Germline-expressed Genes Research Articles

Nuclear Argonaute protein NRDE-3 switches small RNA binding partners during embryogenesis coincident with the formation of SIMR granules.

RNA interference (RNAi) is a conserved gene regulation mechanism that utilizes the Argonaute protein and their associated small RNAs to exert regulatory function on complementary transcripts. While the majority of germline-expressed RNAi pathway components reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here we find that the small RNA biogenesis machinery is spatially and temporally organized during embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. Further, we observe that many other RNAi factors form foci in embryonic cells distinct from SIMR granules, including the Argonaute protein CSR-1, underscoring a potential role for cytoplasmic concentrates of RNAi factors to promote gene regulation in embryos. Curiously, coincident with the appearance of the "SIMR granules", the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during early embryogenesis and switching later in embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions.

Programmed DNA elimination in the parasitic nematode Ascaris.

In most organisms, the whole genome is maintained throughout the life span. However, exceptions occur in some species where the genome is reduced during development through a process known as programmed DNA elimination (PDE). In the human and pig parasite Ascaris, PDE occurs during the 4 to 16 cell stages of embryogenesis, when germline chromosomes are fragmented and specific DNA sequences are reproducibly lost in all somatic cells. PDE was identified in Ascaris over 120 years ago, but little was known about its molecular details until recently. Genome sequencing revealed that approximately 1,000 germline-expressed genes are eliminated in Ascaris, suggesting PDE is a gene silencing mechanism. All germline chromosome ends are removed and remodeled during PDE. In addition, PDE increases the number of chromosomes in the somatic genome by splitting many germline chromosomes. Comparative genomics indicates that these germline chromosomes arose from fusion events. PDE separates these chromosomes at the fusion sites. These observations indicate that PDE plays a role in chromosome karyotype and evolution. Furthermore, comparative analysis of PDE in other parasitic and free-living nematodes illustrates conserved features of PDE, suggesting it has important biological significance. We summarize what is known about PDE in Ascaris and its relatives. We also discuss other potential functions, mechanisms, and the evolution of PDE in these parasites of humans and animals of veterinary importance.

Functional recovery of the germ line following splicing collapse.

Splicing introns from precursor-messenger RNA (pre-mRNA) transcripts is essential for translating functional proteins. Here, we report that the previously uncharacterized Caenorhabditis elegans protein MOG-7 acts as a pre-mRNA splicing factor. Depleting MOG-7 from the C. elegans germ line causes intron retention in most germline-expressed genes, impeding the germ cell cycle, and causing defects in nuclear morphology, germ cell identity and sterility. Despite the deleterious consequences caused by MOG-7 loss, the adult germ line can functionally recover to produce viable and fertile progeny when MOG-7 is restored. Germline recovery is dependent on a burst of apoptosis that likely clears defective germ cells, and viable gametes generated from the proliferation of germ cells in the progenitor zone. Together, these findings reveal that MOG-7 is essential for germ cell development, and that the germ line can functionally recover after a collapse in RNA splicing.

PiRNAs coordinate poly(UG) tailing to prevent aberrant and perpetual gene silencing

Noncoding RNAs have emerged as mediators of transgenerational epigenetic inheritance (TEI) in a number of organisms. A robust example of such RNA-directed TEI is the inheritance of gene-silencing states following RNA interference (RNAi) in the metazoan C.elegans. During RNAi inheritance, gene silencing is transmitted by a self-perpetuating cascade of siRNA-directed poly(UG) tailing of mRNA fragments (pUGylation), followed by siRNA synthesis from poly(UG)-tailed mRNA templates (termed pUG RNA/siRNA cycling). Despite the self-perpetuating nature of pUG RNA/siRNA cycling, RNAi inheritance is finite, suggesting that systems likely exist to prevent indefinite RNAi-triggered gene silencing. Here we show that, in the absence of Piwi-interacting RNAs (piRNAs), an animal-specific class of small noncoding RNA, RNAi-based gene silencing can become essentially permanent, lasting at near 100% penetrance for more than 5 years and hundreds of generations. This perpetual gene silencing is mediated by continuous pUG RNA/siRNA cycling. Further, we find that piRNAs coordinate endogenous RNAi pathways to prevent germline-expressed genes, which are not normally subjected to TEI, from entering a state of continual and irreversible epigenetic silencing also mediated by continuous maintenance of pUG RNA/siRNA cycling. Together, our results show that one function of C.elegans piRNAs is to insulate germline-expressed genes from aberrant and runaway inactivation by the pUG RNA/siRNA epigenetic inheritance system.

