scholarly journals Germ Granules Functions are Memorized by Transgenerationally Inherited Small RNAs

2019 ◽  
Author(s):  
Itamar Lev ◽  
Itai Antoine Toker ◽  
Yael Mor ◽  
Anat Nitzan ◽  
Guy Weintraub ◽  
...  
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Early Embryo ◽  
Small Rna Sequencing ◽  
Wild Type ◽  
P Granules ◽  
Multiple Generations ◽  
C Elegans ◽  
The Core ◽  
Germ Granules

AbstractInC. elegansnematodes, 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 (pptr-1,meg-3/4,pgl-1) can nevertheless inherit potent small RNA-based silencing responses, but some of the mutants lose this ability after many generations of homozygosity. Animals mutated inpptr-1, which is required for stabilization of P granules in the early embryo, display extremely strong heritable RNAi responses, which last for tens of generations, long after the responses in wild type animals peter out. The phenotype of mutants defective in the core germ granules proteins MEG-3 and MEG-4, depends on the genotype of the ancestors: Mutants that derive from maternal lineages that had functional MEG-3 and MEG-4 proteins exhibit enhanced RNAi inheritance for multiple generations. While functional ancestralmeg-3/4alleles correct, and even potentiates the ability of mutant descendants to inherit RNAi, defects in germ granules functions can be memorized as well; Wild type descendants that derive from lineages of mutants show impaired RNAi inheritance for many (>16) generations, although their germ granules are intact. Importantly, while P granules are maternally deposited, wild type progeny derived frommeg-3/4male mutants also show reduced RNAi inheritance. Unlike germ granules, small RNAs are inherited also from the sperm. Moreover, we find that the transgenerational effects that depend on the ancestral germ granules require the argonaute protein HRDE-1, which carries heritable small RNAs in the germline. Indeed, small RNA sequencing reveals imbalanced levels of many endogenous small RNAs in germ granules mutants. Strikingly, we find thathrde-1;meg-3/4triple mutants inherit RNAi, althoughhrde-1was previously thought to be essential for heritable silencing. We propose that germ granules sort and shape the RNA pool, and that small RNA inheritance memorizes this activity for multiple generations.

scholarly journals The Caenorhabditis elegans TDRD5/7-like protein, LOTR-1, interacts with the helicase ZNFX-1 to balance epigenetic signals in the germline

2021 ◽  
Author(s):  
Elisabeth A Marnik ◽  
Miguel Vasconcelos Almeida ◽  
P Giselle Cipriani ◽  
George Chung ◽  
Edoardo Caspani ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Small Rna ◽  
Small Rnas ◽  
Brood Size ◽  
Homologous Proteins ◽  
P Granules ◽  
Transposon Silencing ◽  
Tudor Domain ◽  
Germ Granules

LOTUS and Tudor domain containing proteins have critical roles in the germline. Proteins that contain these domains, such as Tejas/Tapas in Drosophila, help localize Vasa to the germ granules and facilitate piRNA-mediated transposon silencing. The homologous proteins in mammals, TDRD5 and TDRD7, are required during spermiogenesis. Until now, proteins containing both LOTUS and Tudor domains in Caenorhabditis elegans have remained elusive. Here we describe LOTR-1 (D1081.7), which derives its name from its LOTUS and Tudor domains. Interestingly, LOTR-1 docks next to P granules to colocalize with the broadly conserved Z-granule helicase, ZNFX-1. LOTR-1's Z-granule association requires its Tudor domain, but both LOTUS and Tudor deletions affect brood size when coupled with a knockdown of the Vasa homolog glh-1. In addition to interacting with the germ-granule components WAGO-1, PRG-1 and DEPS-1, we identified a Tudor-dependent association with ZNFX-1. Like znfx-1 mutants, lotr-1 mutants lose small RNAs from the 3' ends of WAGO and Mutator targets, reminiscent of the loss of piRNAs from the 3' ends of piRNA precursor transcripts in mouse Tdrd5 mutants. Our work suggests that LOTR-1 acts in a conserved mechanism that brings small RNA generating mechanisms towards the 3' ends of small RNA templates or precursors.

scholarly journals Small RNAs in parasitic nematodes – forms and functions

Parasitology ◽  
2019 ◽  
Vol 147 (8) ◽  
pp. 855-864
Author(s):  
Collette Britton ◽  
Roz Laing ◽  
Eileen Devaney
Keyword(s):  
Gene Expression ◽  
Small Rna ◽  
Small Rnas ◽  
Target Genes ◽  
Parasitic Nematodes ◽  
Small Rna Sequencing ◽  
Small Interfering Rnas ◽  
Rna Sequences ◽  
C Elegans

