The absence of the ribosomal protein Rpl2702 elicits the MAPK-mTOR signaling to modulate mitochondrial morphology and functions.

Ribosomes mediate protein synthesis, which is one of the most energy-demanding activities within the cell, and mitochondria are one of the main sources generating energy. How mitochondrial morphology and functions are adjusted to cope with ribosomal defects, which can impair protein synthesis and affect cell viability, is poorly understood. Here, we used the fission yeast Schizosaccharomyces Pombe as a model organism to investigate the interplay between ribosome and mitochondria. We found that a ribosomal insult, caused by the absence of Rpl2702, activates a signaling pathway involving Sty1/MAPK and mTOR to modulate mitochondrial morphology and functions. Specifically, we demonstrated that Sty1/MAPK induces mitochondrial fragmentation in a mTOR-independent manner while both Sty1/MAPK and mTOR increases the levels of mitochondrial membrane potential and mitochondrial reactive oxygen species (mROS). Moreover, we demonstrated that Sty1/MAPK acts upstream of Tor1/TORC2 and Tor1/TORC2 and is required to activate Tor2/TORC1. The enhancements of mitochondrial membrane potential and mROS function to promote proliferation of cells bearing ribosomal defects. Hence, our study reveals a previously uncharacterized Sty1/MAPK-mTOR signaling axis that regulates mitochondrial morphology and functions in response to ribosomal insults and provides new insights into the molecular and physiological adaptations of cells to impaired protein synthesis.

Early-life stress triggers long-lasting organismal resilience and longevity via tetraspanin.

Early-life stress experiences can produce lasting impacts on organismal adaptation and fitness. How transient stress elicits memory-like physiological effects is largely unknown. Here, we show that early-life thermal stress strongly up-regulates tsp-1, a gene encoding the conserved transmembrane tetraspanin in C. elegans. TSP-1 forms prominent multimers and stable web-like structures critical for membrane barrier functions in adults and during aging. Increased TSP-1 abundance persists even after transient early-life heat stress. Such regulation requires CBP-1, a histone acetyltransferase that facilitates initial tsp-1 transcription. Tetraspanin webs form regular membrane structures and mediate resilience-promoting effects of early-life thermal stress. Gain-of-function TSP-1 confers marked C. elegans longevity extension and thermal resilience in human cells. Together, our results reveal a cellular mechanism by which early-life thermal stress produces long-lasting memory-like impact on organismal resilience and longevity.

xQTLbiolinks: a comprehensive and scalable tool for integrative analysis of molecular QTLs.

Genome-wide association studies (GWAS) have identified thousands of disease-associated non-coding variants, posing urgent needs for functional interpretation. Molecular Quantitative Trait Loci (xQTLs) such as eQTLs serve as an essential intermediate link between these non-coding variants and disease phenotypes and have been widely used to discover disease-risk genes from many population-scale studies. However, mining and analyzing the xQTLs data presents several significant bioinformatics challenges, particularly when it comes to integration with GWAS data. Here, we developed xQTLbiolinks as the first comprehensive and scalable tool for bulk and single-cell xQTLs data retrieval, quality control and pre-processing from public repositories and our integrated resource. In addition, xQTLbiolinks provided a robust colocalization module through integration with GWAS summary statistics. The result generated by xQTLbiolinks can be flexibly visualized or stored in standard R objects that can easily be integrated with other R packages and custom pipelines. We applied xQTLbiolinks to cancer GWAS summary statistics as case studies and demonstrated its robust utility and reproducibility. xQTLbiolinks will profoundly accelerate the interpretation of disease-associated variants, thus promoting a better understanding of disease etiologies. xQTLbiolinks is available at https://github.com/lilab-bioinfo/xQTLbiolinks.

Acquired stress resilience through bacteria-to-nematode interdomain horizontal gene transfer.

Natural selection drives the acquisition of organismal resilience traits to protect against adverse environments. Horizontal gene transfer (HGT) is an important evolutionary mechanism for the acquisition of novel traits, including metazoan acquisitions in immunity, metabolic, and reproduction function via interdomain HGT (iHGT) from bacteria. Here, we report that the nematode gene rml-3 has been acquired by iHGT from bacteria and that it enables exoskeleton resilience and protection against environmental toxins in Caenorhabditis elegans. Phylogenetic analysis reveals that diverse nematode RML-3 proteins form a single monophyletic clade most similar to bacterial enzymes that biosynthesize L-rhamnose, a cell-wall polysaccharide component. C. elegans rml-3 is highly expressed during larval development and upregulated in developing seam cells upon heat stress and during the stress-resistant dauer stage. rml-3 deficiency impairs cuticle integrity, barrier functions, and nematode stress resilience, phenotypes that can be rescued by exogenous L-rhamnose. We propose that interdomain HGT of an ancient bacterial rml-3 homolog has enabled L-rhamnose biosynthesis in nematodes, facilitating cuticle integrity and organismal resilience to environmental stressors during evolution. These findings highlight a remarkable contribution of iHGT on metazoan evolution conferred by the domestication of a bacterial gene.