Arginine methylation promotes siRNA-binding specificity for a spermatogenesis-specific isoform of the Argonaute protein CSR-1

CSR-1 is an essential Argonaute protein that binds to a subclass of 22G-RNAs targeting most germline-expressed genes. Here we show that the two isoforms of CSR-1 have distinct expression patterns; CSR-1B is ubiquitously expressed throughout the germline and during all stages of development while CSR-1A expression is restricted to germ cells undergoing spermatogenesis. Furthermore, CSR-1A associates preferentially with 22G-RNAs mapping to spermatogenesis-specific genes whereas CSR-1B-bound small RNAs map predominantly to oogenesis-specific genes. Interestingly, the exon unique to CSR-1A contains multiple dimethylarginine modifications, which are necessary for the preferential binding of CSR-1A to spermatogenesis-specific 22G-RNAs. Thus, we have discovered a regulatory mechanism for C. elegans Argonaute proteins that allows for specificity of small RNA binding between similar Argonaute proteins with overlapping temporal and spatial localization.

PiRNAs Coordinate Poly(UG) Tailing to Prevent Aberrant and Permanent Gene Silencing

Noncoding RNAs have emerged as mediators of transgenerational epigenetic inheritance (TEI) in a number of organisms. A robust example of RNA-directed TEI is the inheritance of gene silencing states following RNA interference (RNAi) in the metazoan C. elegans. During RNAi inheritance, gene silencing is transmitted by a self-perpetuating cascade of siRNA-directed poly(UG) tailing of mRNA fragments (pUGylation), followed by siRNA synthesis from poly(UG)-tailed mRNA templates (termed pUG RNA/siRNA cycling). Despite the self-perpetuating nature of pUG RNA/siRNA cycling, RNAi inheritance is finite, suggesting that systems likely exist to prevent permanent RNAi-triggered gene silencing. Here we show that, in the absence of Piwi-interacting RNAs (piRNAs), an animal-specific class of small noncoding RNA, RNAi-based gene silencing can become essentially permanent, lasting at near 100% penetrance for more than five years and hundreds of generations. This permanent gene silencing is mediated by perpetual activation of the pUG RNA/siRNA TEI pathway. Further, we find that piRNAs coordinate endogenous RNAi pathways to prevent germline-expressed genes, which are not normally subjected to TEI, from entering a state of permanent and irreversible epigenetic silencing also mediated by perpetual activation of pUG RNA/siRNA cycling. Together, our results show that one function of C. elegans piRNAs is to insulate germline-expressed genes from aberrant and runaway inactivation by the pUG RNA/siRNA epigenetic inheritance system.

