Mechanism for the initiation of spliceosome disassembly.

Precursor-mRNA (pre-mRNA) splicing requires the assembly, remodelling and disassembly of the multi-megadalton ribonucleoprotein complex called the spliceosome1. Recent studies have shed light on spliceosome assembly and remodelling for catalysis2-6, but the mechanism of disassembly remains unclear. Here we report cryo-electron microscopy structures of nematode and human terminal intron lariat spliceosomes along with biochemical and genetic data. Our results uncover how four disassembly factors and the conserved RNA helicase DHX15 initiate spliceosome disassembly. The disassembly factors probe large inner and outer spliceosome surfaces to detect the release of ligated mRNA. Two of these factors, TFIP11 and C19L1, and three general spliceosome subunits, SYF1, SYF2 and SDE2, then dock and activate DHX15 on the catalytic U6 snRNA to initiate disassembly. U6 therefore controls both the start5 and end of pre-mRNA splicing. Taken together, our results explain the molecular basis of the initiation of canonical spliceosome disassembly and provide a framework to understand general spliceosomal RNA helicase control and the discard of aberrant spliceosomes.

Mime-seq 2.0: a method to sequence microRNAs from specific mouse cell types.

Many microRNAs (miRNAs) are expressed with high spatiotemporal specificity during organismal development, with some being limited to rare cell types, often embedded in complex tissues. Yet, most miRNA profiling efforts remain at the tissue and organ levels. To overcome challenges in accessing the microRNomes from tissue-embedded cells, we had previously developed mime-seq (miRNome by methylation-dependent sequencing), a technique in which cell-specific miRNA methylation in C. elegans and Drosophila enabled chemo-selective sequencing without the need for cell sorting or biochemical purification. Here, we present mime-seq 2.0 for profiling miRNAs from specific mouse cell types. We engineered a chimeric RNA methyltransferase that is tethered to Argonaute protein and efficiently methylates miRNAs at their 3'-terminal 2'-OH in mouse and human cell lines. We also generated a transgenic mouse for conditional expression of this methyltransferase, which can be used to direct methylation of miRNAs in a cell type of choice. We validated the use of this mouse model by profiling miRNAs from B cells and bone marrow plasma cells.

Embryonic priming of a miRNA locus predetermines postmitotic neuronal left/right asymmetry in C. elegans.

The mechanisms by which functional left/right asymmetry arises in morphologically symmetric nervous systems are poorly understood. Here, we provide a mechanistic framework for how functional asymmetry in a postmitotic neuron pair is specified in C. elegans. A key feature of this mechanism is a temporally separated, two-step activation of the lsy-6 miRNA locus. The lsy-6 locus is first "primed" by chromatin decompaction in the precursor for the left neuron, but not the right neuron, several divisions before the neurons are born. lsy-6 expression is then "boosted" to functionally relevant levels several divisions later in the mother of the left neuron, through the activity of a bilaterally expressed transcription factor that can only activate lsy-6 in the primed neuron. This study shows how cells can become committed during early developmental stages to execute a specific fate much later in development and provides a conceptual framework for understanding the generation of neuronal diversity.

Development, regeneration and aging: a bizarre love triangle.

The Jacques Monod Conference on 'Growth and regeneration during development and aging' was organized by Claude Desplan and Allison Bardin in May 2023. The conference took place in Roscoff, France, where participants shared recent conceptual advances under the general motto that developmental processes do not end with embryogenesis. The meeting covered various aspects of how development relates to fitness, regeneration and aging across a refreshing diversity of evolutionarily distant organisms.

Genomic surveillance of SARS-CoV-2 evolution by a centralised pipeline and weekly focused sequencing, Austria, January 2021 to March 2023.

