Spliced Leader Trans-splicing Research Articles

A nematode-specific ribonucleoprotein complex mediates interactions between the major nematode spliced leader snRNP and its target pre-mRNAs.

Spliced leader trans-splicing of pre-mRNAs is a critical step in the gene expression of many eukaryotes. How the spliced leader RNA and its target transcripts are brought together to form the trans-spliceosome remains an important unanswered question. Using immunoprecipitation followed by protein analysis via mass spectrometry and RIP-Seq, we show that the nematode-specific proteins, SNA-3 and SUT-1, form a complex with a set of enigmatic non-coding RNAs, the SmY RNAs. Our work redefines the SmY snRNP and shows for the first time that it is essential for nematode viability and is involved in spliced leader trans-splicing. SNA-3 and SUT-1 are associated with the 5' ends of most, if not all, nascent capped RNA polymerase II transcripts, and they also interact with components of the major nematode spliced leader (SL1) snRNP. We show that depletion of SNA-3 impairs the co-immunoprecipitation between one of the SL1 snRNP components, SNA-2, and several core spliceosomal proteins. We thus propose that the SmY snRNP recruits the SL1 snRNP to the 5' ends of nascent pre-mRNAs, an instrumental step in the assembly of the trans-spliceosome.

Trans-splicing in the cestode Hymenolepis microstoma is constitutive across the life cycle and depends on gene structure and composition

Spliced leader (SL) trans-splicing is a key process during mRNA maturation of many eukaryotes, in which a short sequence (SL) is transferred from a precursor SL-RNA into the 5′ region of an immature mRNA. This mechanism is present in flatworms, in which it is known to participate in the resolution of polycistronic transcripts. However, most trans-spliced transcripts are not part of operons, and it is not clear if this process may participate in additional regulatory mechanisms in this group. In this work, we present a comprehensive analysis of SL trans-splicing in the model cestode Hymenolepis microstoma. We identified four different SL-RNAs which are indiscriminately trans-spliced to 622 gene models. SL trans-splicing is enriched in constitutively expressed genes and does not appear to be regulated throughout the life cycle. Operons represented at least 20% of all detected trans-spliced gene models, showed conservation to those of the cestode Echinococcus multilocularis, and included complex loci such as an alternative operon (processed as either a single gene through cis-splicing or as two genes of a polycistron). Most insertion sites were identified in the 5′ untranslated region (UTR) of monocistronic genes. These genes frequently contained introns in the 5′ UTR, in which trans-splicing used the same acceptor sites as cis-splicing. These results suggest that, unlike other eukaryotes, trans-splicing is associated with internal intronic promoters in the 5′ UTR, resulting in transcripts with strong splicing acceptor sites without competing cis-donor sites, pointing towards a simple mechanism driving the evolution of novel SL insertion sites.

A diversified and segregated mRNA spliced-leader system in the parasitic Perkinsozoa.

Spliced-leader trans-splicing (SLTS) has been described in distantly related eukaryotes and acts to mark mRNAs with a short 5' exon, giving different mRNAs identical 5' sequence-signatures. The function of these systems is obscure. Perkinsozoa encompasses a diversity of parasitic protists that infect bivalves, toxic-tide dinoflagellates, fish and frog tadpoles. Here, we report considerable sequence variation in the SLTS-system across the Perkinsozoa and find that multiple variant SLTS-systems are encoded in parallel in the ecologically important Perkinsozoa parasite Parvilucifera sinerae. These results demonstrate that the transcriptome of P. sinerae is segregated based on the addition of different spliced-leader (SL) exons. This segregation marks different gene categories, suggesting that SL-segregation relates to functional differentiation of the transcriptome. By contrast, both sets of gene categories are present in the single SL-transcript type sampled from Maranthos, implying that the SL-segregation of the Parvilucifera transcriptome is a recent evolutionary innovation. Furthermore, we show that the SLTS-system marks a subsection of the transcriptome with increased mRNA abundance and includes genes that encode the spliceosome system necessary for SLTS-function. Collectively, these data provide a picture of how the SLTS-systems can vary within a major evolutionary group and identify how additional transcriptional-complexity can be achieved through SL-segregation.

