Selecting genes for analysis using historically contingent progress: from RNA changes to protein-protein interactions.

Progress in biology has generated numerous lists of genes that share some property. But, advancing from these lists of genes to understanding their roles is slow and unsystematic. Here we use RNA silencing in C. elegans to illustrate an approach for prioritizing genes for detailed study given limited resources. The partially subjective relationships between genes forged by both deduced functional relatedness and biased progress in the field was captured as mutual information and used to cluster genes that were frequently identified yet remain understudied. Studied genes in these clusters suggest regulatory links connecting RNA silencing with other processes like the cell cycle. Many proteins encoded by the understudied genes are predicted to physically interact with known regulators of RNA silencing. These predicted influencers of RNA-regulated expression could be used for feedback regulation, which is essential for the homeostasis observed in all living systems. Thus, among the gene products altered when a process is perturbed are regulators of that process, providing a way to use RNA sequencing to identify candidate protein-protein interactions. Together, the analysis of perturbed transcripts and potential interactions of the proteins they encode could help prioritize candidate regulators of any process.

Heritable epigenetic changes are constrained by the dynamics of regulatory architectures.

Interacting molecules create regulatory architectures that can persist despite turnover of molecules. Although epigenetic changes occur within the context of such architectures, there is limited understanding of how they can influence the heritability of changes. Here, I develop criteria for the heritability of regulatory architectures and use quantitative simulations of interacting regulators parsed as entities, their sensors, and the sensed properties to analyze how architectures influence heritable epigenetic changes. Information contained in regulatory architectures grows rapidly with the number of interacting molecules and its transmission requires positive feedback loops. While these architectures can recover after many epigenetic perturbations, some resulting changes can become permanently heritable. Architectures that are otherwise unstable can become heritable through periodic interactions with external regulators, which suggests that mortal somatic lineages with cells that reproducibly interact with the immortal germ lineage could make a wider variety of architectures heritable. Differential inhibition of the positive feedback loops that transmit regulatory architectures across generations can explain the gene-specific differences in heritable RNA silencing observed in the nematode Caenorhabditis elegans. More broadly, these results provide a foundation for analyzing the inheritance of epigenetic changes within the context of the regulatory architectures implemented using diverse molecules in different living systems.

Evidence for multiple forms of heritable RNA silencing.

Heritable gene silencing has been proposed to rely on DNA methylation, histone modifications, and/or non-coding RNAs in different organisms. Here we demonstrate that multiple RNA-mediated mechanisms with distinct and easily detectable molecular signatures can underlie heritable silencing of the same open-reading frame in the nematode C. elegans. Using two-gene operons, we reveal three cases of gene-selective silencing that provide support for the transmission of heritable epigenetic changes through different mechanisms of RNA silencing independent of changes in chromatin that would affect all genes of an operon equally. Different heritable epigenetic states of a gene were associated with distinct populations of stabilized mRNA fragments with untemplated poly-UG (pUG) tails, which are known intermediates of RNA silencing. These 'pUG signatures' provide a way to distinguish the multiple mechanisms that can drive heritable RNA silencing of a single gene.

Target-specific requirements for RNA interference can arise through restricted RNA amplification despite the lack of specialized pathways.

Since double-stranded RNA (dsRNA) is effective for silencing a wide variety of genes, all genes are typically considered equivalent targets for such RNA interference (RNAi). Yet, loss of some regulators of RNAi in the nematode C. elegans can selectively impair the silencing of some genes. Here we show that such selective requirements can be explained by an intersecting network of regulators acting on genes with differences in their RNA metabolism. In this network, the Maelstrom domain-containing protein RDE-10, the intrinsically disordered protein MUT-16, and the Argonaute protein NRDE-3 work together so that any two are required for silencing one somatic gene, but each is singly required for silencing another somatic gene, where only the requirement for NRDE-3 can be overcome by enhanced dsRNA processing. Quantitative models and their exploratory simulations led us to find that (1) changing cis-regulatory elements of the target gene can reduce the dependence on NRDE-3, (2) animals can recover from silencing in non-dividing cells and (3) cleavage and tailing of mRNAs with UG dinucleotides, which makes them templates for amplifying small RNAs, is enriched within 'pUG zones' matching the dsRNA. Similar crosstalk between pathways and restricted amplification could result in apparently selective silencing by endogenous RNAs.

