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.

CMTr mediated 2'-O-ribose methylation status of cap-adjacent nucleotides across animals.

Cap methyltransferases (CMTrs) O methylate the 2' position of the ribose (cOMe) of cap-adjacent nucleotides of animal, protist, and viral mRNAs. Animals generally have two CMTrs, whereas trypanosomes have three, and many viruses encode one in their genome. In the splice leader of mRNAs in trypanosomes, the first four nucleotides contain cOMe, but little is known about the status of cOMe in animals. Here, we show that cOMe is prominently present on the first two cap-adjacent nucleotides with species- and tissue-specific variations in Caenorhabditis elegans, honeybees, zebrafish, mouse, and human cell lines. In contrast, Drosophila contains cOMe primarily on the first cap-adjacent nucleotide. De novo RoseTTA modeling of CMTrs reveals close similarities of the overall structure and near identity for the catalytic tetrad, and for cap and cofactor binding for human, Drosophila and C. elegans CMTrs. Although viral CMTrs maintain the overall structure and catalytic tetrad, they have diverged in cap and cofactor binding. Consistent with the structural similarity, both CMTrs from Drosophila and humans methylate the first cap-adjacent nucleotide of an AGU consensus start. Because the second nucleotide is also methylated upon heat stress in Drosophila, these findings argue for regulated cOMe important for gene expression regulation.

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.

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.

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

BACKGROUND: Spliced 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. RESULTS: Here 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. CONCLUSIONS: SLIDR 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.

An in vivo genetic screen for genes involved in spliced leader trans-splicing indicates a crucial role for continuous de novo spliced leader RNP assembly.

Spliced leader (SL) trans-splicing is a critical element of gene expression in a number of eukaryotic groups. This process is arguably best understood in nematodes, where biochemical and molecular studies in Caenorhabditis elegans and Ascaris suum have identified key steps and factors involved. Despite this, the precise details of SL trans-splicing have yet to be elucidated. In part, this is because the systematic identification of the molecules involved has not previously been possible due to the lack of a specific phenotype associated with defects in this process. We present here a novel GFP-based reporter assay that can monitor SL1 trans-splicing in living C. elegans. Using this assay, we have identified mutants in sna-1 that are defective in SL trans-splicing, and demonstrate that reducing function of SNA-1, SNA-2 and SUT-1, proteins that associate with SL1 RNA and related SmY RNAs, impairs SL trans-splicing. We further demonstrate that the Sm proteins and pICln, SMN and Gemin5, which are involved in small nuclear ribonucleoprotein assembly, have an important role in SL trans-splicing. Taken together these results provide the first in vivo evidence for proteins involved in SL trans-splicing, and indicate that continuous replacement of SL ribonucleoproteins consumed during trans-splicing reactions is essential for effective trans-splicing.

Mutation of genes controlling mRNA metabolism and protein synthesis predisposes to neurodevelopmental disorders.

Brain development is a tightly controlled process that depends upon differentiation and function of neurons to allow for the formation of functional neural networks. Mutation of genes encoding structural proteins is well recognized as causal for neurodevelopmental disorders (NDDs). Recent studies have shown that aberrant gene expression can also lead to disorders of neural development. Here we summarize recent evidence implicating in the aetiology of NDDs mutation of factors acting at the level of mRNA splicing, mRNA nuclear export, translation and mRNA degradation. This highlights the importance of these fundamental processes for human health and affords new strategies and targets for therapeutic intervention.

Center domains and their phenomenological consequences.

We argue that the domain structure of deconfined QCD matter, which can be inferred from the properties of the Polyakov loop, can simultaneously explain the two most prominent experimentally verified features of the quark-gluon plasma, namely its large opacity as well as its near ideal fluid properties.

SL2-like spliced leader RNAs in the basal nematode Prionchulus punctatus: New insight into the evolution of nematode SL2 RNAs.

Spliced-leader (SL) trans-splicing has been found in all molecularly characterized nematode species to date, and it is likely to be a nematode synapomorphy. Most information regarding SL trans-splicing has come from the study of nematodes from a single monophyletic group, the Rhabditida, all of which employ SL RNAs that are identical to, or variants of, the SL1 RNA first characterized in Caenorhabditis elegans. In contrast, the more distantly related Trichinella spiralis, belonging to the subclass Dorylaimia, utilizes a distinct set of SL RNAs that display considerable sequence diversity. To investigate whether this is true of other members of the Dorylaimia, we have characterized SL RNAs from Prionchulus punctatus. Surprisingly, this revealed the presence of a set of SLs that show clear sequence similarity to the SL2 family of spliced leaders, which have previously only been found within the rhabditine group (which includes C. elegans). Expression of one of the P. punctatus SL RNAs in C. elegans reveals that it can compete specifically with the endogenous C. elegans SL2 spliced leaders, being spliced to the pre-mRNAs derived from downstream genes in operons, but does not compete with the SL1 spliced leaders. This discovery raises the possibility that SL2-like spliced leaders were present in the last common ancestor of the nematode phylum.