Comprehensive Chromosome End Remodeling during Programmed DNA Elimination

Germline and somatic genomes are in general the same in a multicellular organism. However, programmed DNA elimination leads to a reduced somatic genome compared to germline cells. Previous work on the parasitic nematode Ascaris demonstrated that programmed DNA elimination encompasses high-fidelity chromosomal breaks and loss of specific genome sequences including a major tandem repeat of 120bp and ~1,000 germline-expressed genes. However, the precise chromosomal locations of these repeats, breaks regions, and eliminated genes remained unknown. We used PacBio long-read sequencing and chromosome conformation capture (Hi-C) to obtain fully assembled chromosomes of Ascaris germline and somatic genomes, enabling a complete chromosomal view of DNA elimination. We found that all 24 germline chromosomes undergo comprehensive chromosome end remodeling with DNA breaks in their subtelomeric regions and loss of distal sequences including the telomeres at both chromosome ends. All new Ascaris somatic chromosome ends are recapped by de novo telomere healing. We provide an ultrastructural analysis of Ascaris DNA elimination and show that eliminated DNA is incorporated into double membrane-bound structures, similar to micronuclei, during telophase of a DNA elimination mitosis. These micronuclei undergo dynamic changes including loss of active histone marks and localize to the cytoplasm following daughter nuclei formation and cytokinesis where they form autophagosomes. Comparative analysis of nematode chromosomes suggests that chromosome fusions occurred, forming Ascaris sex chromosomes that become independent chromosomes following DNA elimination breaks in somatic cells. These studies provide the first chromosomal view and define novel features and functions of metazoan programmed DNA elimination.

Defend Thyself and Thy Offspring.

Three recent studies (Dodson and Kennedy, 2019; Lev etal., 2019; Ouyang etal., 2019) reveal that germ granule formation is necessary to protect germline-expressed genes from improper small RNA-mediated silencing. Loss of this protection leads to accumulation of small RNAs, impacting gene expression in multiple subsequent generations.

Germ Granules Coordinate RNA-Based Epigenetic Inheritance Pathways.

Germ granules are biomolecular condensates that promote germ cell totipotency in animals. In C.elegans, MEG-3 and MEG-4 function redundantly to assemble germ granules in germline blastomeres. Here, we show that meg-3/4 mutant animals exhibit defects in RNA interference (RNAi) that are transgenerationally disconnected from the meg-3/4 genotype. Similar non-Mendelian inheritance is associated with other mutations disrupting germ granule formation, indicating that loss of germ granules is the likely cause of the observed disconnects between genotype and phenotype. meg-3/4 animals produce aberrant siRNAs that are propagated for≅10 generations in wild-type descendants of meg-3/4 ancestors. Aberrant siRNAs inappropriately and heritably silence germline-expressed genes including the RNAi gene sid-1, suggesting that transgenerational silencing of sid-1 underlies inherited defects in RNAi. We conclude that one function of germ granules is to organize RNA-based epigenetic inheritance pathways and that germ granule loss has consequences that persist for many generations.

Mutational Landscape of Spontaneous Base Substitutions and Small Indels in Experimental Caenorhabditis elegans Populations of Differing Size.

Experimental investigations into the rates and fitness effects of spontaneous mutations are fundamental to our understanding of the evolutionary process. To gain insights into the molecular and fitness consequences of spontaneous mutations, we conducted a mutation accumulation (MA) experiment at varying population sizes in the nematode Caenorhabditis elegans, evolving 35 lines in parallel for 409 generations at three population sizes (N = 1, 10, and 100 individuals). Here, we focus on nuclear SNPs and small insertion/deletions (indels) under minimal influence of selection, as well as their accrual rates in larger populations under greater selection efficacy. The spontaneous rates of base substitutions and small indels are 1.84 (95% C.I. ± 0.14) × 10-9 substitutions and 6.84 (95% C.I. ± 0.97) × 10-10 changes/site/generation, respectively. Small indels exhibit a deletion bias with deletions exceeding insertions by threefold. Notably, there was no correlation between the frequency of base substitutions, nonsynonymous substitutions, or small indels with population size. These results contrast with our previous analysis of mitochondrial DNA mutations and nuclear copy-number changes in these MA lines, and suggest that nuclear base substitutions and small indels are under less stringent purifying selection compared to the former mutational classes. A transition bias was observed in exons as was a near universal base substitution bias toward A/T. Strongly context-dependent base substitutions, where 5'-Ts and 3'-As increase the frequency of A/T → T/A transversions, especially at the boundaries of A or T homopolymeric runs, manifest as higher mutation rates in (i) introns and intergenic regions relative to exons, (ii) chromosomal cores vs. arms and tips, and (iii) germline-expressed genes.

UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase is indispensable for oogenesis, oocyte-to-embryo transition, and larval development of the nematode Caenorhabditis elegans.

N-linked glycosylation of proteins is the most common post-translational modification of proteins. The enzyme UDP-N-acetylglucosamine-dolichyl-phosphate N-acetylglucosaminephosphotransferase (DPAGT1) catalyses the first step of N-glycosylation, and DPAGT1 knockout is embryonic lethal in mice. In this study, we identified the sole orthologue (algn-7) of the human DPAGT1 in the nematode C. elegans. The gene activity was disrupted by RNAi and deletion mutagenesis, which resulted in larval lethality, defects in oogenesis and oocyte-to-embryo transition. Endomitotic oocytes, abnormal fusion of pronuclei, abnormal AB cell rotation, disruption of permeation barriers of eggs, and abnormal expression of chitin and chitin synthase in oocytes and eggs were the typical phenotypes observed. The results indicate that N-glycosylation is indispensable for these processes. We further screened an N-glycosylated protein database of C. elegans, and identified 456 germline-expressed genes coding N-glycosylated proteins. By examining RNAi phenotypes, we identified five germline-expressed genes showing similar phenotypes to the algn-7 (RNAi) animals. They were ribo-1, stt-3, ptc-1, ptc-2, and vha-19. We identified known congenital disorders of glycosylation (CDG) genes (ribo-1 and stt-3) and a recently found CDG gene (vha-19). The results show that phenotype analyses using the nematode could be a powerful tool to detect new CDG candidate genes and their associated gene networks.

The piRNA targeting rules and the resistance to piRNA silencing in endogenous genes.

Piwi-interacting RNAs (piRNAs) silence transposons to safeguard genome integrity in animals. However, the functions of the many piRNAs that do not map to transposons remain unknown. Here, we show that piRNA targeting in Caenorhabditis elegans can tolerate a few mismatches but prefer perfect pairing at the seed region. The broad targeting capacity of piRNAs underlies the germline silencing of transgenes in C. elegans Transgenes engineered to avoid piRNA recognition are stably expressed. Many endogenous germline-expressed genes also contain predicted piRNA targeting sites, and periodic An/Tn clusters (PATCs) are an intrinsic signal that provides resistance to piRNA silencing. Together, our study revealed the piRNA targeting rules and highlights a distinct strategy that C. elegans uses to distinguish endogenous from foreign nucleic acids.

The Germline-Specific Factor OEF-1 Facilitates Coordinated Progression Through Germ Cell Development in Caenorhabditis elegans.

The purpose of germ cells is to ensure the faithful transmission of genetic material to the next generation. To develop into mature gametes, germ cells must pass through cell cycle checkpoints while maintaining totipotency and genomic integrity. How germ cells coordinate developmental events while simultaneously protecting their unique fate is not well understood. Here, we characterize a novel nuclear protein, Oocyte-Excluded Factor-1 (OEF-1), with highly specific germline expression in Caenorhabditis elegans OEF-1 is initially detected early in embryogenesis and is expressed in the nuclei of all germ cells during larval stages. In adults, OEF-1 expression abruptly decreases just prior to oocyte differentiation. In oef-1 mutants, the developmental progression of germ cells is accelerated, resulting in subtle defects at multiple stages of germ cell development. Lastly, OEF-1 is primarily associated with the bodies of germline-expressed genes, and as such is excluded from the X chromosome. We hypothesize that OEF-1 may regulate the rate of progression through germ cell development, providing insight into how these critical maturation events are coordinated.