BackgroundThe COVID-19 pandemic was largely driven by genetic mutations of SARS-CoV-2, leading in some instances to enhanced infectiousness of the virus or its capacity to evade the host immune system. To closely monitor SARS-CoV-2 evolution and resulting variants at genomic-level, an innovative pipeline termed SARSeq was developed in Austria.AimWe discuss technical aspects of the SARSeq pipeline, describe its performance and present noteworthy results it enabled during the pandemic in Austria.MethodsThe SARSeq pipeline was set up as a collaboration between private and public clinical diagnostic laboratories, a public health agency, and an academic institution. Representative SARS-CoV-2 positive specimens from each of the nine Austrian provinces were obtained from SARS-CoV-2 testing laboratories and processed centrally in an academic setting for S-gene sequencing and analysis.ResultsSARS-CoV-2 sequences from up to 2,880 cases weekly resulted in 222,784 characterised case samples in January 2021-March 2023. Consequently, Austria delivered the fourth densest genomic surveillance worldwide in a very resource-efficient manner. While most SARS-CoV-2 variants during the study showed comparable kinetic behaviour in all of Austria, some, like Beta, had a more focused spread. This highlighted multifaceted aspects of local population-level acquired immunity. The nationwide surveillance system enabled reliable nowcasting. Measured early growth kinetics of variants were predictive of later incidence peaks.ConclusionWith low automation, labour, and cost requirements, SARSeq is adaptable to monitor other pathogens and advantageous even for resource-limited countries. This multiplexed genomic surveillance system has potential as a rapid response tool for future emerging threats.

Neuron type-specific miRNA represses two broadly expressed genes to modulate an avoidance behavior in C. elegans.

Two broad gene classes are distinguished within multicellular organisms: cell type-specific genes, which confer particular cellular properties, and ubiquitous genes that support general cellular functions. However, certain so-called ubiquitous genes show functionally relevant cell type-specific repression. How such repression is achieved is poorly understood. MicroRNAs (miRNAs) are repressors, many of which are expressed with high cell type specificity. Here we show that mir-791, expressed exclusively in the CO2-sensing neurons in Caenorhabditis elegans, represses two otherwise broadly expressed genes. This repression is necessary for normal neuronal function and behavior of the animals toward CO2 miRNA-mediated repression of broadly transcribed genes is a previously unappreciated strategy for cellular specialization.

Two distinct types of neuronal asymmetries are controlled by the Caenorhabditis elegans zinc finger transcription factor die-1.

Left/right asymmetric features of animals are either randomly distributed on either the left or right side within a population ("antisymmetries") or found stereotypically on one particular side of an animal ("directional asymmetries"). Both types of asymmetries can be found in nervous systems, but whether the regulatory programs that establish these asymmetries share any mechanistic features is not known. We describe here an unprecedented molecular link between these two types of asymmetries in Caenorhabditis elegans. The zinc finger transcription factor die-1 is expressed in a directionally asymmetric manner in the gustatory neuron pair ASE left (ASEL) and ASE right (ASER), while it is expressed in an antisymmetric manner in the olfactory neuron pair AWC left (AWCL) and AWC right (AWCR). Asymmetric die-1 expression is controlled in a fundamentally distinct manner in these two neuron pairs. Importantly, asymmetric die-1 expression controls the directionally asymmetric expression of gustatory receptor proteins in the ASE neurons and the antisymmetric expression of olfactory receptor proteins in the AWC neurons. These asymmetries serve to increase the ability of the animal to discriminate distinct chemosensory inputs.

Cell-type specific sequencing of microRNAs from complex animal tissues.

MicroRNAs (miRNAs) play an essential role in the post-transcriptional regulation of animal development and physiology. However, in vivo studies aimed at linking miRNA function to the biology of distinct cell types within complex tissues remain challenging, partly because in vivo miRNA-profiling methods lack cellular resolution. We report microRNome by methylation-dependent sequencing (mime-seq), an in vivo enzymatic small-RNA-tagging approach that enables high-throughput sequencing of tissue- and cell-type-specific miRNAs in animals. The method combines cell-type-specific 3'-terminal 2'-O-methylation of animal miRNAs by a genetically encoded, plant-specific methyltransferase (HEN1), with chemoselective small-RNA cloning and high-throughput sequencing. We show that mime-seq uncovers the miRNomes of specific cells within Caenorhabditis elegans and Drosophila at unprecedented specificity and sensitivity, enabling miRNA profiling with single-cell resolution in whole animals. Mime-seq overcomes current challenges in cell-type-specific small-RNA profiling and provides novel entry points for understanding the function of miRNAs in spatially restricted physiological settings.