Heritable epigenetic changes are constrained by the dynamics of regulatory architectures.

Interacting molecules create regulatory architectures that can persist despite turnover of molecules. Although epigenetic changes occur within the context of such architectures, there is limited understanding of how they can influence the heritability of changes. Here I develop criteria for the heritability of regulatory architectures and use quantitative simulations of interacting regulators parsed as entities, their sensors and the sensed properties to analyze how architectures influence heritable epigenetic changes. Information contained in regulatory architectures grows rapidly with the number of interacting molecules and its transmission requires positive feedback loops. While these architectures can recover after many epigenetic perturbations, some resulting changes can become permanently heritable. Such stable changes can (1) alter steady-state levels while preserving the architecture, (2) induce different architectures that persist for many generations, or (3) collapse the entire architecture. Architectures that are otherwise unstable can become heritable through periodic interactions with external regulators, which suggests that the evolution of mortal somatic lineages with cells that reproducibly interact with the immortal germ lineage could make a wider variety of regulatory architectures heritable. Differential inhibition of the positive feedback loops that transmit regulatory architectures across generations can explain the gene-specific differences in heritable RNA silencing observed in the nematode C. elegans, which range from permanent silencing to recovery from silencing within a few generations and subsequent resistance to silencing. More broadly, these results provide a foundation for analyzing the inheritance of epigenetic changes within the context of the regulatory architectures implemented using diverse molecules in different living systems.

Heritable epigenetic changes at single genes: challenges and opportunities in Caenorhabditis elegans.

Organisms rely on stereotyped patterns of gene expression for similar form and function in every generation. The analysis of epigenetic changes in the expression of different genes across generations can provide the rationale for measured actions in one generation that consider impact on future generations.

Mating can initiate stable RNA silencing that overcomes epigenetic recovery.

Stable epigenetic changes appear uncommon, suggesting that changes typically dissipate or are repaired. Changes that stably alter gene expression across generations presumably require particular conditions that are currently unknown. Here we report that a minimal combination of cis-regulatory sequences can support permanent RNA silencing of a single-copy transgene and its derivatives in C. elegans simply upon mating. Mating disrupts competing RNA-based mechanisms to initiate silencing that can last for >300 generations. This stable silencing requires components of the small RNA pathway and can silence homologous sequences in trans. While animals do not recover from mating-induced silencing, they often recover from and become resistant to trans silencing. Recovery is also observed in most cases when double-stranded RNA is used to silence the same coding sequence in different regulatory contexts that drive germline expression. Therefore, we propose that regulatory features can evolve to oppose permanent and potentially maladaptive responses to transient change.

The FDA-approved drugs ticlopidine, sertaconazole, and dexlansoprazole can cause morphological changes in C. elegans.

Urgent need for treatments limit studies of therapeutic drugs before approval by regulatory agencies. Analyses of drugs after approval can therefore improve our understanding of their mechanism of action and enable better therapies. We screened a library of 1443 Food and Drug Administration (FDA)-approved drugs using a simple assay in the nematode C. elegans and found three compounds that caused morphological changes. While the anticoagulant ticlopidine and the antifungal sertaconazole caused both accumulations that resulted in distinct distortions of pharyngeal anatomy and lethality upon acute exposure, the proton-pump inhibitor dexlansoprazole caused molting defects and required exposure during larval development. Such easily detectable defects in a powerful genetic model system advocate the continued exploration of current medicines using a variety of model organisms to better understand drugs already prescribed to millions of patients.

The analysis of living systems can generate both knowledge and illusions.

Life relies on phenomena that range from changes in molecules that occur within nanoseconds to changes in populations that occur over millions of years. Researchers have developed a vast range of experimental techniques to analyze living systems, but a given technique usually only works over a limited range of length or time scales. Therefore, gaining a full understanding of a living system usually requires the integration of information obtained at multiple different scales by two or more techniques. This approach has undoubtedly led to a much better understanding of living systems but, equally, the staggering complexity of these systems, the sophistication and limitations of the techniques available in modern biology, and the need to use two or more techniques, can lead to persistent illusions of knowledge. Here, in an effort to make better use of the experimental techniques we have at our disposal, I propose a broad classification of techniques into six complementary approaches: perturbation, visualization, substitution, characterization, reconstitution, and simulation. Such a taxonomy might also help increase the reproducibility of inferences and improve peer review.