AbstractSmall RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes – microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host–parasite interface.

scholarly journals Protein-based condensation mechanisms drive the assembly of RNA-rich P granules

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Helen Schmidt ◽  
Andrea Putnam ◽  
Dominique Rasoloson ◽  
Geraldine Seydoux
Keyword(s):  
Intrinsically Disordered Protein ◽  
P Granules ◽  
C Elegans ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
The Core ◽  
C Terminus ◽  
Germ Granules

Germ granules are protein-RNA condensates that segregate with the embryonic germline. In C. elegans embryos, germ (P) granule assembly requires MEG-3, an intrinsically-disordered protein that forms RNA-rich condensates on the surface of PGL condensates at the core of P granules. MEG-3 is related to the GCNA family and contains an N-terminal disordered region (IDR) and a predicted ordered C-terminus featuring an HMG-like motif (HMGL). We find that MEG-3 is modular protein that uses its IDR to bind RNA and its C-terminus to drive condensation. The HMGL motif mediates binding to PGL-3 and is required for co-assembly of MEG-3 and PGL-3 condensates in vivo. Mutations in HMGL cause MEG-3 and PGL-3 to form separate condensates that no longer co-segregate to the germline or recruit RNA. Our findings highlight the importance of protein-based condensation mechanisms and condensate-condensate interactions in the assembly of RNA-rich germ granules.

scholarly journals RppH can faithfully replace TAP to allow cloning of 5’-triphosphate carrying small RNAs

2018 ◽  
Author(s):  
Miguel Vasconcelos Almeida ◽  
António Miguel de Jesus Domingues ◽  
Hanna Lukas ◽  
Maria Mendez-Lago ◽  
René F. Ketting
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Small Rna Sequencing ◽  
Cloning Efficiency ◽  
Library Preparation ◽  
Small Interfering Rnas ◽  
Rdrp Activity ◽  
C Elegans ◽  
Rna Pathways ◽  
Small Rna Species

AbstractRNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans, maintain the 5’ triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5’PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5’ pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.

scholarly journals Genome-Wide Analysis of Single Non-Templated Nucleotides in Plant Endogenous siRNAs and miRNAs

2016 ◽  
Author(s):  
Feng Wang ◽  
Nathan R. Johnson ◽  
Ceyda Coruh ◽  
Michael J. Axtell
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Small Rna Sequencing ◽  
Plant Mirnas ◽  
Wild Type ◽  
Type Specimens ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
Heterochromatic Sirna ◽  
Very High

AbstractPlant small RNAs are subject to various modifications. Previous reports revealed widespread 3' modifications (truncations and non-templated tailing) of plant miRNAs when the 2'-C-methyltransferase HEN1 is absent. However, non-templated nucleotides in plant heterochromatic siRNAs have not been deeply studied, especially in wild-type plants. We systematically studied non-templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing libraries from Arabidopsis. tomato, Medicago, rice, maize, and Physcomitrella. Elevated rates of non-templated nucleotides were observed at the 3' ends of both miRNAs and endogenous siRNAs from wild-type specimens of all species. 'Off-sized' small RNAs, such as 25 and 23 nt siRNAs arising from loci dominated by 24 nt siRNAs, often had very high rates of 3'-non-templated nucleotides. The same pattern was observed in all species that we studied. Further analysis of 24 nt siRNA clusters in Arabidopsis revealed distinct patterns of 3'-non-templated nucleotides of 23 nt siRNAs arising from heterochromatic siRNA loci. This pattern of non-templated 3' nucleotides on 23 nt siRNAs is not affected by loss of known small RNA 3'-end modifying enzymes, and may result from modifications added to longer heterochromatic siRNA precursors.

scholarly journals P granules protect RNA interference genes from silencing by piRNAs

2019 ◽  
Author(s):  
John Paul T. Ouyang ◽  
Andrew Folkmann ◽  
Lauren Bernard ◽  
Chih-Yung Lee ◽  
Uri Seroussi ◽  
...  
Keyword(s):  
Rna Interference ◽  
Germ Cells ◽  
Small Rnas ◽  
Primordial Germ Cells ◽  
Safe Harbor ◽  
Wild Type ◽  
P Granules ◽  
C Elegans ◽  
Rna Biogenesis ◽  
Endogenous Genes