A framework for understanding the roles of miRNAs in animal development.

MicroRNAs (miRNAs) contribute to the progressive changes in gene expression that occur during development. The combined loss of all miRNAs results in embryonic lethality in all animals analyzed, illustrating the crucial role that miRNAs play collectively. However, although the loss of some individual miRNAs also results in severe developmental defects, the roles of many other miRNAs have been challenging to uncover. This has been mostly attributed to their proposed function as tuners of gene expression or providers of robustness. Here, we present a view of miRNAs in the context of development as a hierarchical and canalized series of gene regulatory networks. In this scheme, only a fraction of embryonic miRNAs act at the top of this hierarchy, with their loss resulting in broad developmental defects, whereas most other miRNAs are expressed with high cellular specificity and play roles at the periphery of development, affecting the terminal features of specialized cells. This view could help to shed new light on our understanding of miRNA function in development, disease and evolution.

A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans.

One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo.

Neurogenesis in Caenorhabditis elegans.

Animals rely on their nervous systems to process sensory inputs, integrate these with internal signals, and produce behavioral outputs. This is enabled by the highly specialized morphologies and functions of neurons. Neuronal cells share multiple structural and physiological features, but they also come in a large diversity of types or classes that give the nervous system its broad range of functions and plasticity. This diversity, first recognized over a century ago, spurred classification efforts based on morphology, function, and molecular criteria. Caenorhabditis elegans, with its precisely mapped nervous system at the anatomical level, an extensive molecular description of most of its neurons, and its genetic amenability, has been a prime model for understanding how neurons develop and diversify at a mechanistic level. Here, we review the gene regulatory mechanisms driving neurogenesis and the diversification of neuron classes and subclasses in C. elegans. We discuss our current understanding of the specification of neuronal progenitors and their differentiation in terms of the transcription factors involved and ensuing changes in gene expression and chromatin landscape. The central theme that has emerged is that the identity of a neuron is defined by modules of gene batteries that are under control of parallel yet interconnected regulatory mechanisms. We focus on how, to achieve these terminal identities, cells integrate information along their developmental lineages. Moreover, we discuss how neurons are diversified postembryonically in a time-, genetic sex-, and activity-dependent manner. Finally, we discuss how the understanding of neuronal development can provide insights into the evolution of neuronal diversity.

Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring.

Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5' tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Injection of sperm small RNA fractions from MTX-treated males into normal fertilized eggs generated cranial cartilage defects in the offspring. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.

Neuron-type specific regulation of a 3'UTR through redundant and combinatorially acting cis-regulatory elements.

3' Untranslated region (UTR)-dependent post-transcriptional regulation has emerged as a critical mechanism of controlling gene expression in various physiological contexts, including cellular differentiation events. Here, we examine the regulation of the 3'UTR of the die-1 transcription factor in a single neuron of the nematode C. elegans. This 3'UTR shows the intriguing feature of being differentially regulated across the animal's left/right axis. In the left gustatory neuron, ASEL, in which DIE-1 protein is normally expressed in adult animals, the 3'UTR confers no regulatory information, while in the right gustatory neuron, ASER, where DIE-1 is normally not expressed, this 3'UTR confers negative regulatory information. Here, we systematically analyze the cis-regulatory architecture of the die-1 3'UTR using a transgenic, in vivo assay system. Through extensive mutagenesis and sequence insertions into heterologous 3'UTR contexts, we describe three 25-base-pair (bp) sequence elements that are both required and sufficient to mediate the ASER-specific down-regulation of the die-1 3'UTR. These three 25-bp sequence elements operate in both a redundant and combinatorial manner. Moreover, there are not only redundant elements within the die-1 3'UTR regulating its left/right asymmetric activity but asymmetric 3'UTR regulation is itself redundant with other regulatory mechanisms to achieve asymmetric DIE-1 protein expression and function in ASEL versus ASER. The features of 3'UTR regulation we describe here may apply to some of the vast number of genes in animal genomes whose expression is predicted to be regulated through their 3'UTR.