A framework for parsing heritable information.

Living systems transmit heritable information using the replicating gene sequences and the cycling regulators assembled around gene sequences. Here, I develop a framework for heredity and development that includes the cycling regulators parsed in terms of what an organism can sense about itself and its environment by defining entities, their sensors and the sensed properties. Entities include small molecules (ATP, ions, metabolites, etc.), macromolecules (individual proteins, RNAs, polysaccharides, etc.) and assemblies of molecules. While concentration may be the only relevant property measured by sensors for small molecules, multiple properties that include concentration, sequence, conformation and modification may all be measured for macromolecules and assemblies. Each configuration of these entities and sensors that is recreated in successive generations in a given environment thus specifies a potentially vast amount of information driving complex development in each generation. This entity-sensor-property framework explains how sensors limit the number of distinguishable states, how distinct molecular configurations can be functionally equivalent and how regulation of sensors prevents detection of some perturbations. Overall, this framework is a useful guide for understanding how life evolves and how the storage of information has itself evolved with complexity since before the origin of life.

Heritable Epigenetic Changes Alter Transgenerational Waveforms Maintained by Cycling Stores of Information.

Our view of heredity can potentially be distorted by the ease of introducing heritable changes in the replicating gene sequences but not in the cycling assembly of regulators around gene sequences. Here, key experiments that have informed the understanding of heredity are reinterpreted to highlight this distortion and the possible variety of heritable changes are considered. Unlike heritable genetic changes, which are always associated with mutations in gene sequence, heritable epigenetic changes can be associated with physical or chemical changes in molecules or only changes in the system. The transmission of cycling stores along the continuous lineage of cells that connects successive generations creates waves of activity and localization of the molecules that together form the cell code for development in each generation. As a result, heritable epigenetic changes can include any that can alter a wave such as changes in form, midline, frequency, amplitude, or phase. Testing this integrated view of all heritable information will require the concerted application of multiple experimental approaches across generations.

Gene silencing by double-stranded RNA from C. elegans neurons reveals functional mosaicism of RNA interference.

Delivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the nematode Caenorhabditis elegans, dsRNA from multiple sources can trigger the amplification of silencing signals. Amplification occurs through the production of small RNAs by two RNA-dependent RNA polymerases (RdRPs) that are thought to be tissue-specific - EGO-1 in the germline and RRF-1 in somatic cells. Here we demonstrate that EGO-1 can compensate for the lack of RRF-1 when dsRNA from neurons is used to silence genes in intestinal cells. However, the lineal origins of cells that can use EGO-1 varies. This variability could be because random sets of cells can either receive different amounts of dsRNA from the same source or use different RdRPs to perform the same function. Variability is masked in wild-type animals, which show extensive silencing by neuronal dsRNA. As a result, cells appear similarly functional despite underlying differences that vary from animal to animal. This functional mosaicism cautions against inferring uniformity of mechanism based on uniformity of outcome. We speculate that functional mosaicism could contribute to escape from targeted therapies and could allow developmental systems to drift over evolutionary time.

Replicating and Cycling Stores of Information Perpetuate Life.

Life is perpetuated through a single-cell bottleneck between generations in many organisms. Here, I highlight that this cell holds information in two distinct stores: in the linear DNA sequence that is replicated during cell divisions, and in the three-dimensional arrangement of molecules that can change during development but is recreated at the start of each generation. These two interdependent stores of information - one replicating with each cell division and the other cycling with a period of one generation - coevolve while perpetuating an organism. Unlike the genome sequence, the arrangement of molecules, including DNA, RNAs, proteins, sugars, lipids, etc., is not well understood. Because this arrangement and the genome sequence are transmitted together from one generation to the next, analysis of both is necessary to understand evolution and origins of inherited diseases. Recent developments suggest that tools are in place to examine how all the information to build an organism is encoded within a single cell, and how this cell code is reproduced in every generation. See also the video abstract here: https://youtu.be/IdWEL-T6TPU.

The double-stranded RNA binding protein RDE-4 can act cell autonomously during feeding RNAi in C. elegans.