A novel, essential trans-splicing protein connects the nematode SL1 snRNP to the CBC-ARS2 complex.

Spliced leader trans-splicing is essential for gene expression in many eukaryotes. To elucidate the molecular mechanism of this process, we characterise the molecules associated with the Caenorhabditis elegans major spliced leader snRNP (SL1 snRNP), which donates the spliced leader that replaces the 5′ untranslated region of most pre-mRNAs. Using a GFP-tagged version of the SL1 snRNP protein SNA-1 created by CRISPR-mediated genome engineering, we immunoprecipitate and identify RNAs and protein components by RIP-Seq and mass spectrometry. This reveals the composition of the SL1 snRNP and identifies associations with spliceosome components PRP-8 and PRP-19. Significantly, we identify a novel, nematode-specific protein required for SL1 trans-splicing, which we designate SNA-3. SNA-3 is an essential, nuclear protein with three NADAR domains whose function is unknown. Mutation of key residues in NADAR domains inactivates the protein, indicating that domain function is required for activity. SNA-3 interacts with the CBC-ARS2 complex and other factors involved in RNA metabolism, including SUT-1 protein, through RNA or protein-mediated contacts revealed by yeast two-hybrid assays, localisation studies and immunoprecipitations. Our data are compatible with a role for SNA-3 in coordinating trans-splicing with target pre-mRNA transcription or in the processing of the Y-branch product of the trans-splicing reaction.

Hypothesis: Trans-splicing Generates Evolutionary Novelty in the Photosynthetic Amoeba Paulinella.

Plastid primary endosymbiosis has occurred twice, once in the Archaeplastida ancestor and once in the Paulinella (Rhizaria) lineage. Both events precipitated massive evolutionary changes, including the recruitment and activation of genes that are horizontally acquired (HGT) and the redeployment of existing genes and pathways in novel contexts. Here we address the latter aspect in Paulinella micropora KR01 (hereafter, KR01) that has independently evolved spliced leader (SL) trans‐splicing (SLTS) of nuclear–derived transcripts. We investigated the role of this process in gene regulation, novel gene origination, and endosymbiont integration. Our analysis shows that 20% of KR01 genes give rise to transcripts with at least one (but in some cases, multiple) sites of SL addition. This process, which often occurs at canonical cis‐splicing acceptor sites (internal introns), results in shorter transcripts that may produce 5′‐truncated proteins with novel functions. SL–truncated transcripts fall into four categories that may show: (i) altered protein localization, (ii) altered protein function, structure, or regulation, (iii) loss of valid alternative start codons, preventing translation, or (iv) multiple SL addition sites at the 5′‐terminus. The SL RNA genes required for SLTS are putatively absent in the heterotrophic sister lineage of photosynthetic Paulinella species. Moreover, a high proportion of transcripts derived from genes of endosymbiotic gene transfer (EGT) and HGT origin contain SL sequences. We hypothesize that truncation of transcripts by SL addition may facilitate the generation and expression of novel gene variants and that SLTS may have enhanced the activation and fixation of foreign genes in the host genome of the photosynthetic lineages, playing a key role in primary endosymbiont integration.

The SNAPc complex mediates starvation-induced trans-splicing in Caenorhabditis elegans

Dietary restriction usually suppresses biosynthesis but activates catabolic pathways in animals. However, the short-term starvation enhances biosynthetic activities and promotes ribosomal biogenesis in adult Caenorhabditis elegans. The mechanism underlying the processes remains largely unknown. Here, we find that the short-term starvation enhances the SL1 trans-splicing of translation-related genes in adult C. elegans by transcriptome analysis. The small nuclear RNA-activating protein complex (SNAPc) promotes SL RNA production and mediates starvation-induced trans-splicing. TOFU-5, a core factor in the upstream sequence transcription complex (USTC) essential for piRNA production, is also involved in the starvation-induced trans-splicing processes. Knocking down components of the SNAPc complex and tofu-5 extends worm survival under starvation conditions. Taken together, our study highlights the importance of SL trans-splicing in the nutrition response and reveals a mechanism of the survival regulation by food deprivation via SNAPc and TOFU-5.