Acquired stress resilience through bacteria-to-nematode horizontal gene transfer.

Natural selection drives acquisition of organismal resilience traits to protect against adverse environments. Horizontal gene transfer (HGT) is an important evolutionary mechanism for the acquisition of novel traits, including metazoan acquisition of functions in immunity, metabolism, and reproduction via interdomain HGT (iHGT) from bacteria. We report that the nematode gene rml-3, which was acquired by iHGT from bacteria, enables exoskeleton resilience and protection against environmental toxins in C. elegans. Phylogenetic analysis reveals that diverse nematode RML-3 proteins form a single monophyletic clade most highly similar to bacterial enzymes that biosynthesize L-rhamnose to build cell wall polysaccharides. C. elegans rml-3 is regulated in developing seam cells by heat stress and stress-resistant dauer stage. Importantly, rml-3 deficiency impairs cuticle integrity, barrier functions and organismal stress resilience, phenotypes that are rescued by exogenous L-rhamnose. We propose that iHGT of an ancient bacterial rml-3 homolog enables L-rhamnose biosynthesis in nematodes that facilitates cuticle integrity and organismal resilience in adaptation to environmental stresses during evolution. These findings highlight the remarkable contribution of iHGT on metazoan evolution that is conferred by the domestication of bacterial genes.

Early-life stress triggers long-lasting organismal resilience and longevity via tetraspanin.

Early-life stress experiences can produce lasting impacts on organismal adaptation and fitness. How transient stress elicits memory-like physiological effects is largely unknown. Here we show that early-life thermal stress strongly up-regulates tsp-1, a gene encoding the conserved transmembrane tetraspanin in C. elegans. TSP-1 forms prominent multimers and stable web-like structures critical for membrane barrier functions in adults and during aging. The up-regulation of TSP-1 persists even after transient early-life stress. Such regulation requires CBP-1, a histone acetyl-transferase that facilitates initial tsp-1 transcription. Tetraspanin webs form regular membrane structures and mediate resilience-promoting effects of early-life thermal stress. Gain-of-function TSP-1 confers marked C. elegans longevity extension and thermal resilience in human cells. Together, our results reveal a cellular mechanism by which early-life thermal stress produces long-lasting memory-like impact on organismal resilience and longevity.

rRNA intermediates coordinate the formation of nucleolar vacuoles in C. elegans.

The nucleolus is the most prominent membraneless organelle within the nucleus. How the nucleolar structure is regulated is poorly understood. Here, we identified two types of nucleoli in C. elegans. Type I nucleoli are spherical and do not have visible nucleolar vacuoles (NoVs), and rRNA transcription and processing factors are evenly distributed throughout the nucleolus. Type II nucleoli contain vacuoles, and rRNA transcription and processing factors exclusively accumulate in the periphery rim. The NoV contains nucleoplasmic proteins and is capable of exchanging contents with the nucleoplasm. The high-order structure of the nucleolus is dynamically regulated in C. elegans. Faithful rRNA processing is important to prohibit NoVs. The depletion of 27SA2 rRNA processing factors resulted in NoV formation. The inhibition of RNA polymerase I (RNAPI) transcription and depletion of two conserved nucleolar factors, nucleolin and fibrillarin, prohibits the formation of NoVs. This finding provides a mechanism to coordinate structure maintenance and gene expression.

Global histone H2B degradation regulates insulin/IGF signaling-mediated nutrient stress.

Eukaryotic organisms adapt to environmental fluctuations by altering their epigenomic landscapes and transcriptional programs. Nucleosomal histones carry vital epigenetic information and regulate gene expression, yet the mechanisms underlying chromatin-bound histone exchange remain elusive. Here, we found that histone H2Bs are globally degraded in Caenorhabditis elegans during starvation. Our genetic screens identified mutations in ubiquitin and ubiquitin-related enzymes that block H2B degradation in starved animals, identifying lysine 31 as the crucial residue for chromatin-bound H2B ubiquitination and elimination. Retention of aberrant nucleosomal H2B increased the association of the FOXO transcription factor DAF-16 with chromatin, generating an ectopic gene expression profile detrimental to animal viability when insulin/IGF signaling was reduced in well-fed animals. Furthermore, we show that the ubiquitin-proteasome system regulates chromosomal histone turnover in human cells. During larval development, C. elegans epidermal cells undergo H2B turnover after fusing with the epithelial syncytium. Thus, histone degradation may be a widespread mechanism governing dynamic changes of the epigenome.

