Illuminating Genetic Mysteries of the Dead Sea Scrolls.

The discovery of the 2,000-year-old Dead Sea Scrolls had an incomparable impact on the historical understanding of Judaism and Christianity. "Piecing together" scroll fragments is like solving jigsaw puzzles with an unknown number of missing parts. We used the fact that most scrolls are made from animal skins to "fingerprint" pieces based on DNA sequences. Genetic sorting of the scrolls illuminates their textual relationship and historical significance. Disambiguating the contested relationship between Jeremiah fragments supplies evidence that some scrolls were brought to the Qumran caves from elsewhere; significantly, they demonstrate that divergent versions of Jeremiah circulated in parallel throughout Israel (ancient Judea). Similarly, patterns discovered in non-biblical scrolls, particularly the Songs of the Sabbath Sacrifice, suggest that the Qumran scrolls represent the broader cultural milieu of the period. Finally, genetic analysis divorces debated fragments from the Qumran scrolls. Our study demonstrates that interdisciplinary approaches enrich the scholar's toolkit.

Neuronal Small RNAs Control Behavior Transgenerationally.

It is unknown whether the activity of the nervous system can be inherited. In Caenorhabditis elegans nematodes, parental responses can transmit heritable small RNAs that regulate gene expression transgenerationally. In this study, we show that a neuronal process can impact the next generations. Neurons-specific synthesis of RDE-4-dependent small RNAs regulates germline amplified endogenous small interfering RNAs (siRNAs) and germline gene expression for multiple generations. Further, the production of small RNAs in neurons controls the chemotaxis behavior of the progeny for at least three generations via the germline Argonaute HRDE-1. Among the targets of these small RNAs, we identified the conserved gene saeg-2, which is transgenerationally downregulated in the germline. Silencing of saeg-2 following neuronal small RNA biogenesis is required for chemotaxis under stress. Thus, we propose a small-RNA-based mechanism for communication of neuronal processes transgenerationally.

A dual program for translation regulation in cellular proliferation and differentiation.

A dichotomous choice for metazoan cells is between proliferation and differentiation. Measuring tRNA pools in various cell types, we found two distinct subsets, one that is induced in proliferating cells, and repressed otherwise, and another with the opposite signature. Correspondingly, we found that genes serving cell-autonomous functions and genes involved in multicellularity obey distinct codon usage. Proliferation-induced and differentiation-induced tRNAs often carry anticodons that correspond to the codons enriched among the cell-autonomous and the multicellularity genes, respectively. Because mRNAs of cell-autonomous genes are induced in proliferation and cancer in particular, the concomitant induction of their codon-enriched tRNAs suggests coordination between transcription and translation. Histone modifications indeed change similarly in the vicinity of cell-autonomous genes and their corresponding tRNAs, and in multicellularity genes and their tRNAs, suggesting the existence of transcriptional programs coordinating tRNA supply and demand. Hence, we describe the existence of two distinct translation programs that operate during proliferation and differentiation.

RNAlysis: analyze your RNA sequencing data without writing a single line of code.

BACKGROUND: Among the major challenges in next-generation sequencing experiments are exploratory data analysis, interpreting trends, identifying potential targets/candidates, and visualizing the results clearly and intuitively. These hurdles are further heightened for researchers who are not experienced in writing computer code since most available analysis tools require programming skills. Even for proficient computational biologists, an efficient and replicable system is warranted to generate standardized results. RESULTS: We have developed RNAlysis, a modular Python-based analysis software for RNA sequencing data. RNAlysis allows users to build customized analysis pipelines suiting their specific research questions, going all the way from raw FASTQ files (adapter trimming, alignment, and feature counting), through exploratory data analysis and data visualization, clustering analysis, and gene set enrichment analysis. RNAlysis provides a friendly graphical user interface, allowing researchers to analyze data without writing code. We demonstrate the use of RNAlysis by analyzing RNA sequencing data from different studies using C. elegans nematodes. We note that the software applies equally to data obtained from any organism with an existing reference genome. CONCLUSIONS: RNAlysis is suitable for investigating various biological questions, allowing researchers to more accurately and reproducibly run comprehensive bioinformatic analyses. It functions as a gateway into RNA sequencing analysis for less computer-savvy researchers, but can also help experienced bioinformaticians make their analyses more robust and efficient, as it offers diverse tools, scalability, automation, and standardization between analyses.

Systematic analysis of long intergenic non-coding RNAs in C. elegans germline uncovers roles in somatic growth.