The control of histone gene expression.

Histone proteins are essential for the packaging of DNA into chromosomes. Histone gene expression is cell-cycle-regulated and coupled to DNA replication. Control of histone gene expression occurs at the transcriptional and post-transcriptional level and ensures that a fine balance between histone abundance and DNA replication is maintained for the correct packaging of newly replicated DNA into chromosomes. In the present paper, we review histone gene expression, highlighting the control mechanisms and key molecules involved in this process.

All that glitters is not gold: trustworthy and ethical AI principles.

Ethics of technology systems have become an area of interest in academic research as well as international policy in recent years. Several organisation have consequently published principles of ethical artificial intelligence (AI) in line with this trend. The documents identify principles, values, and other abstract requirements for AI development and deployment. Critics raise concerns about whether these documents are in fact constructive, or if they are produced as a higher form of virtue signalling. A theme that is beginning to become apparent in the academic literature regarding these documents is the inherent lack of effective and practical methods and processes for producing ethical AI. This article attempts a critical analysis which draws upon ethical AI documents from a range of contexts including company, organisational, governmental, and academic perspectives. Both the theoretical and practical components of AI guidelines are explored and analysed, consequently bringing to light the necessity of introducing a measurable component to such documents for the purpose of ensuring a positive outcome of deploying AI systems based on ethical principles. We propose a minimal framework for stakeholders to develop AI in an ethical and human-centred manner.

A Bioluminescence Reporter Assay for Retinoic Acid Control of Translation of the GluR1 Subunit of the AMPA Glutamate Receptor.

The present protocol describes a bioluminescence reporter assay developed to quantify the ability of synthetic agonists of retinoic acid receptors (RARs) to activate glutamate receptor subunit 1 (GluR1) translation. The reporter assay uses firefly luciferase under the control of the GluR1 5' untranslated region (5' UTR) which is bound by RARs to regulate its translation. This method is used to demonstrate the role of RARα in retinoic acid regulation of GluR1 translation. This method may also be used to screen drugs that influence RAR induction of GluR1 translation as an important mechanism controlling learning and memory in the brain.

Explanation of dijet asymmetry in Pb-Pb collisions at the Large Hadron Collider.

We investigate the medium modification of a partonic jet shower traversing in a hot quark-gluon plasma. We derive and solve a differential equation that governs the evolution of the radiated gluon distribution as the jet propagates through the medium. Energy contained inside the jet cone is lost by dissipation through elastic collisions with the medium and by scattering of shower partons to larger angles. We find that the jet energy loss at early times is dominated by medium effects on the vacuum radiation, and by medium-induced radiation effects at late times. We compare our numerical results for the nuclear modification of the dijet asymmetry with that recently reported by the ATLAS Collaboration.

Spliced leader trans-splicing in the nematode Trichinella spiralis uses highly polymorphic, noncanonical spliced leaders.

The trans-splicing of short spliced leader (SL) RNAs onto the 5' ends of mRNAs occurs in a diverse range of taxa. In nematodes, all species so far characterized utilize a characteristic, conserved spliced leader, SL1, as well as variants that are employed in the resolution of operons. Here we report the identification of spliced leader trans-splicing in the basal nematode Trichinella spiralis, and show that this nematode does not possess a canonical SL1, but rather has at least 15 distinct spliced leaders, encoded by at least 19 SL RNA genes. The individual spliced leaders vary in both size and primary sequence, showing a much higher degree of diversity compared to other known trans-spliced leaders. In a survey of T. spiralis mRNAs, individual mRNAs were found to be trans-spliced to a number of different spliced leader sequences. These data provide the first indication that the last common ancestor of the phylum Nematoda utilized spliced leader trans-splicing and that the canonical spliced leader, SL1, found in Caenorhabditis elegans, evolved after the divergence of the major nematode clades. This discovery sheds important light on the nature and evolution of mRNA processing in the Nematoda.

The evolution of spliced leader trans-splicing in nematodes.

Spliced leader trans-splicing occurs in many primitive eukaryotes including nematodes. Most of our knowledge of trans-splicing in nematodes stems from the model organism Caenorhabditis elegans and relatives, and from work with Ascaris. Our investigation of spliced leader trans-splicing in distantly related Dorylaimia nematodes indicates that spliced-leader trans-splicing arose before the nematode phylum and suggests that the spliced leader RNA gene complements in extant nematodes have evolved from a common ancestor with a diverse set of spliced leader RNA genes.

Hadron mass spectrum from lattice QCD.