Monodopsis and Vischeria Genomes Shed New Light on the Biology of Eustigmatophyte Algae.

Members of eustigmatophyte algae, especially Nannochloropsis and Microchloropsis, have been tapped for biofuel production owing to their exceptionally high lipid content. Although extensive genomic, transcriptomic, and synthetic biology toolkits have been made available for Nannochloropsis and Microchloropsis, very little is known about other eustigmatophytes. Here we present three near-chromosomal and gapless genome assemblies of Monodopsis strains C73 and C141 (60 Mb) and Vischeria strain C74 (106 Mb), which are the sister groups to Nannochloropsis and Microchloropsis in the order Eustigmatales. These genomes contain unusually high percentages of simple repeats, ranging from 12% to 21% of the total assembly size. Unlike Nannochloropsis and Microchloropsis, long interspersed nuclear element repeats are abundant in Monodopsis and Vischeria and might constitute the centromeric regions. We found that both mevalonate and nonmevalonate pathways for terpenoid biosynthesis are present in Monodopsis and Vischeria, which is different from Nannochloropsis and Microchloropsis that have only the latter. Our analysis further revealed extensive spliced leader trans-splicing in Monodopsis and Vischeria at 36–61% of genes. Altogether, the high-quality genomes of Monodopsis and Vischeria not only serve as the much-needed outgroups to advance Nannochloropsis and Microchloropsis research, but also shed new light on the biology and evolution of eustigmatophyte algae.

SLIDR and SLOPPR: flexible identification of spliced leader trans-splicing and prediction of eukaryotic operons from RNA-Seq data

BackgroundSpliced leader (SL) trans-splicing replaces the 5′ end of pre-mRNAs with the spliced leader, an exon derived from a specialised non-coding RNA originating from elsewhere in the genome. This process is essential for resolving polycistronic pre-mRNAs produced by eukaryotic operons into monocistronic transcripts. SL trans-splicing and operons may have independently evolved multiple times throughout Eukarya, yet our understanding of these phenomena is limited to only a few well-characterised organisms, most notably C. elegans and trypanosomes. The primary barrier to systematic discovery and characterisation of SL trans-splicing and operons is the lack of computational tools for exploiting the surge of transcriptomic and genomic resources for a wide range of eukaryotes.ResultsHere we present two novel pipelines that automate the discovery of SLs and the prediction of operons in eukaryotic genomes from RNA-Seq data. SLIDR assembles putative SLs from 5′ read tails present after read alignment to a reference genome or transcriptome, which are then verified by interrogating corresponding SL RNA genes for sequence motifs expected in bona fide SL RNA molecules. SLOPPR identifies RNA-Seq reads that contain a given 5′ SL sequence, quantifies genome-wide SL trans-splicing events and predicts operons via distinct patterns of SL trans-splicing events across adjacent genes. We tested both pipelines with organisms known to carry out SL trans-splicing and organise their genes into operons, and demonstrate that (1) SLIDR correctly detects expected SLs and often discovers novel SL variants; (2) SLOPPR correctly identifies functionally specialised SLs, correctly predicts known operons and detects plausible novel operons.ConclusionsSLIDR and SLOPPR are flexible tools that will accelerate research into the evolutionary dynamics of SL trans-splicing and operons throughout Eukarya and improve gene discovery and annotation for a wide range of eukaryotic genomes. Both pipelines are implemented in Bash and R and are built upon readily available software commonly installed on most bioinformatics servers. Biological insight can be gleaned even from sparse, low-coverage datasets, implying that an untapped wealth of information can be retrieved from existing RNA-Seq datasets as well as from novel full-isoform sequencing protocols as they become more widely available.

Computational analysis of spliced leader trans-splicing in the regenerative flatworm Macrostomum lignano reveals its prevalence in conserved and stem cell related genes.