Evidence for a Temporal Window of Argonaute Surveillance Prior to mRNA Translation in the C. Elegans Germline

Piwi Argonautes and their piRNA co-factors silence transposons in animal germlines. However, little is known about whether and how piRNAs regulate other germline-expressed mRNAs. Here we explore Piwi-mediated gene regulation in C. elegans. We show that mRNAs expressed within a common cytoplasm and with identical piRNA-targeted regions, can nevertheless differ in their sensitivity to piRNA silencing. Sensitivity was largely determined by the coding region of the mRNA, exclusive of the promoter, introns, and untranslated regions (UTRs). We show that piRNA scanning occurs independently, or upstream, of nonsense-mediated-decay (NMD), a pathway that mediates surveillance of transcripts for premature stop codons. Our findings reveal that C. elegans piRNAs provide incremental regulation on germline-expressed genes and suggest that piRNA scan mRNAs prior to ribo some access, likely during transit through peri-nuclear germline nuage.

Identification of Cis-Regulatory Modules that Function in the Male Germline of Flowering Plants.

The male germline of flowering plants develops within the vegetative cell of the male gametophyte and displays a distinct transcriptional profile. Key to understanding the development of this unique cell lineage is determining how gene expression is regulated within germline cells. This knowledge impacts upon our understanding of cell specification, differentiation, and plant fertility. Here, we describe methods to identify cis-regulatory modules (CRMs) that act as key regulatory regions in the promoters of germline-expressed genes. We detail the complimentary techniques of phylogenetic footprinting and the use of fluorescent reporters in pollen for the identification and verification of CRMs.

Opposing Activities of DRM and MES-4 Tune Gene Expression and X-Chromosome Repression inCaenorhabditis elegansGerm Cells

During animal development, gene transcription is tuned to tissue-appropriate levels. Here we uncover antagonistic regulation of transcript levels in the germline of Caenorhabditis elegans hermaphrodites. The histone methyltransferase MES-4 (Maternal Effect Sterile-4) marks genes expressed in the germline with methylated lysine on histone H3 (H3K36me) and promotes their transcription; MES-4 also represses genes normally expressed in somatic cells and genes on the X chromosome. The DRM transcription factor complex, named for its Dp/E2F, Retinoblastoma-like, and MuvB subunits, affects germline gene expression and prevents excessive repression of X-chromosome genes. Using genome-scale analyses of germline tissue, we show that common germline-expressed genes are activated by MES-4 and repressed by DRM, and that MES-4 and DRM co-bind many germline-expressed genes. Reciprocally, MES-4 represses and DRM activates a set of autosomal soma-expressed genes and overall X-chromosome gene expression. Mutations in mes-4 and the DRM subunit lin-54 oppositely skew the transcript levels of their common targets and cause sterility. A double mutant restores target gene transcript levels closer to wild type, and the concomitant loss of lin-54 suppresses the severe germline proliferation defect observed in mes-4 single mutants. Together, “yin-yang” regulation by MES-4 and DRM ensures transcript levels appropriate for germ-cell function, elicits robust but not excessive dampening of X-chromosome-wide transcription, and may poise genes for future expression changes. Our study reveals that conserved transcriptional regulators implicated in development and cancer counteract each other to fine-tune transcript dosage.

Multiple small RNA pathways regulate the silencing of repeated and foreign genes in C. elegans