Finite temperature lattice simulations of quantum chromodynamics (QCD) are sensitive to the hadronic mass spectrum for temperatures below the "critical" temperature T(c) ≈ 160 MeV. We show that a recent precision determination of the QCD trace anomaly shows evidence for the existence of a large number of hadron states beyond those known from experiment. The lattice results are well represented by an exponentially growing mass spectrum up to a temperature T=155 MeV. Using simple parametrizations of the hadron mass spectrum we show how one may estimate the total spectral weight in these yet undermined states.

A Bioluminescence Reporter Assay for Retinoic Acid Control of Translation of the GluR1 Subunit of the AMPA Glutamate Receptor.

Retinoic acid (RA) regulates numerous aspects of central nervous system function through modulation of gene transcription via retinoic acid receptors (RARs). However, RA has important roles independent of gene transcription (non-genomic actions) and in the brain a crucial regulator of homeostatic plasticity is RAR control of glutamate receptor subunit 1 (GluR1) translation. An assay to quantify RAR regulation of GluR1 translation would be beneficial both to study the molecular components regulating this system and screen drugs that influence this critical mechanism for learning and memory in the brain. A bioluminescence reporter assay was developed that expresses firefly luciferase under the control of the GluR1 5' untranslated region bound by RAR. This assay was introduced into SH-SY5Y cells and used to demonstrate the role of RARα in RA regulation of GluR1 translation. A screen of synthetic RAR and RXR ligands indicated that only a subset of these ligands activated GluR1 translation. The results demonstrate the practicality of this assay to explore the contribution of RARα to this pathway and that the capacity of RAR ligands to activate translation is a quality restricted to a limited number of compounds, with implications for their RAR selectivity and potentially their specificity in drug use.

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.

Histone gene expression and histone mRNA 3' end structure in Caenorhabditis elegans.

BACKGROUND: Histone protein synthesis is essential for cell proliferation and required for the packaging of DNA into chromatin. In animals, histone proteins are provided by the expression of multicopy replication-dependent histone genes. Histone mRNAs that are processed by a histone-specific mechanism to end after a highly conserved RNA hairpin element, and lack a poly(A) tail. In vertebrates and Drosophila, their expression is dependent on HBP/SLBP that binds to the RNA hairpin element. We showed previously that these cis and trans acting regulators of histone gene expression are conserved in C. elegans. Here we report the results of an investigation of the histone mRNA 3' end structure and of histone gene expression during C. elegans development. RESULTS: Sequence analysis of replication-dependent histone genes revealed the presence of several highly conserved sequence elements in the 3' untranslated region of histone pre-mRNAs, including an RNA hairpin element and a polyadenylation signal. To determine whether in C. elegans histone mRNA 3' end formation occurs at this polyadenylation signal and results in polyadenylated histone mRNA, we investigated the mRNA 3' end structure of histone mRNA. Using poly(A) selection, RNAse protection and sequencing of histone mRNA ends, we determined that a majority of C. elegans histone mRNAs lack a poly(A) tail and end three to six nucleotides after the hairpin structure, after an A or a U, and have a 3' OH group. RNAi knock down of CDL-1, the C. elegans HBP/SLBP, does not significantly affect histone mRNA levels but severely depletes histone protein levels. Histone gene expression varies during development and is reduced in L3 animals compared to L1 animals and adults. In adults, histone gene expression is restricted to the germ line, where cell division occurs. CONCLUSION: Our findings indicate that the expression of C. elegans histone genes is subject to control mechanisms similar to the ones in other animals: the structure of C. elegans histone mRNA 3' ends is compatible with histone-specific mRNA 3' end processing; CDL-1 functions in post-transcriptional control of histone gene expression; and C. elegans histone mRNA levels are elevated at periods of active cell division, indicating that histone gene expression is linked to DNA replication.

Modelling Robust Feedback Control Mechanisms That Ensure Reliable Coordination of Histone Gene Expression with DNA Replication.

Histone proteins are key elements in the packing of eukaryotic DNA into chromosomes. A little understood control system ensures that histone gene expression is balanced with DNA replication so that histone proteins are produced in appropriate amounts. Disturbing or disrupting this system affects genome stability and gene expression, and has detrimental consequences for human development and health. It has been proposed that feedback control involving histone proteins contributes to this regulation and there is evidence implicating cell cycle checkpoint molecules activated when DNA synthesis is impaired in this control. We have developed mathematical models that incorporate these control modes in the form of inhibitory feedback of histone gene expression from free histone proteins, and alternatively a direct link that couples histone RNA synthesis to DNA synthesis. Using our experimental evidence and related published data we provide a simplified description of histone protein synthesis during S phase. Both models reproduce the coordination of histone gene expression with DNA replication during S phase and the down-regulation of histone RNA when DNA synthesis is interrupted, but only the model incorporating histone protein feedback control was able to effectively simulate the coordinate expression of a simplified histone gene family. Our combined theoretical and experimental approach supports the hypothesis that the regulation of histone gene expression involves feedback control.