Mutational analysis reveals two independent molecular requirements during transfer RNA selection on the ribosome.

Accurate discrimination between cognate and near-cognate aminoacyl-tRNAs during translation relies on the specific acceleration of forward rate constants for cognate tRNAs. Such specific rate enhancement correlates with conformational changes in the tRNA and small ribosomal subunit that depend on an RNA-specific type of interaction, the A-minor motif, between universally conserved 16S ribosomal RNA nucleotides and the cognate codon-anticodon helix. We show that perturbations of these two components of the A-minor motif, the conserved rRNA bases and the codon-anticodon helix, result in distinct outcomes. Although both cause decreases in the rates of tRNA selection that are rescued by aminoglycoside antibiotics, only disruption of the codon-anticodon helix is overcome by a miscoding tRNA variant. On this basis, we propose that two independent molecular requirements must be met to allow tRNAs to proceed through the selection pathway, providing a mechanism for exquisite control of fidelity during this step in gene expression.

Concepts and functions of small RNA pathways in C. elegans.

A diversity of gene regulatory mechanisms drives the changes in gene expression required for animal development. Here, we discuss the developmental roles of a class of gene regulatory factors composed of a core protein subunit of the Argonaute family and a 21-26-nucleotide RNA cofactor. These represent ancient regulatory complexes, originally evolved to repress genomic parasites such as transposons, viruses and retroviruses. However, over the course of evolution, small RNA-guided pathways have expanded and diversified, and they play multiple roles across all eukaryotes. Pertinent to this review, Argonaute and small RNA-mediated regulation has acquired numerous functions that affect all aspects of animal life. The regulatory function is provided by the Argonaute protein and its interactors, while the small RNA provides target specificity, guiding the Argonaute to a complementary RNA. C. elegans has 19 different, functional Argonautes, defining distinct yet interconnected pathways. Each Argonaute binds a relatively well-defined class of small RNA with distinct molecular properties. A broad classification of animal small RNA pathways distinguishes between two groups: (i) the microRNA pathway is involved in repressing relatively specific endogenous genes and (ii) the other small RNA pathways, which effectively act as a genomic immune system to primarily repress expression of foreign or "non-self" RNA while maintaining correct endogenous gene expression. microRNAs play prominent direct roles in all developmental stages, adult physiology and lifespan. The other small RNA pathways act primarily in the germline, but their impact extends far beyond, into embryogenesis and adult physiology, and even to subsequent generations. Here, we review the mechanisms and developmental functions of the diverse small RNA pathways of C. elegans.

miR-1 sustains muscle physiology by controlling V-ATPase complex assembly.

Muscle function requires unique structural and metabolic adaptations that can render muscle cells selectively vulnerable, with mutations in some ubiquitously expressed genes causing myopathies but sparing other tissues. We uncovered a muscle cell vulnerability by studying miR-1, a deeply conserved, muscle-specific microRNA whose ablation causes various muscle defects. Using Caenorhabditis elegans, we found that miR-1 represses multiple subunits of the ubiquitous vacuolar adenosine triphosphatase (V-ATPase) complex, which is essential for internal compartment acidification and metabolic signaling. V-ATPase subunits are predicted miR-1 targets in animals ranging from C. elegans to humans, and we experimentally validated this in Drosophila. Unexpectedly, up-regulation of V-ATPase subunits upon miR-1 deletion causes reduced V-ATPase function due to defects in complex assembly. These results reveal V-ATPase assembly as a conserved muscle cell vulnerability and support a previously unknown role for microRNAs in the regulation of protein complexes.

Pax5 regulates B cell immunity by promoting PI3K signaling via PTEN down-regulation.