Long intergenic non-coding RNAs (lincRNAs) are transcripts longer than 200 nucleotides that are transcribed from non-coding loci yet undergo biosynthesis similar to coding mRNAs. The disproportional number of lincRNAs expressed in testes suggests that lincRNAs are important during gametogenesis, but experimental evidence has implicated very few lincRNAs in this process. We took advantage of the relatively limited number of lincRNAs in the genome of the nematode Caenorhabditis elegans to systematically analyse the functions of lincRNAs during meiosis. We deleted six lincRNA genes that are highly and dynamically expressed in the C. elegans gonad and tested the effects on central meiotic processes. Surprisingly, whereas the lincRNA deletions did not strongly impact fertility, germline apoptosis, crossovers, or synapsis, linc-4 was required for somatic growth. Slower growth was observed in linc-4-deletion mutants and in worms depleted of linc-4 using RNAi, indicating that linc-4 transcripts are required for this post-embryonic process. Unexpectedly, analysis of worms depleted of linc-4 in soma versus germline showed that the somatic role stems from linc-4 expression in germline cells. This unique feature suggests that some lincRNAs, like some small non-coding RNAs, are required for germ-soma interactions.

Tissue- and Time-Specific Expression of Otherwise Identical tRNA Genes.

Codon usage bias affects protein translation because tRNAs that recognize synonymous codons differ in their abundance. Although the current dogma states that tRNA expression is exclusively regulated by intrinsic control elements (A- and B-box sequences), we revealed, using a reporter that monitors the levels of individual tRNA genes in Caenorhabditis elegans, that eight tryptophan tRNA genes, 100% identical in sequence, are expressed in different tissues and change their expression dynamically. Furthermore, the expression levels of the sup-7 tRNA gene at day 6 were found to predict the animal's lifespan. We discovered that the expression of tRNAs that reside within introns of protein-coding genes is affected by the host gene's promoter. Pairing between specific Pol II genes and the tRNAs that are contained in their introns is most likely adaptive, since a genome-wide analysis revealed that the presence of specific intronic tRNAs within specific orthologous genes is conserved across Caenorhabditis species.

Identifying the ligated amino acid of archaeal tRNAs based on positions outside the anticodon.

Proper recognition of tRNAs by their aminoacyl-tRNA synthetase is essential for translation accuracy. Following evidence that the enzymes can recognize the correct tRNA even when anticodon information is masked, we search for additional nucleotide positions within the tRNA molecule that potentially contain information for amino acid identification. Analyzing 3936 sequences of tRNA genes from 86 archaeal species, we show that the tRNAs' cognate amino acids can be identified by the information embedded in the tRNAs' nucleotide positions without relying on the anticodon information. We present a small set of six to 10 informative positions along the tRNA, which allow for amino acid identification accuracy of 90.6% to 97.4%, respectively. We inspected tRNAs for each of the 20 amino acid types for such informative positions and found that tRNA genes for some amino acids are distinguishable from others by as few as one or two positions. The informative nucleotide positions are in agreement with nucleotide positions that were experimentally shown to affect the loaded amino acid identity. Interestingly, the knowledge gained from the tRNA genes of one archaeal phylum does not extrapolate well to another phylum. Furthermore, each species has a unique ensemble of nucleotides in the informative tRNA positions, and the similarity between the sets of positions of two distinct species reflects their evolutionary distance. Hence, we term this set of informative positions a "tRNA cipher." It is tempting to suggest that the diverging code identified here might also serve the aminoacyl tRNA synthetase in the task of tRNA recognition.

A comprehensive tRNA deletion library unravels the genetic architecture of the tRNA pool.

Deciphering the architecture of the tRNA pool is a prime challenge in translation research, as tRNAs govern the efficiency and accuracy of the process. Towards this challenge, we created a systematic tRNA deletion library in Saccharomyces cerevisiae, aimed at dissecting the specific contribution of each tRNA gene to the tRNA pool and to the cell's fitness. By harnessing this resource, we observed that the majority of tRNA deletions show no appreciable phenotype in rich medium, yet under more challenging conditions, additional phenotypes were observed. Robustness to tRNA gene deletion was often facilitated through extensive backup compensation within and between tRNA families. Interestingly, we found that within tRNA families, genes carrying identical anti-codons can contribute differently to the cellular fitness, suggesting the importance of the genomic surrounding to tRNA expression. Characterization of the transcriptome response to deletions of tRNA genes exposed two disparate patterns: in single-copy families, deletions elicited a stress response; in deletions of genes from multi-copy families, expression of the translation machinery increased. Our results uncover the complex architecture of the tRNA pool and pave the way towards complete understanding of their role in cell physiology.

Regulatory mechanisms and networks couple the different phases of gene expression.

Gene expression comprises multiple stages, from transcription to protein degradation. Although much is known about the regulation of each stage separately, an understanding of the regulatory coupling between the different stages is only beginning to emerge. For example, there is a clear crosstalk between translation and transcription, and the localization and stability of an mRNA in the cytoplasm could already be determined during transcription in the nucleus. We review a diversity of mechanisms discovered in recent years that couple the different stages of gene expression. We then speculate on the functional and evolutionary significance of this coupling and suggest certain systems-level functionalities that might be optimized via the various coupling modes. In particular, we hypothesize that coupling is often an economic strategy that allows biological systems to respond robustly and precisely to genetic and environmental perturbations.