In eukaryotes, trans-splicing is a process of nuclear pre-mRNA maturation where two different RNA molecules are joined together by the spliceosomal machinery utilizing mechanisms similar to cis-splicing. In diverse taxa of lower eukaryotes, spliced leader (SL) trans-splicing is the most frequent type of trans-splicing, when the same sequence derived from short small nuclear RNA molecules, called SL RNAs, is attached to the 5’ ends of different non-processed pre-mRNAs. One of the functions of SL trans-splicing is processing polycistronic pre-mRNA molecules transcribed from operons, when several genes are transcribed as one pre-mRNA molecule. However, only a fraction of trans-spliced genes reside in operons, suggesting that SL trans-splicing must also have some other, less understood functions. Regenerative flatworms are informative model organisms which hold the keys to understand the mechanism of stem cell regulation and specialization during regeneration and homeostasis. Their ability to regenerate is fueled by the division and differentiation of the adult somatic stem cell population called neoblasts. Macrostomum lignano is a flatworm model organism where substantial technological advances have been achieved in recent years, including the development of transgenesis. Although a large fraction of genes in M. lignano were estimated to be SL trans-spliced, SL trans-splicing was not studied in detail in M. lignano before. Here, we performed the first comprehensive study of SL trans-splicing in M. lignano. By reanalyzing the existing genome and transcriptome data of M. lignano, we estimate that 30 % of its genes are SL trans-spliced, 15 % are organized in operons, and almost 40 % are both SL trans-spliced and in operons. We annotated and characterized the sequence of SL RNA and characterized conserved cis- and SL transsplicing motifs. Finally, we found that a majority of SL trans-spliced genes are evolutionarily conserved and significantly over-represented in neoblast-specific genes. Our findings suggest an important role of SL trans-splicing in the regulation and maintenance of neoblasts in M. lignano.

RNA polymerase II CTD S2P is dispensable for embryogenesis but mediates exit from developmental diapause in C. elegans

Serine 2 phosphorylation (S2P) within the CTD of RNA polymerase II is considered a Cdk9/Cdk12-dependent mark required for 3'-end processing. However, the relevance of CTD S2P in metazoan development is unknown. We show that cdk-12 lesions or a full-length CTD S2A substitution results in an identical phenotype in Caenorhabditis elegans Embryogenesis occurs in the complete absence of S2P, but the hatched larvae arrest development, mimicking the diapause induced when hatching occurs in the absence of food. Genome-wide analyses indicate that when CTD S2P is inhibited, only a subset of growth-related genes is not properly expressed. These genes correspond to SL2 trans-spliced mRNAs located in position 2 and over within operons. We show that CDK-12 is required for maximal occupancy of cleavage stimulatory factor necessary for SL2 trans-splicing. We propose that CTD S2P functions as a gene-specific signaling mark ensuring the nutritional control of the C. elegans developmental program.

Evaluation of various RNA-seq approaches for identification of gene outrons in the flatworm Opisthorchis felineus.

The parasitic flatworm Opisthorchis felineus is one of the causative agents of opisthorchiasis in humans. Recently, we assembled the O. felineus genome, but the correct genome annotation by means of standard methods was hampered by the presence of spliced leader trans-splicing (SLTS). As a result of SLTS, the original 5’-end (outron) of the transcripts is replaced by a short spliced leader sequence donated from a specialized SL RNA. SLTS is involved in the RNA processing of more than half of O. felineus genes, making it hard to determine the structure of outrons and bona fide transcription start sites of the corresponding genes and operons, being based solely on mRNA-seq data. In the current study, we tested various experimental approaches for identifying the sequences of outrons in O. felineus using massive parallel sequencing. Two of them were developed by us for targeted sequencing of already processed branched outrons. One was based on sequence-specific reverse transcription from the SL intron toward the 5’-end of the Y-branched outron. The other used outron hybridization with an immobilized single-stranded DNA probe complementary to the SL intron. Additionally, two approaches to the sequencing of rRNA-depleted total RNA were used, allowing the identification of a wider range of transcripts compared to mRNAseq. One is based on the enzymatic elimination of overrepresented cDNAs, the other utilizes exonucleolytic degradation of uncapped RNA by Terminator enzyme. By using the outron-targeting methods, we were not able to obtain the enrichment of RNA preparations by processed outrons, which is most likely indicative of a rapid turnover of these trans-splicing intermediate products. Of the two rRNA depletion methods, a method based on the enzymatic normalization of cDNA (Zymo-Seq RiboFree) showed high efficiency. Compared to mRNA-seq, it provides an approximately twofold increase in the fraction of reads originating from outrons and introns. The results suggest that unprocessed nascent transcripts are the main source of outron sequences in the RNA pool of O. felineus.

