. Obtained data provide the basis for future fundamental research into genes associated with parasitic lifestyle but also applied research like vaccine and/or drug development.

mut-16 and other mutator class genes modulate 22G and 26G siRNA pathways in Caenorhabditis elegans.

Argonaute-associated siRNAs and Piwi-associated piRNAs have overlapping roles in silencing mobile genetic elements in animals. In Caenorhabditis elegans, mutator (mut) class genes mediate siRNA-guided repression of transposons as well as exogenous RNAi, but their roles in endogenous RNA silencing pathways are not well-understood. To characterize the endogenous small RNAs dependent on mut class genes, small RNA populations from a null allele of mut-16 as well as a regulatory mut-16(mg461) allele that disables only somatic RNAi were subjected to deep sequencing. Additionally, each of the mut class genes was tested for a requirement in 26G siRNA pathways. The results indicate that mut-16 is an essential factor in multiple endogenous germline and somatic siRNA pathways involving several distinct Argonautes and RNA-dependent RNA polymerases. The results also reveal essential roles for mut-2 and mut-7 in the ERGO-1 class 26G siRNA pathway and less critical roles for mut-8, mut-14, and mut-15. We show that transposons are hypersusceptible to mut-16-dependent silencing and identify a requirement for the siRNA machinery in piRNA biogenesis from Tc1 transposons. We also show that the soma-specific mut-16(mg461) mutant allele is present in multiple C. elegans laboratory strains.

Computational prediction of Caenorhabditis box H/ACA snoRNAs using genomic properties of their host genes.

Identification of small nucleolar RNAs (snoRNAs) in genomic sequences has been challenging due to the relative paucity of sequence features. Many current prediction algorithms rely on detection of snoRNA motifs complementary to target sites in snRNAs and rRNAs. However, recent discovery of snoRNAs without apparent targets requires development of alternative prediction methods. We present an approach that combines rule-based filters and a Bayesian Classifier to identify a class of snoRNAs (H/ACA) without requiring target sequence information. It takes advantage of unique attributes of their genomic organization and improved species-specific motif characterization to predict snoRNAs that may otherwise be difficult to discover. Searches in the genomes of Caenorhabditis elegans and the closely related Caenorhabditis briggsae suggest that our method performs well compared to recent benchmark algorithms. Our results illustrate the benefits of training gene discovery engines on features restricted to particular phylogenetic groups and the utility of incorporating diverse data types in gene prediction.

26G endo-siRNAs regulate spermatogenic and zygotic gene expression in Caenorhabditis elegans.

Endogenous small interfering RNAs (endo-siRNAs) regulate diverse gene expression programs in eukaryotes by either binding and cleaving mRNA targets or mediating heterochromatin formation; however, the mechanisms of endo-siRNA biogenesis, sorting, and target regulation remain poorly understood. Here we report the identification and function of a specific class of germline-generated endo-siRNAs in Caenorhabditis elegans that are 26 nt in length and contain a guanine at the first nucleotide position (i.e., 26G RNAs). 26G RNAs regulate gene expression during spermatogenesis and zygotic development, and their biogenesis requires the ERI-1 exonuclease and the RRF-3 RNA-dependent RNA polymerase (RdRP). Remarkably, we identified two nonoverlapping subclasses of 26G RNAs that sort into specific RNA-induced silencing complexes (RISCs) and differentially regulate distinct mRNA targets. Class I 26G RNAs target genes are expressed during spermatogenesis, whereas class II 26G RNAs are maternally inherited and silence gene expression during zygotic development. These findings implicate a class of endo-siRNAs in the global regulation of transcriptional programs required for fertility and development.

Evolution of small nucleolar RNAs in nematodes.

In contrast to mRNAs, which are templates for translating proteins, non-protein coding (npc) RNAs (also known as 'non-coding' RNA, ncRNA), exhibit various functions in different compartments and developmental stages of the cell. Small nucleolar RNAs (snoRNAs), one of the largest classes of npcRNAs, guide post-transcriptional modifications of other RNAs that are crucial for appropriate RNA folding as well as for RNA-RNA and RNA-protein interactions. Although snoRNA genes comprise a significant fraction of the eutherian genome, identifying and characterizing large numbers of them is not sufficiently accessible by classical computer searches alone. Furthermore, most previous investigations of snoRNAs yielded only limited indications of their evolution. Using data obtained by a combination of high-throughput cDNA library screening and computational search strategies based on a modified DNAMAN program, we characterized 151 npcRNAs, and in particular 121 snoRNAs, from Caenorhabditis elegans and extensively compared them with those in the related, Caenorhabditis briggsae. Detailed comparisons of paralog snoRNAs in the two nematodes revealed, in addition to trans-duplication, a novel, cis-duplication distribution strategy with insertions near to the original loci. Some snoRNAs coevolved with their modification target sites, demonstrating the close interaction of complementary regions. Some target sites modified by snoRNAs were changed, added or lost, documenting a high degree of evolutionary plasticity of npcRNAs.