Dynamic changes in translational efficiency are deduced from codon usage of the transcriptome.

Translation of a gene is assumed to be efficient if the supply of the tRNAs that translate it is high. Yet high-abundance tRNAs are often also at high demand since they correspond to preferred codons in genomes. Thus to fully model translational efficiency one must gauge the supply-to-demand ratio of the tRNAs that are required by the transcriptome at a given time. The tRNAs' supply is often approximated by their gene copy number in the genome. Yet neither the demand for each tRNA nor the extent to which its concentration changes across environmental conditions has been extensively examined. Here we compute changes in the codon usage of the transcriptome across different conditions in several organisms by inspecting conventional mRNA expression data. We find recurring dynamics of codon usage in the transcriptome in multiple stressful conditions. In particular, codons that are translated by rare tRNAs become over-represented in the transcriptome in response to stresses. These results raise the possibility that the tRNA pool might dynamically change upon stress to support efficient translation of stress-transcribed genes. Alternatively, stress genes may be typically translated with low efficiency, presumably due to lack of sufficient evolutionary optimization pressure on their codon usage.

Sex-specific developmental gene expression atlas unveils dimorphic gene networks in C. elegans.

Sex-specific traits and behaviors emerge during development by the acquisition of unique properties in the nervous system of each sex. However, the genetic events responsible for introducing these sex-specific features remain poorly understood. In this study, we create a comprehensive gene expression atlas of pure populations of hermaphrodites and males of the nematode Caenorhabditis elegans across development. We discover numerous differentially expressed genes, including neuronal gene families like transcription factors, neuropeptides, and G protein-coupled receptors. We identify INS-39, an insulin-like peptide, as a prominent male-biased gene expressed specifically in ciliated sensory neurons. We show that INS-39 serves as an early-stage male marker, facilitating the effective isolation of males in high-throughput experiments. Through complex and sex-specific regulation, ins-39 plays pleiotropic sexually dimorphic roles in various behaviors, while also playing a shared, dimorphic role in early life stress. This study offers a comparative sexual and developmental gene expression database for C. elegans. Furthermore, it highlights conserved genes that may underlie the sexually dimorphic manifestation of different human diseases.

Nucleus-independent transgenerational small RNA inheritance in Caenorhabditis elegans.

In Caenorhabditis elegans worms, epigenetic information transmits transgenerationally. Still, it is unknown whether the effects transfer to the next generation inside or outside of the nucleus. Here, we use the tractability of gene-specific double-stranded RNA-induced silencing to demonstrate that RNA interference can be inherited independently of any nuclear factors via mothers that are genetically engineered to transmit only their ooplasm but not the oocytes' nuclei to the next generation. We characterize the mechanisms and, using RNA sequencing, chimeric worms, and sequence polymorphism between different isolates, identify endogenous small RNAs which, similarly to exogenous siRNAs, are inherited in a nucleus-independent manner. From a historical perspective, these results might be regarded as partial vindication of discredited cytoplasmic inheritance theories from the 19th century, such as Darwin's "pangenesis" theory.

Determinants of translation efficiency and accuracy.

Proper functioning of biological cells requires that the process of protein expression be carried out with high efficiency and fidelity. Given an amino-acid sequence of a protein, multiple degrees of freedom still remain that may allow evolution to tune efficiency and fidelity for each gene under various conditions and cell types. Particularly, the redundancy of the genetic code allows the choice between alternative codons for the same amino acid, which, although 'synonymous,' may exert dramatic effects on the process of translation. Here we review modern developments in genomics and systems biology that have revolutionized our understanding of the multiple means by which translation is regulated. We suggest new means to model the process of translation in a richer framework that will incorporate information about gene sequences, the tRNA pool of the organism and the thermodynamic stability of the mRNA transcripts. A practical demonstration of a better understanding of the process would be a more accurate prediction of the proteome, given the transcriptome at a diversity of biological conditions.

Transgenerational inheritance of sexual attractiveness via small RNAs enhances evolvability in C. elegans.

It is unknown whether transient transgenerational epigenetic responses to environmental challenges affect the process of evolution, which typically unfolds over many generations. Here, we show that in C. elegans, inherited small RNAs control genetic variation by regulating the crucial decision of whether to self-fertilize or outcross. We found that under stressful temperatures, younger hermaphrodites secrete a male-attracting pheromone. Attractiveness transmits transgenerationally to unstressed progeny via heritable small RNAs and the Argonaute Heritable RNAi Deficient-1 (HRDE-1). We identified an endogenous small interfering RNA pathway, enriched in endo-siRNAs that target sperm genes, that transgenerationally regulates sexual attraction, male prevalence, and outcrossing rates. Multigenerational mating competition experiments and mathematical simulations revealed that over generations, animals that inherit attractiveness mate more and their alleles spread in the population. We propose that the sperm serves as a "stress-sensor" that, via small RNA inheritance, promotes outcrossing in challenging environments when increasing genetic variation is advantageous.