Complete representation of a tapeworm genome reveals chromosomes capped by centromeres, necessitating a dual role in segregation and protection

BackgroundChromosome-level assemblies are indispensable for accurate gene prediction, synteny assessment, and understanding higher-order genome architecture. Reference and draft genomes of key helminth species have been published, but little is yet known about the biology of their chromosomes. Here, we present the complete genome of the tapeworm Hymenolepis microstoma, providing a reference quality, end-to-end assembly that represents the first fully assembled genome of a spiralian/lophotrochozoan, revealing new insights into chromosome evolution.ResultsLong-read sequencing and optical mapping data were added to previous short-read data enabling complete re-assembly into six chromosomes, consistent with karyology. Small genome size (169 Mb) and lack of haploid variation (1 SNP/3.2 Mb) contributed to exceptionally high contiguity with only 85 gaps remaining in regions of low complexity sequence. Resolution of repeat regions reveals novel gene expansions, micro-exon genes, and spliced leader trans-splicing, and illuminates the landscape of transposable elements, explaining observed length differences in sister chromatids. Syntenic comparison with other parasitic flatworms shows conserved ancestral linkage groups indicating that the H. microstoma karyotype evolved through fusion events. Strikingly, the assembly reveals that the chromosomes terminate in centromeric arrays, indicating that these motifs play a role not only in segregation, but also in protecting the linear integrity and full lengths of chromosomes.ConclusionsDespite strong conservation of canonical telomeres, our results show that they can be substituted by more complex, species-specific sequences, as represented by centromeres. The assembly provides a robust platform for investigations that require complete genome representation.

Resolution of polycistronic RNA by SL2 trans-splicing is a widely conserved nematode trait.

Spliced leader trans-splicing is essential for the processing and translation of polycistronic RNAs generated by eukaryotic operons. In C. elegans, a specialized spliced leader, SL2, provides the 5′ end for uncapped pre-mRNAs derived from polycistronic RNAs. Studies of other nematodes suggested that SL2-type trans-splicing is a relatively recent innovation, confined to Rhabditina, the clade containing C. elegans and its close relatives. Here we conduct a survey of transcriptome-wide spliced leader trans-splicing in Trichinella spiralis, a distant relative of C. elegans with a particularly diverse repertoire of 15 spliced leaders. By systematically comparing the genomic context of trans-splicing events for each spliced leader, we identified a subset of T. spiralis spliced leaders that are specifically used to process polycistronic RNAs—the first examples of SL2-type spliced leaders outside of Rhabditina. These T. spiralis spliced leader RNAs possess a perfectly conserved stem–loop motif previously shown to be essential for SL2-type trans-splicing in C. elegans. We show that genes trans-spliced to these SL2-type spliced leaders are organized in operonic fashion, with short intercistronic distances. A subset of T. spiralis operons show conservation of synteny with C. elegans operons. Our work substantially revises our understanding of nematode spliced leader trans-splicing, showing that SL2 trans-splicing is a major mechanism for nematode polycistronic RNA processing, which may have evolved prior to the radiation of the Nematoda. This work has important implications for the improvement of genome annotation pipelines in nematodes and other eukaryotes with operonic gene organization.

SLFinder, a pipeline for the novel identification of splice-leader sequences: a\xa0good enough solution for a complex problem

BackgroundSpliced Leader trans-splicing is an important mechanism for the maturation of mRNAs in several lineages of eukaryotes, including several groups of parasites of great medical and economic importance. Nevertheless, its study across the tree of life is severely hindered by the problem of identifying the SL sequences that are being trans-spliced.ResultsIn this paper we present SLFinder, a four-step pipeline meant to identify de novo candidate SL sequences making very few assumptions regarding the SL sequence properties. The pipeline takes transcriptomic de novo assemblies and a reference genome as input and allows the user intervention on several points to account for unexpected features of the dataset. The strategy and its implementation were tested on real RNAseq data from species with and without SL Trans-Splicing.ConclusionsSLFinder is capable to identify SL candidates with good precision in a reasonable amount of time. It is especially suitable for species with unknown SL sequences, generating candidate sequences for further refining and experimental validation.