Long double-stranded RNA (dsRNA) can silence genes of matching sequence upon ingestion in many invertebrates and is therefore being developed as a pesticide. Such feeding RNA interference (RNAi) is best understood in the worm Caenorhabditis elegans, where the dsRNA-binding protein RDE-4 initiates silencing by recruiting an endonuclease to process long dsRNA into short dsRNA. These short dsRNAs are thought to move between cells because muscle-specific rescue of rde-4 using repetitive transgenes enables silencing in other tissues. Here, we extend this observation using additional promoters, report an inhibitory effect of repetitive transgenes, and discover conditions for cell-autonomous silencing in animals with tissue-specific rescue of rde-4. While expression of rde-4(+) in intestine, hypodermis, or neurons using a repetitive transgene can enable silencing also in unrescued tissues, silencing can be inhibited wihin tissues that express a repetitive transgene. Single-copy transgenes that express rde-4(+) in body-wall muscles or hypodermis, however, enable silencing selectively in the rescued tissue but not in other tissues. These results suggest that silencing by the movement of short dsRNA between cells is not an obligatory feature of feeding RNAi in C. elegans. We speculate that similar control of dsRNA movement could modulate tissue-specific silencing by feeding RNAi in other invertebrates.

Removing bias against short sequences enables northern blotting to better complement RNA-seq for the study of small RNAs.

Changes in small non-coding RNAs such as micro RNAs (miRNAs) can serve as indicators of disease and can be measured using next-generation sequencing of RNA (RNA-seq). Here, we highlight the need for approaches that complement RNA-seq, discover that northern blotting of small RNAs is biased against short sequences and develop a protocol that removes this bias. We found that multiple small RNA-seq datasets from the worm Caenorhabditis elegans had shorter forms of miRNAs that appear to be degradation products that arose during the preparatory steps required for RNA-seq. When using northern blotting during these studies, we discovered that miRNA-length probes can have ∼1000-fold bias against detecting even synthetic sequences that are 8 nt shorter. By using shorter probes and by performing hybridization and washes at low temperatures, we greatly reduced this bias to enable nearly equivalent detection of 24 to 14 nt RNAs. Our protocol can discriminate RNAs that differ by a single nucleotide and can detect specific miRNAs present in total RNA from C. elegans and pRNAs in total RNA from bacteria. This improved northern blotting is particularly useful to analyze products of RNA processing or turnover, and functional RNAs that are shorter than typical miRNAs.

Extracellular RNA is transported from one generation to the next in Caenorhabditis elegans.

Experiences during the lifetime of an animal have been proposed to have consequences for subsequent generations. Although it is unclear how such intergenerational transfer of information occurs, RNAs found extracellularly in animals are candidate molecules that can transfer gene-specific regulatory information from one generation to the next because they can enter cells and regulate gene expression. In support of this idea, when double-stranded RNA (dsRNA) is introduced into some animals, the dsRNA can silence genes of matching sequence and the silencing can persist in progeny. Such persistent gene silencing is thought to result from sequence-specific interaction of the RNA within parents to generate chromatin modifications, DNA methylation, and/or secondary RNAs, which are then inherited by progeny. Here, we show that dsRNA can be directly transferred between generations in the worm Caenorhabditis elegans Intergenerational transfer of dsRNA occurs even in animals that lack any DNA of matching sequence, and dsRNA that reaches progeny can spread between cells to cause gene silencing. Surprisingly, extracellular dsRNA can also reach progeny without entry into the cytosol, presumably within intracellular vesicles. Fluorescently labeled dsRNA is imported from extracellular space into oocytes along with yolk and accumulates in punctate structures within embryos. Subsequent entry into the cytosol of early embryos causes gene silencing in progeny. These results demonstrate the transport of extracellular RNA from one generation to the next to regulate gene expression in an animal and thus suggest a mechanism for the transmission of experience-dependent effects between generations.

Tissue homogeneity requires inhibition of unequal gene silencing during development.