Systematic characterization of chromodomain proteins reveals an H3K9me1/2 reader regulating aging in C. elegans.

The chromatin organization modifier domain (chromodomain) is an evolutionally conserved motif across eukaryotic species. The chromodomain mainly functions as a histone methyl-lysine reader to modulate gene expression, chromatin spatial conformation and genome stability. Mutations or aberrant expression of chromodomain proteins can result in cancer and other human diseases. Here, we systematically tag chromodomain proteins with green fluorescent protein (GFP) using CRISPR/Cas9 technology in C. elegans. By combining ChIP-seq analysis and imaging, we delineate a comprehensive expression and functional map of chromodomain proteins. We then conduct a candidate-based RNAi screening and identify factors that regulate the expression and subcellular localization of the chromodomain proteins. Specifically, we reveal an H3K9me1/2 reader, CEC-5, both by in vitro biochemistry and in vivo ChIP assays. MET-2, an H3K9me1/2 writer, is required for CEC-5 association with heterochromatin. Both MET-2 and CEC-5 are required for the normal lifespan of C. elegans. Furthermore, a forward genetic screening identifies a conserved Arginine124 of CEC-5's chromodomain, which is essential for CEC-5's association with chromatin and life span regulation. Thus, our work will serve as a reference to explore chromodomain functions and regulation in C. elegans and allow potential applications in aging-related human diseases.

A ZTF-7/RPS-2 complex mediates the cold-warm response in C. elegans.

Temperature greatly affects numerous biological processes in all organisms. How multicellular organisms respond to and are impacted by hypothermic stress remains elusive. Here, we found that cold-warm stimuli induced depletion of the RNA exosome complex in the nucleoli but enriched it in the nucleoplasm. To further understand the function and mechanism of cold-warm stimuli, we conducted forward genetic screening and identified ZTF-7, which is required for RNA exosome depletion from nucleoli upon transient cold-warm exposure in C. elegans. ZTF-7 is a putative ortholog of human ZNF277 that may contribute to language impairments. Immunoprecipitation followed by mass spectrometry (IP-MS) found that ZTF-7 interacted with RPS-2, which is a ribosomal protein of the small subunit and participates in pre-rRNA processing. A partial depletion of RPS-2 and other proteins of the small ribosomal subunit blocked the cold-warm stimuli-induced reduction of exosome subunits from the nucleoli. These results established a novel mechanism by which C. elegans responds to environmental cold-warm exposure.

ipaQTL-atlas: an atlas of intronic polyadenylation quantitative trait loci across human tissues.

Functional interpretation of disease-associated non-coding variants remains a significant challenge in the post-GWAS era. Our recent study has identified 3'UTR alternative polyadenylation (APA) quantitative trait loci (3'aQTLs) and connects APA events with QTLs as a major driver of human traits and diseases. Besides 3'UTR, APA events can also occur in intron regions, and increasing evidence has connected intronic polyadenylation with disease risk. However, systematic investigation of the roles of intronic polyadenylation in human diseases remained challenging due to the lack of a comprehensive database across a variety of human tissues. Here, we developed ipaQTL-atlas (http://bioinfo.szbl.ac.cn/ipaQTL) as the first comprehensive portal for intronic polyadenylation. The ipaQTL-atlas is based on the analysis of 15 170 RNA-seq data from 838 individuals across 49 Genotype-Tissue Expression (GTEx v8) tissues and contains ∼0.98 million SNPs associated with intronic APA events. It provides an interface for ipaQTLs search, genome browser, boxplots, and data download, as well as the visualization of GWAS and ipaQTL colocalization results. ipaQTL-atlas provides a one-stop portal to access intronic polyadenylation information and could significantly advance the discovery of APA-associated disease susceptibility genes.

H3K9me1/2 methylation limits the lifespan of daf-2 mutants in C. elegans.