Organization of the Caenorhabditis elegans small non-coding transcriptome: genomic features, biogenesis, and expression.

Recent evidence points to considerable transcription occurring in non-protein-coding regions of eukaryote genomes. However, their lack of conservation and demonstrated function have created controversy over whether these transcripts are functional. Applying a novel cloning strategy, we have cloned 100 novel and 61 known or predicted Caenorhabditis elegans full-length ncRNAs. Studying the genomic environment and transcriptional characteristics have shown that two-thirds of all ncRNAs, including many intronic snoRNAs, are independently transcribed under the control of ncRNA-specific upstream promoter elements. Furthermore, the transcription levels of at least 60% of the ncRNAs vary with developmental stages. We identified two new classes of ncRNAs, stem-bulge RNAs (sbRNAs) and snRNA-like RNAs (snlRNAs), both featuring distinct internal motifs, secondary structures, upstream elements, and high and developmentally variable expression. Most of the novel ncRNAs are conserved in Caenorhabditis briggsae, but only one homolog was found outside the nematodes. Preliminary estimates indicate that the C. elegans transcriptome contains approximately 2700 small non-coding RNAs, potentially acting as regulatory elements in nematode development.

RSEARCH: finding homologs of single structured RNA sequences.

BACKGROUND: For many RNA molecules, secondary structure rather than primary sequence is the evolutionarily conserved feature. No programs have yet been published that allow searching a sequence database for homologs of a single RNA molecule on the basis of secondary structure. RESULTS: We have developed a program, RSEARCH, that takes a single RNA sequence with its secondary structure and utilizes a local alignment algorithm to search a database for homologous RNAs. For this purpose, we have developed a series of base pair and single nucleotide substitution matrices for RNA sequences called RIBOSUM matrices. RSEARCH reports the statistical confidence for each hit as well as the structural alignment of the hit. We show several examples in which RSEARCH outperforms the primary sequence search programs BLAST and SSEARCH. The primary drawback of the program is that it is slow. The C code for RSEARCH is freely available from our lab's website. CONCLUSION: RSEARCH outperforms primary sequence programs in finding homologs of structured RNA sequences.

Analysis and functional classification of transcripts from the nematode Meloidogyne incognita.

BACKGROUND: Plant parasitic nematodes are major pathogens of most crops. Molecular characterization of these species as well as the development of new techniques for control can benefit from genomic approaches. As an entrée to characterizing plant parasitic nematode genomes, we analyzed 5,700 expressed sequence tags (ESTs) from second-stage larvae (L2) of the root-knot nematode Meloidogyne incognita. RESULTS: From these, 1,625 EST clusters were formed and classified by function using the Gene Ontology (GO) hierarchy and the Kyoto KEGG database. L2 larvae, which represent the infective stage of the life cycle before plant invasion, express a diverse array of ligand-binding proteins and abundant cytoskeletal proteins. L2 are structurally similar to Caenorhabditis elegans dauer larva and the presence of transcripts encoding glyoxylate pathway enzymes in the M. incognita clusters suggests that root-knot nematode larvae metabolize lipid stores while in search of a host. Homology to other species was observed in 79% of translated cluster sequences, with the C. elegans genome providing more information than any other source. In addition to identifying putative nematode-specific and Tylenchida-specific genes, sequencing revealed previously uncharacterized horizontal gene transfer candidates in Meloidogyne with high identity to rhizobacterial genes including homologs of nodL acetyltransferase and novel cellulases. CONCLUSIONS: With sequencing from plant parasitic nematodes accelerating, the approaches to transcript characterization described here can be applied to more extensive datasets and also provide a foundation for more complex genome analyses.

[The molecular genetic typification of planarians in the genus Bdellocephala (Dendrocoelidae, Tricladida, Turbellaria) from Lake Baikal with an assessment of their species diversity]. / Molekuliarno-geneticheskaia tipizatsiia planarii roda Bdellocephala (Dendrocoelidae, Tricladida, Turbellaria) ozera Baikal s otsenkoi vidovogo raznoobraziia.

Baikal planaria from genus Bdellocephala were typified using rDNA locus coding 5'--end domain of 18S ribosome RNA. Five colour forms of 24 possible variants that differ in diapason 0-1.3% of genotype were determined by comparative analysis of nucleotide sequences. The authors use back colour--one of the most variable and typical character in the given group--to collect material for investigation. It allows to minimize the size of investigation sample and at the same time to cover maximum variability of Bdellocephala. One of the positive result of molecular typification of colour forms was a discovery of unique individuals that belong to new species. Karyological analysis of colour forms shows variations in chromosome numbers that divide planaria into 3 groups (2n = 20, 24, 26). Comparative analysis of morphological and ecological characters and karyotypes of some forms united by the same genotype allows to distinguish them as separate species. Criteria of modern phenetic system of Baikal planaria are discussed.