Multicellular organisms can generate and maintain homogenous populations of cells that make up individual tissues. However, cellular processes that can disrupt homogeneity and how organisms overcome such disruption are unknown. We found that ∼100-fold differences in expression from a repetitive DNA transgene can occur between intestinal cells in Caenorhabditis elegans These differences are caused by gene silencing in some cells and are actively suppressed by parental and zygotic factors such as the conserved exonuclease ERI-1. If unsuppressed, silencing can spread between some cells in embryos but can be repeat specific and independent of other homologous loci within each cell. Silencing can persist through DNA replication and nuclear divisions, disrupting uniform gene expression in developed animals. Analysis at single-cell resolution suggests that differences between cells arise during early cell divisions upon unequal segregation of an initiator of silencing. Our results suggest that organisms with high repetitive DNA content, which include humans, could use similar developmental mechanisms to achieve and maintain tissue homogeneity.

Reproducible features of small RNAs in C. elegans reveal NU RNAs and provide insights into 22G RNAs and 26G RNAs.

Small RNAs regulate gene expression and most genes in the worm Caenorhabditis elegans are subject to their regulation. Here, we analyze small RNA data sets and use reproducible features of RNAs present in multiple data sets to discover a new class of small RNAs and to reveal insights into two known classes of small RNAs--22G RNAs and 26G RNAs. We found that reproducibly detected 22-nt RNAs, although are predominantly RNAs with a G at the 5' end, also include RNAs with A, C, or U at the 5' end. These RNAs are synthesized downstream from characteristic sequence motifs on mRNA and have U-tailed derivatives. Analysis of 26G RNAs revealed that they are processed from a blunt end of double-stranded RNAs and that production of one 26G RNA generates a hotspot immediately downstream for production of another. To our surprise, analysis of RNAs shorter than 18 nt revealed a new class of RNAs, which we call NU RNAs (pronounced "new RNAs") because they have a NU bias at the 5' end, where N is any nucleotide. NU RNAs are antisense to genes and originate downstream from U bases on mRNA. Although many genes have complementary NU RNAs, their genome-wide distribution is distinct from that of previously known classes of small RNAs. Our results suggest that current approaches underestimate reproducibly detected RNAs that are shorter than 18 nt, and theoretical considerations suggest that such shorter RNAs could be used for sequence-specific gene regulation in organisms like C. elegans that have small genomes.

Movement of regulatory RNA between animal cells.

Recent studies suggest that RNA can move from one cell to another and regulate genes through specific base-pairing. Mechanisms that modify or select RNA for secretion from a cell are unclear. Secreted RNA can be stable enough to be detected in the extracellular environment and can enter the cytosol of distant cells to regulate genes. Mechanisms that import RNA into the cytosol of an animal cell can enable uptake of RNA from many sources including other organisms. This role of RNA is akin to that of steroid hormones, which cross cell membranes to regulate genes. The potential diagnostic use of RNA in human extracellular fluids has ignited interest in understanding mechanisms that enable the movement of RNA between animal cells. Genetic model systems will be essential to gain more confidence in proposed mechanisms of RNA transport and to connect an extracellular RNA with a specific biological function. Studies in the worm C. elegans and in other animals have begun to reveal parts of this novel mechanism of cell-to-cell communication. Here, I summarize the current state of this nascent field, highlight the many unknowns, and suggest future directions.

Double-stranded RNA made in C. elegans neurons can enter the germline and cause transgenerational gene silencing.

An animal that can transfer gene-regulatory information from somatic cells to germ cells may be able to communicate changes in the soma from one generation to the next. In the worm Caenorhabditis elegans, expression of double-stranded RNA (dsRNA) in neurons can result in the export of dsRNA-derived mobile RNAs to other distant cells. Here, we show that neuronal mobile RNAs can cause transgenerational silencing of a gene of matching sequence in germ cells. Consistent with neuronal mobile RNAs being forms of dsRNA, silencing of target genes that are expressed either in somatic cells or in the germline requires the dsRNA-selective importer SID-1. In contrast to silencing in somatic cells, which requires dsRNA expression in each generation, silencing in the germline is heritable after a single generation of exposure to neuronal mobile RNAs. Although initiation of inherited silencing within the germline requires SID-1, a primary Argonaute RDE-1, a secondary Argonaute HRDE-1, and an RNase D homolog MUT-7, maintenance of inherited silencing is independent of SID-1 and RDE-1, but requires HRDE-1 and MUT-7. Inherited silencing can persist for >25 generations in the absence of the ancestral source of neuronal dsRNA. Therefore, our results suggest that sequence-specific regulatory information in the form of dsRNA can be transferred from neurons to the germline to cause transgenerational silencing.