Histone methylation plays crucial roles in the development, gene regulation, and maintenance of stem cell pluripotency in mammals. Recent work shows that histone methylation is associated with aging, yet the underlying mechanism remains unclear. In this work, we identified a class of putative histone 3 lysine 9 mono/dimethyltransferase genes (met-2, set-6, set-19, set-20, set-21, set-32, and set-33), mutations in which induce synergistic lifespan extension in the long-lived DAF-2 (insulin growth factor 1 [IGF-1] receptor) mutant in Caenorhabditis elegans. These putative histone methyltransferase plus daf-2 double mutants not only exhibited an average lifespan nearly three times that of wild-type animals and a maximal lifespan of approximately 100 days, but also significantly increased resistance to oxidative and heat stress. Synergistic lifespan extension depends on the transcription factor DAF-16 (FOXO). mRNA-seq experiments revealed that the mRNA levels of DAF-16 Class I genes, which are activated by DAF-16, were further elevated in the daf-2;set double mutants. Among these genes, tts-1, F35E8.7, ins-35, nhr-62, sod-3, asm-2, and Y39G8B.7 are required for the lifespan extension of the daf-2;set-21 double mutant. In addition, treating daf-2 animals with the H3K9me1/2 methyltransferase G9a inhibitor also extends lifespan and increases stress resistance. Therefore, investigation of DAF-2 and H3K9me1/2 deficiency-mediated synergistic longevity will contribute to a better understanding of the molecular mechanisms of aging and therapeutic applications.

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.

Molecular basis for PICS-mediated piRNA biogenesis and cell division.

By incorporating two mutually exclusive factors, PID-1 and TOST-1, C. elegans PICS complex plays important roles in piRNA biogenesis, chromosome segregation and cell division. We firstly map the interaction network between PICS subunits, then uncover the mechanisms underlying the interactions between PICS subunits by solving several complex structures, including those of TOFU-6/PICS-1, ERH-2/PICS-1, and ERH-2/TOST-1. Our biochemical experiment also demonstrates that PICS exists as an octamer consisting of two copies of each subunit. Combining structural analyses with mutagenesis experiments, we identify interfacial residues of PICS subunits that are critical for maintaining intact PICS complex in vitro. Furthermore, using genetics, cell biology and imaging experiments, we find that those mutants impairing the in vitro interaction network within PICS, also lead to dysfunction of PICS in vivo, including mislocalization of PICS, and reduced levels of piRNAs or aberrant chromosome segregation and cell division. Therefore, our work provides structural insights into understanding the PICS-mediated piRNA biogenesis and cell division.

Structural recognition of the mRNA 3' UTR by PUF-8 restricts the lifespan of C. elegans.

The molecular mechanisms of aging are unsolved fundamental biological questions. Caenorhabditis elegans is an ideal model organism for investigating aging. PUF-8, a PUF (Pumilio and FBF) protein in C. elegans, is crucial for germline development through binding with the 3' untranslated regions (3' UTR) in the target mRNAs. Recently, PUF-8 was reported to alter mitochondrial dynamics and mitophagy by regulating MFF-1, a mitochondrial fission factor, and subsequently regulated longevity. Here, we determined the crystal structure of the PUF domain of PUF-8 with an RNA substrate. Mutagenesis experiments were performed to alter PUF-8 recognition of its target mRNAs. Those mutations reduced the fertility and extended the lifespan of C. elegans. Deep sequencing of total mRNAs from wild-type and puf-8 mutant worms as well as in vivo RNA Crosslinking and Immunoprecipitation (CLIP) experiments identified six PUF-8 regulated genes, which contain at least one PUF-binding element (PBE) at the 3' UTR. One of the six genes, pqm-1, is crucial for lipid storage and aging process. Knockdown of pqm-1 could revert the lifespan extension of puf-8 mutant animals. We conclude that PUF-8 regulate the lifespan of C. elegans may not only via MFF but also via modulating pqm-1-related pathways.

Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans.

Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that function in the nucleus. We previously found that erroneous rRNAs induce the generation of antisense ribosomal siRNAs (risiRNAs) which silence the expression of rRNAs via the nuclear RNAi defective (Nrde) pathway. To further understand the biological roles and mechanisms of this class of small regulatory RNAs, we conducted forward genetic screening to identify factors involved in risiRNA generation in Caenorhabditis elegans. We found that risiRNAs accumulated in the RNA exosome mutants. risiRNAs directed the association of NRDE proteins with pre-rRNAs and the silencing of pre-rRNAs. In the presence of risiRNAs, NRDE-2 accumulated in the nucleolus and colocalized with RNA polymerase I. risiRNAs inhibited the transcription elongation of RNA polymerase I by decreasing RNAP I occupancy downstream of the RNAi-targeted site. Meanwhile, exosomes mislocalized from the nucleolus to nucleoplasm in suppressor of siRNA (susi) mutants, in which erroneous rRNAs accumulated. These results established a novel model of rRNA surveillance by combining ribonuclease-mediated RNA degradation with small RNA-directed nucleolar RNAi system.