Stress resets ancestral heritable small RNA responses.

Transgenerational inheritance of small RNAs challenges basic concepts of heredity. In Caenorhabditis elegans nematodes, small RNAs are transmitted across generations to establish a transgenerational memory trace of ancestral environments and distinguish self-genes from non-self-elements. Carryover of aberrant heritable small RNA responses was shown to be maladaptive and to lead to sterility. Here, we show that various types of stress (starvation, high temperatures, and high osmolarity) induce resetting of ancestral small RNA responses and a genome-wide reduction in heritable small RNA levels. We found that mutants that are defective in various stress pathways exhibit irregular RNAi inheritance dynamics even in the absence of stress. Moreover, we discovered that resetting of ancestral RNAi responses is specifically orchestrated by factors that function in the p38 MAPK pathway and the transcription factor SKN-1/Nrf2. Stress-dependent termination of small RNA inheritance could protect from run-on of environment-irrelevant heritable gene regulation.

H3K9me3 is required for inheritance of small RNAs that target a unique subset of newly evolved genes.

In Caenorhabditis elegans, RNA interference (RNAi) responses can transmit across generations via small RNAs. RNAi inheritance is associated with Histone-3-Lysine-9 tri-methylation (H3K9me3) of the targeted genes. In other organisms, maintenance of silencing requires a feed-forward loop between H3K9me3 and small RNAs. Here, we show that in C. elegans not only is H3K9me3 unnecessary for inheritance, the modification's function depends on the identity of the RNAi-targeted gene. We found an asymmetry in the requirement for H3K9me3 and the main worm H3K9me3 methyltransferases, SET-25 and SET-32. Both methyltransferases promote heritable silencing of the foreign gene gfp, but are dispensable for silencing of the endogenous gene oma-1. Genome-wide examination of heritable endogenous small interfering RNAs (endo-siRNAs) revealed that endo-siRNAs that depend on SET-25 and SET-32 target newly acquired and highly H3K9me3 marked genes. Thus, 'repressive' chromatin marks could be important specifically for heritable silencing of genes which are flagged as 'foreign', such as gfp. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Germ Granules Govern Small RNA Inheritance.

In C. elegans nematodes, components of liquid-like germ granules were shown to be required for transgenerational small RNA inheritance. Surprisingly, we show here that mutants with defective germ granules can nevertheless inherit potent small RNA-based silencing responses, but some of the mutants lose this ability after many generations of homozygosity. Animals mutated in pptr-1, which is required for stabilization of P granules in the early embryo, display extraordinarily strong heritable RNAi responses, lasting for tens of generations. Intriguingly, the RNAi capacity of descendants derived from mutants defective in the core germ granule proteins MEG-3 and MEG-4 is determined by the genotype of the ancestors and changes transgenerationally. Further, whether the meg-3/4 mutant alleles were present in the paternal or maternal lineages leads to different transgenerational consequences. Small RNA inheritance, rather than maternal contribution of the germ granules themselves, mediates the transgenerational defects in RNAi of meg-3/4 mutants and their progeny. Accordingly, germ granule defects lead to heritable genome-wide mis-expression of endogenous small RNAs. Upon disruption of germ granules, hrde-1 mutants can inherit RNAi, although HRDE-1 was previously thought to be absolutely required for RNAi inheritance. We propose that germ granules sort and shape the RNA pool, and that small RNA inheritance maintains this activity for multiple generations.

Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules.

The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.

MET-2-Dependent H3K9 Methylation Suppresses Transgenerational Small RNA Inheritance.

In C. elegans, alterations to chromatin produce transgenerational effects, such as inherited increase in lifespan and gradual loss of fertility. Inheritance of histone modifications can be induced by double-stranded RNA-derived heritable small RNAs. Here, we show that the mortal germline phenotype, which is typical of met-2 mutants, defective in H3K9 methylation, depends on HRDE-1, an argonaute that carries small RNAs across generations, and is accompanied by accumulated transgenerational misexpression of heritable small RNAs. We discovered that MET-2 inhibits small RNA inheritance, and, as a consequence, induction of RNAi in met-2 mutants leads to permanent RNAi responses that do not terminate even after more than 30 generations. We found that potentiation of heritable RNAi in met-2 animals results from global hyperactivation of the small RNA inheritance machinery. Thus, changes in histone modifications can give rise to drastic transgenerational epigenetic effects, by controlling the overall potency of small RNA inheritance.