Rapid block of pre-mRNA splicing by chemical inhibition of analog-sensitive CRK9 in Trypanosoma brucei.

Trypanosoma brucei CRK9 is an essential cyclin-dependent kinase for the parasite-specific mode of pre-mRNA processing. In trypanosomes, protein coding genes are arranged in directional arrays that are transcribed polycistronically, and individual mRNAs are generated by spliced leader trans-splicing and polyadenylation, processes that are functionally linked. Since CRK9 silencing caused a decline of mRNAs, a concomitant increase of unspliced pre-mRNAs and the disappearance of the trans-splicing Y structure intermediate, CRK9 is essential for the first step of splicing. CRK9 depletion also caused a loss of phosphorylation in RPB1, the largest subunit of RNA polymerase (pol) II. Here, we established cell lines that exclusively express analog-sensitive CRK9 (CRK9AS ). Inhibition of CRK9AS in these cells by the ATP-competitive inhibitor 1-NM-PP1 reproduced the splicing defects and proved that it is the CKR9 kinase activity that is required for pre-mRNA processing. Since defective trans-splicing was detected as early as 5min after inhibitor addition, CRK9 presumably carries out reversible phosphorylation on the pre-mRNA processing machinery. Loss of RPB1 phosphorylation, however, took 12-24hr. Surprisingly, RNA pol II-mediated RNA synthesis in 24hr-treated cells was upregulated, indicating that, in contrast to other eukaryotes, RPB1 phosphorylation is not a prerequisite for transcription in trypanosomes.

Trans-splicing of mRNAs links gene transcription to translational control regulated by mTOR

BackgroundIn phylogenetically diverse organisms, the 5′ ends of a subset of mRNAs are trans-spliced with a spliced leader (SL) RNA. The functions of SL trans-splicing, however, remain largely enigmatic.ResultsWe quantified translation genome-wide in the marine chordate, Oikopleura dioica, under inhibition of mTOR, a central growth regulator. Translation of trans-spliced TOP mRNAs was suppressed, consistent with a role of the SL sequence in nutrient-dependent translational control of growth-related mRNAs. Under crowded, nutrient-limiting conditions, O. dioica continued to filter-feed, but arrested growth until favorable conditions returned. Upon release from unfavorable conditions, initial recovery was independent of nutrient-responsive, trans-spliced genes, suggesting animal density sensing as a first trigger for resumption of development.ConclusionOur results are consistent with a proposed role of trans-splicing in the coordinated translational down-regulation of nutrient-responsive genes under growth-limiting conditions.

On the Function of Trans-Splicing: No Evidence for Widespread Proteome Diversification in Trypanosomes.

A long-standing mystery of genomic/transcriptomic structure involves spliced leader trans-splicing (SLTS), in which short RNA “tags” transcribed from a distinct genomic locus is added near the 5′ end of RNA transcripts by the spliceosome. SLTS has been observed in diverse eukaryotes in a phylogenetic pattern implying recurrent independent evolution. This striking convergence suggests important functions for SLTS, however no general novel function is known. Recent findings of frequent alternative SLTS (ALT-TS) suggest that ALT-TS could impart widespread functionality. Here, we tested the hypothesis that ALT-TS diversifies proteomes by comparing splicing patterns in orthologous genes between two deeply diverged trypanosome parasites. We also tested proteome diversification functions of ALT-TS by utilizing ribosome profiling sequence data. Finally, we investigated ALT-TS as a mechanism to regulate the expression of unproductive transcripts. Although our results indicate the functional importance of some cases of trans-splicing, we find no evidence for the hypothesis that proteome diversification is a general function of trans-splicing.