A chromodomain protein mediates heterochromatin-directed piRNA expression.

PIWI-interacting RNAs (piRNAs) play significant roles in suppressing transposons, maintaining genome integrity, and defending against viral infections. How piRNA source loci are efficiently transcribed is poorly understood. Here, we show that in Caenorhabditis elegans, transcription of piRNA clusters depends on the chromatin microenvironment and a chromodomain-containing protein, UAD-2. piRNA clusters form distinct focus in germline nuclei. We conducted a forward genetic screening and identified UAD-2 that is required for piRNA focus formation. In the absence of histone 3 lysine 27 methylation or proper chromatin-remodeling status, UAD-2 is depleted from the piRNA focus. UAD-2 recruits the upstream sequence transcription complex (USTC), which binds the Ruby motif to piRNA promoters and promotes piRNA generation. Vice versa, the USTC complex is required for UAD-2 to associate with the piRNA focus. Thus, transcription of heterochromatic small RNA source loci relies on coordinated recruitment of both the readers of histone marks and the core transcriptional machinery to DNA.

Functional Proteomics Identifies a PICS Complex Required for piRNA Maturation and Chromosome Segregation.

piRNAs play significant roles in suppressing transposons and nonself nucleic acids, maintaining genome integrity, and defending against viral infections. In C. elegans, piRNA precursors are transcribed in the nucleus and are subjected to a number of processing and maturation steps. The biogenesis of piRNAs is not fully understood. We use functional proteomics in C. elegans and identify a piRNA biogenesis and chromosome segregation (PICS) complex. The PICS complex contains TOFU-6, PID-1, PICS-1, TOST-1, and ERH-2, which exhibit dynamic localization among different subcellular compartments. In the germlines, the PICS complex contains TOFU-6/PICS-1/ERH-2/PID-1, is largely concentrated at the perinuclear granule zone, and engages in piRNA processing. During embryogenesis, the TOFU-6/PICS-1/ERH-2/TOST-1 complex accumulates in the nucleus and plays essential roles in chromosome segregation. The functions of these factors in mediating chromosome segregation are independent of piRNA production. We speculate that differential compositions of PICS factors may help cells coordinate distinct cellular processes.

The Functions of Non-coding RNAs in rRNA Regulation.

Ribosomes are ribonucleoprotein machines that decode the genetic information embedded in mRNAs into polypeptides. Ribosome biogenesis is tightly coordinated and controlled from the transcription of pre-rRNAs to the assembly of ribosomes. Defects or disorders in rRNA production result in a number of human ribosomopathy diseases. During the processes of rRNA synthesis, non-coding RNAs, especially snoRNAs, play important roles in pre-rRNA transcription, processing, and maturation. Recent research has started to reveal that other long and short non-coding RNAs, including risiRNA, LoNA, and SLERT (among others), are also involved in pre-rRNA transcription and rRNA production. Here, we summarize the current understanding of the mechanisms of non-coding RNA-mediated rRNA generation and regulation and their biological roles.

The USTC co-opts an ancient machinery to drive piRNA transcription in C. elegans.

Piwi-interacting RNAs (piRNAs) engage Piwi proteins to suppress transposons and nonself nucleic acids and maintain genome integrity and are essential for fertility in a variety of organisms. In Caenorhabditis elegans, most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units. While a few genes have been shown to be required for piRNA biogenesis, the mechanism of piRNA transcription remains elusive. Here we used functional proteomics approaches to identify an upstream sequence transcription complex (USTC) that is essential for piRNA biogenesis. The USTC contains piRNA silencing-defective 1 (PRDE-1), SNPC-4, twenty-one-U fouled-up 4 (TOFU-4), and TOFU-5. The USTC forms unique piRNA foci in germline nuclei and coats the piRNA cluster genomic loci. USTC factors associate with the Ruby motif just upstream of type I piRNA genes. USTC factors are also mutually dependent for binding to the piRNA clusters and forming the piRNA foci. Interestingly, USTC components bind differentially to piRNAs in the clusters and other noncoding RNA genes. These results reveal the USTC as a striking example of the repurposing of a general transcription factor complex to aid in genome defense against transposons.