SUMMARYP granules are perinuclear condensates in C. elegans germ cells proposed to serve as hubs for self/non-self RNA discrimination by Argonautes. We report that a mutant (meg-3 meg-4) that does not assemble P granules in primordial germ cells loses competence for RNA-interference over several generations and accumulates silencing small RNAs against hundreds of endogenous genes, including the RNA-interference genes rde-11 and sid-1. In wild-type, rde-11 and sid-1 transcripts are heavily targeted by piRNAs, accumulate in P granules, but maintain expression. In the primordial germ cells of meg-3 meg-4 mutants, rde-11 and sid-1 transcripts disperse in the cytoplasm with the small RNA biogenesis machinery, become hyper-targeted by secondary sRNAs, and are eventually silenced. Silencing requires the PIWI-class Argonaute PRG-1 and the nuclear Argonaute HRDE-1 that maintains trans-generational silencing of piRNA targets. These observations support a “safe harbor” model for P granules in protecting germline transcripts from piRNA-initiated silencing.

scholarly journals The Influence of Competition Among C. elegans Small RNA Pathways on Development

Genes ◽  
2012 ◽  
Vol 3 (4) ◽  
pp. 671-685 ◽  
Author(s):  
Jimmy J. Zhuang ◽  
Craig P. Hunter
Keyword(s):  
Rna Interference ◽  
Caenorhabditis Elegans ◽  
Small Rna ◽  
Small Rnas ◽  
Wild Type ◽  
C Elegans ◽  
Exogenous Rna ◽  
Rna Pathways ◽  
Sirna Pathway ◽  
The Relationship

Small RNAs play a variety of regulatory roles, including highly conserved developmental functions. Caenorhabditis elegans not only possesses most known small RNA pathways, it is also an easy system to study their roles and interactions during development. It has been proposed that in C. elegans, some small RNA pathways compete for access to common limiting resources. The strongest evidence supporting this model is that disrupting the production or stability of endogenous short interfering RNAs (endo-siRNAs) enhances sensitivity to experimentally induced exogenous RNA interference (exo-RNAi). Here, we examine the relationship between the endo-siRNA and microRNA (miRNA) pathways, and find that, consistent with competition among these endogenous small RNA pathways, endo-siRNA pathway mutants may enhance miRNA efficacy. Furthermore, we show that exo-RNAi may also compete with both endo-siRNAs and miRNAs. Our data thus provide support that all known Dicer-dependent small RNA pathways may compete for limiting common resources. Finally, we observed that both endo-siRNA mutants and animals experiencing exo-RNAi have increased expression of miRNA-regulated stage-specific developmental genes. These observations suggest that perturbing the small RNA flux and/or the induction of exo-RNAi, even in wild-type animals, may impact development via effects on the endo-RNAi and microRNA pathways.

scholarly journals Altered Fecal Small RNA Profiles in Colorectal Cancer Reflect Gut Microbiome Composition in Stool Samples

mSystems ◽  
2019 ◽  
Vol 4 (5) ◽  
Author(s):  
Sonia Tarallo ◽  
Giulio Ferrero ◽  
Gaetano Gallo ◽  
Antonio Francavilla ◽  
Giuseppe Clerico ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Gut Microbiota ◽  
Small Rna ◽  
Gut Microbiome ◽  
Small Rnas ◽  
Area Under The Curve ◽  
Small Rna Sequencing ◽  
Predictive Tool ◽  
Cross Sectional ◽  
Stool Samples