A high-throughput screen for the identification of compounds that inhibit nematode gene expression by targeting spliced leader trans-splicing

Infections with parasitic nematodes are among the most significant of the neglected tropical diseases affecting about a billion people living mainly in tropical regions with low economic activity.The most effective current strategy to control nematode infections involves large scale treatment programs with anthelmintic drugs. This strategy is at risk from the emergence of drug resistant parasites. Parasitic nematodes also affect livestock, which are treated with the same limited group of anthelmintic drugs. Livestock parasites resistant to single drugs, and even multi-drug resistant parasites, are appearing in many areas. There is therefore a pressing need for new anthelmintic drugs.Here we use the nematode Caenorhabditis elegans as a model for parasitic nematodes and demonstrate that sinefungin, a competitive inhibitor of methyltransferases, causes a delay in development and reduced fecundity, and inhibits spliced leader trans-splicing. Spliced leader trans-splicing is an essential step in gene expression that does not occur in the hosts of parasitic nematodes, and is therefore a potential target for new anthelmintic drugs.We have exploited the ability of sinefungin to inhibit spliced leader trans-splicing to adapt a green fluorescent protein based reporter gene assay that monitors spliced leader trans-splicing for high-throughput screening for new anthelmintic compounds. We have established a protocol for robust high-throughput screening, combining mechanical dispensing of living C. elegans into 384- or 1536- well plates with addition of compounds using an acoustic liquid dispenser, and the detection of the inhibition of SL trans-splicing using a microplate reader. We have tested this protocol in a first pilot screen and envisage that this assay will be a valuable tool in the search for new anthelmintic drugs.

Heterodera glycines utilizes promiscuous spliced leaders and demonstrates a unique preference for a species-specific spliced leader over C. elegans SL1

Spliced leader trans-splicing (SLTS) plays a part in the maturation of pre-mRNAs in select species across multiple phyla but is particularly prevalent in Nematoda. The role of spliced leaders (SL) within the cell is unclear and an accurate assessment of SL occurrence within an organism is possible only after extensive sequencing data are available, which is not currently the case for many nematode species. SL discovery is further complicated by an absence of SL sequences from high-throughput sequencing results due to incomplete sequencing of the 5’-ends of transcripts during RNA-seq library preparation, known as 5′-bias. Existing datasets and novel methodology were used to identify both conserved SLs and unique hypervariable SLs within Heterodera glycines, the soybean cyst nematode. In H. glycines, twenty-one distinct SL sequences were found on 2,532 unique H. glycines transcripts. The SL sequences identified on the H. glycines transcripts demonstrated a high level of promiscuity, meaning that some transcripts produced as many as nine different individual SL-transcript combinations. Most uniquely, transcriptome analysis revealed that H. glycines is the first nematode to demonstrate a higher SL trans-splicing rate using a species-specific SL over well-conserved Caenorhabditis elegans SL-like sequences.

Characterization of spliced leader trans-splicing in a photosynthetic rhizarian amoeba, Paulinella micropora, and its possible role in functional gene transfer.

Paulinella micropora is a rhizarian thecate amoeba, belonging to a photosynthetic Paulinella species group that has a unique organelle termed chromatophore, whose cyanobacterial origin is distinct from that of plant and algal chloroplasts. Because acquisition of the chromatophore was quite a recent event compared with that of the chloroplast ancestor, the Paulinella species are thought to be model organisms for studying the early process of primary endosymbiosis. To obtain insight into how endosymbiotically transferred genes acquire expression competence in the host nucleus, here we analyzed the 5′ end sequences of the mRNAs of P. micropora MYN1 strain with the aid of a cap-trapper cDNA library. As a result, we found that mRNAs of 27 genes, including endosymbiotically transferred genes, possessed the common 5′ end sequence of 28–33 bases that were posttranscriptionally added by spliced leader (SL) trans-splicing. We also found two subtypes of SL RNA genes encoded by the P. micropora MYN1 genome. Differing from the other SL trans-splicing organisms that usually possess poly(A)-less SL RNAs, this amoeba has polyadenylated SL RNAs. In this study, we characterize the SL trans-splicing of this unique organism and discuss the putative merits of SL trans-splicing in functional gene transfer and genome evolution.