The transcription factor Pax5 controls B cell development, but its role in mature B cells is largely enigmatic. Here, we demonstrated that the loss of Pax5 by conditional mutagenesis in peripheral B lymphocytes led to the strong reduction of B-1a, marginal zone (MZ), and germinal center (GC) B cells as well as plasma cells. Follicular (FO) B cells tolerated the loss of Pax5 but had a shortened half-life. The Pax5-deficient FO B cells failed to proliferate upon B cell receptor or Toll-like receptor stimulation due to impaired PI3K-AKT signaling, which was caused by increased expression of PTEN, a negative regulator of the PI3K pathway. Pax5 restrained PTEN protein expression at the posttranscriptional level, likely involving Pten-targeting microRNAs. Additional PTEN loss in Pten,Pax5 double-mutant mice rescued FO B cell numbers and the development of MZ B cells but did not restore GC B cell formation. Hence, the posttranscriptional down-regulation of PTEN expression is an important function of Pax5 that facilitates the differentiation and survival of mature B cells, thereby promoting humoral immunity.

Multiplexed detection of SARS-CoV-2 and other respiratory infections in high throughput by SARSeq.

The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.

MicroRNAs: From Mechanism to Organism.

MicroRNAs (miRNAs) are short, regulatory RNAs that act as post-transcriptional repressors of gene expression in diverse biological contexts. The emergence of small RNA-mediated gene silencing preceded the onset of multicellularity and was followed by a drastic expansion of the miRNA repertoire in conjunction with the evolution of complexity in the plant and animal kingdoms. Along this process, miRNAs became an essential feature of animal development, as no higher metazoan lineage tolerated loss of miRNAs or their associated protein machinery. In fact, ablation of the miRNA biogenesis machinery or the effector silencing factors results in severe embryogenesis defects in every animal studied. In this review, we summarize recent mechanistic insight into miRNA biogenesis and function, while emphasizing features that have enabled multicellular organisms to harness the potential of this broad class of repressors. We first discuss how different mechanisms of regulation of miRNA biogenesis are used, not only to generate spatio-temporal specificity of miRNA production within an animal, but also to achieve the necessary levels and dynamics of expression. We then explore how evolution of the mechanism for small RNA-mediated repression resulted in a diversity of silencing complexes that cause different molecular effects on their targets. Multicellular organisms have taken advantage of this variability in the outcome of miRNA-mediated repression, with differential use in particular cell types or even distinct subcellular compartments. Finally, we present an overview of how the animal miRNA repertoire has evolved and diversified, emphasizing the emergence of miRNA families and the biological implications of miRNA sequence diversification. Overall, focusing on selected animal models and through the lens of evolution, we highlight canonical mechanisms in miRNA biology and their variations, providing updated insight that will ultimately help us understand the contribution of miRNAs to the development and physiology of multicellular organisms.

Two MicroRNAs Are Sufficient for Embryonic Patterning in C. elegans.

MicroRNAs (miRNAs) are a class of post-transcriptional repressors with diverse roles in animal development and physiology [1]. The Microprocessor complex, composed of Drosha and Pasha/DGCR8, is necessary for the biogenesis of all canonical miRNAs and essential for the early stages of animal embryogenesis [2-8]. However, the cause for this requirement is largely unknown. Animals often express hundreds of miRNAs, and it remains unclear whether the Microprocessor is required to produce one or few essential miRNAs or many individually non-essential miRNAs. Additionally, both Drosha and Pasha/DGCR8 bind and cleave a variety of non-miRNA substrates [9-15], and it is unknown whether these activities account for the Microprocessor's essential requirement. To distinguish between these possibilities, we developed a system in C. elegans to stringently deplete embryos of Microprocessor activity. Using a combination of auxin-inducible degradation (AID) and RNA interference (RNAi), we achieved Drosha and Pasha/DGCR8 depletion starting in the maternal germline, resulting in Microprocessor and miRNA-depleted embryos, which fail to undergo morphogenesis or form organs. Using a Microprocessor-bypass strategy, we show that this early embryonic arrest is rescued by the addition of just two miRNAs, one miR-35 and one miR-51 family member, resulting in morphologically normal larvae. Thus, just two out of ∼150 canonical miRNAs are sufficient for morphogenesis and organogenesis, and the processing of these miRNAs accounts for the essential requirement for Drosha and Pasha/DGCR8 during the early stages of C. elegans embryonic development. VIDEO ABSTRACT.