Gene segments from other organisms, such as viruses, are detected as foreign and targeted for silencing by RNAi pathways. A deep-sequencing map of the small RNA response to repeated transgenes introduced to Caenorhabditis elegans revealed that specific segments are targeted by siRNAs. Silencing of the foreign gene segments depends on an antiviral response that involves changes in active and silent chromatin modifications and altered levels of antisense siRNAs. Distinct Argonaute proteins target foreign genes for silencing or protection against silencing. We used a repeated transgene in a genome-wide screen to identify gene disruptions that enhance silencing of foreign genetic elements and identified 69 genes. These genes cluster in four groups based on overlapping sets of coexpressed genes, including a group of germline-expressed genes that are likely coregulated by the E2F transcription factor. Many of the gene inactivations enhance exogenous RNAi. About half of the 69 genes have roles in endogenous RNAi pathways that regulate diverse processes, including silencing of duplicated genes and transposons and chromosome segregation. Of these newly identified genes, several are required for siRNA biogenesis or stability in the oocyte-specific ERGO-1 pathway, including eri-12, encoding an interactor of the RNAi-defective protein RDE-10, and ntl-9/CNOT9, one of several CCR4/NOT complex genes that we identified. The conserved ARF-like small GTPase ARL-8 is required specifically for primary siRNA biogenesis or stability in the sperm-specific ALG-3/4 endogenous RNAi pathway.

Protection of Germline Gene Expression by the C. elegans Argonaute CSR-1

In Caenorhabditis elegans, the Piwi-interacting small RNA (piRNA)-mediated germline surveillance system encodes more than 30,000 unique 21-nucleotide piRNAs, which silence a variety of foreign nucleic acids. What mechanisms allow endogenous germline-expressed transcripts to evade silencing by the piRNA pathway? One likely candidate in a protective mechanism is the Argonaute CSR-1, which interacts with 22G-small RNAs that are antisense to nearly all germline-expressed genes. Here, we use an in vivo RNA tethering assay to demonstrate that the recruitment of CSR-1 to a transcript licenses expression of the transcript, protecting it from piRNA-mediated silencing. Licensing occurs mainly at the level of transcription, as we observe changes in pre-mRNA levels consistent with transcriptional activation when CSR-1 is tethered. Furthermore, the recruitment of CSR-1 to a previously silenced locus transcriptionally activates its expression. Together, these results demonstrate a rare positive role for an endogenous Argonaute pathway in heritably licensing and protecting germline transcripts.

Silencing of Germline-Expressed Genes by DNA Elimination in Somatic Cells

Chromatin diminution is the programmed elimination of specific DNA sequences during development. Itoccurs in diverse species, but the function(s) of diminution and the specificity of sequence loss remain largely unknown. Diminution in the nematode Ascaris suum occurs during early embryonic cleavages and leads to the loss of germline genome sequences and the formation of a distinct genome in somatic cells. We found that ∼43 Mb (∼13%) of genome sequence is eliminated in A.suum somatic cells, including ∼12.7 Mb of unique sequence. The eliminated sequences and location of the DNA breaks are the same in all somatic lineages from a single individual and between different individuals. At least 685 genes are eliminated. These genes arepreferentially expressed in the germline and during early embryogenesis. We propose that diminution is a mechanism of germline gene regulation that specifically removes a large number of genes involved in gametogenesis and early embryogenesis.

Antagonism between MES-4 and Polycomb Repressive Complex 2 Promotes Appropriate Gene Expression in C. elegans Germ Cells

The Caenorhabditis elegans MES proteins are key chromatin regulators of the germline. MES-2, MES-3, and MES-6 form the C.elegans Polycomb repressive complex 2 and generate repressive H3K27me3. MES-4 generates H3K36me3 on germline-expressed genes. Transcript profiling of dissected mutant germlines revealed that MES-2/3/6 and MES-4 cooperate to promote the expression of germline genes and repress the X chromosomes and somatic genes. Results from genome-wide chromatin immunoprecipitation showed that H3K27me3 and H3K36me3 occupy mutually exclusive domains on the autosomes and that H3K27me3 is enriched on the X. Loss of MES-4 from germline genes causes H3K27me3 to spread to germline genes, resulting in reduced H3K27me3 elsewhere on the autosomes and especially on the X. Our findings support a model in which H3K36me3 repels H3K27me3 from germline genes and concentrates it on other regions of the genome. This antagonism ensures proper patterns of gene expression for germ cells, which includes repression of somatic genes and the X chromosomes.