ABSTRACT Dysbiotic configurations of the human gut microbiota have been linked to colorectal cancer (CRC). Human small noncoding RNAs are also implicated in CRC, and recent findings suggest that their release in the gut lumen contributes to shape the gut microbiota. Bacterial small RNAs (bsRNAs) may also play a role in carcinogenesis, but their role has been less extensively explored. Here, we performed small RNA and shotgun sequencing on 80 stool specimens from patients with CRC or with adenomas and from healthy subjects collected in a cross-sectional study to evaluate their combined use as a predictive tool for disease detection. We observed considerable overlap and a correlation between metagenomic and bsRNA quantitative taxonomic profiles obtained from the two approaches. We identified a combined predictive signature composed of 32 features from human and microbial small RNAs and DNA-based microbiome able to accurately classify CRC samples separately from healthy and adenoma samples (area under the curve [AUC] = 0.87). In the present study, we report evidence that host-microbiome dysbiosis in CRC can also be observed by examination of altered small RNA stool profiles. Integrated analyses of the microbiome and small RNAs in the human stool may provide insights for designing more-accurate tools for diagnostic purposes. IMPORTANCE The characteristics of microbial small RNA transcription are largely unknown, while it is of primary importance for a better identification of molecules with functional activities in the gut niche under both healthy and disease conditions. By performing combined analyses of metagenomic and small RNA sequencing (sRNA-Seq) data, we characterized both the human and microbial small RNA contents of stool samples from healthy individuals and from patients with colorectal carcinoma or adenoma. With the integrative analyses of metagenomic and sRNA-Seq data, we identified a human and microbial small RNA signature which can be used to improve diagnosis of the disease. Our analysis of human and gut microbiome small RNA expression is relevant to generation of the first hypotheses about the potential molecular interactions occurring in the gut of CRC patients, and it can be the basis for further mechanistic studies and clinical tests.

scholarly journals A stress-induced Tyrosine tRNA depletion response mediates codon-based translational repression and growth suppression

2018 ◽  
Author(s):  
Doowon Huh ◽  
Maria C. Passarelli ◽  
Jenny Gao ◽  
Shahnoza N Dusmatova ◽  
Clara Goin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Small Rna ◽  
Small Rnas ◽  
Oxidative Stress Response ◽  
Translational Repression ◽  
Dependent Manner ◽  
Transfer Rnas ◽  
C Elegans ◽  
Metabolic Genes ◽  
Rna Fragments

SUMMARYEukaryotic transfer RNAs can become selectively fragmented upon various stresses, generating tRNA-derived small RNA fragments. Such fragmentation has been reported to impact a small fraction of the tRNA pool and thus presumed to not directly impact translation. We report that oxidative stress can rapidly generate tyrosine tRNAGUA fragments in human cells—causing significant depletion of the precursor tRNA. Tyrosine tRNAGUA depletion impaired translation of growth and metabolic genes enriched in cognate tyrosine codons. Depletion of tyrosine tRNAGUA or its translationally regulated targets USP3 and SCD repressed proliferation—revealing a dedicated tRNA-regulated growth suppressive pathway for oxidative stress response. Tyrosine fragments are generated in a DIS3L2 exoribonuclease-dependent manner and inhibit hnRNPA1-mediated transcript destabilization. Moreover, tyrosine fragmentation is conserved in C. elegans. Thus, tRNA fragmentation can coordinately generate trans-acting small-RNAs and functionally deplete a tRNA. Our findings reveal the existence of an underlying adaptive codon-based regulatory response inherent to the genetic code.

scholarly journals Massively differential bias between two widely used Illumina library preparation methods for small RNA sequencing

2013 ◽  
Author(s):  
Jeanette Baran-Gale ◽  
Michael R Erdos ◽  
Christina Sison ◽  
Alice Young ◽  
Emily E Fannin ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
Small Rnas ◽  
Small Rna Sequencing ◽  
Library Preparation ◽  
Sequencing Technology ◽  
Preparation Methods ◽  
Sequencing Platforms ◽  
Small Rna Library ◽  
Illumina Library

Recent advances in sequencing technology have helped unveil the unexpected complexity and diversity of small RNAs. A critical step in small RNA library preparation for sequencing is the ligation of adapter sequences to both the 5’ and 3’ ends of small RNAs. Two widely used protocols for small RNA library preparation, Illumina v1.5 and Illumina TruSeq, use different pairs of adapter sequences. In this study, we compare the results of small RNA-sequencing between v1.5 and TruSeq and observe a striking differential bias. Nearly 100 highly expressed microRNAs (miRNAs) are >5-fold differentially detected and 48 miRNAs are >10-fold differentially detected between the two methods of library preparation. In fact, some miRNAs, such as miR-24-3p, are over 30-fold differentially detected. The results are reproducible across different sequencing centers (NIH and UNC) and both major Illumina sequencing platforms, GAIIx and HiSeq. While some level of bias in library preparation is not surprising, the apparent massive differential bias between these two widely used adapter sets is not well appreciated. As increasingly more laboratories transition to the newer TruSeq-based library preparation for small RNAs, researchers should be aware of the extent to which the results may differ from previously published results using v1.5.

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