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

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.

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Small RNAs in parasitic nematodes – forms and functions

Parasitology ◽

10.1017/s0031182019001689 ◽

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.

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Modeling small RNA competition in C. elegans

10.1101/021576 ◽

2015 ◽

Author(s):

Joshua Elkington

Keyword(s):

Small Rna ◽

Random Sampling ◽

Small Rnas ◽

Small Interfering Rnas ◽

System Of Differential Equations ◽

Cell Dynamics ◽

Rna Dynamics ◽

C Elegans ◽

Normal Behavior ◽

Rna Pathways

Small RNAs have been determined to have an essential role in gene regulation. However, competition between small RNAs is a poorly understood aspect of small RNA dynamics. Recent evidence has suggested that competition between small RNA pathways arises from a scarcity of common resources essential for small RNA activity. In order to understand how competition affects small RNAs in C. elegans, a system of differential equations was used. The model recreates normal behavior of small RNAs and uses random sampling in order to determine the coefficients of competition for each small RNA class. The model includes endogenous small-interfering RNAs (endo-siRNA), exogenous small-interfering RNAs (exo-siRNA), and microRNAs (miRNA). The model predicts that exo-siRNAs is dominated by competition between endo-siRNAs and miRNAs. Furthermore, the model predicts that competition is required for normal levels of endogenous small RNAs to be maintained. Although the model makes several assumptions about cell dynamics, the model is still useful in order to understand competition between small RNA pathways.

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

10.1101/001479 ◽

2013 ◽

Cited By ~ 3

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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Germ Granules Functions are Memorized by Transgenerationally Inherited Small RNAs

10.1101/576934 ◽

2019 ◽

Cited By ~ 2

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.

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The interplay between small RNA pathways shapes chromatin landscapes in C. elegans

10.1101/320713 ◽

2018 ◽

Author(s):

Ekaterina Gushchanskaia ◽

Ruben Esse ◽

Qicheng Ma ◽

Nelson Lau ◽

Alla Grishok

Keyword(s):

Small Rnas ◽

Target Genes ◽

Noncoding Rnas ◽

Small Interfering Rnas ◽

Loss Of Function ◽

Partial Loss ◽

Rna Dependent Rna Polymerase ◽

C Elegans ◽

Highly Expressed Genes ◽

Rna Pathways

ABSTRACTThe nematode C. elegans contains several types of endogenous small interfering RNAs (endo-siRNAs) produced by RNA-dependent RNA polymerase (RdRP) complexes. Both “silencing” siRNAs bound by Worm-specific Argonautes (WAGO) and “activating” siRNAs bound by the CSR-1 Argonaute require the DRH-3 helicase, an RdRP component. Here we show that, in the drh-3(ne4253) mutant deficient in RdRP-produced secondary endo-siRNAs, the silencing histone mark H3K9me3 is largely depleted, whereas in the csr-1 partial loss-of-function mutant this mark is ectopically deposited on CSR-1 target genes. Moreover, we observe ectopic H3K9me3 at enhancer elements in both drh-3 and csr-1 partial loss-of-function mutants and describe small RNAs matching enhancers. Finally, we detect accumulation of H3K27me3 at highly expressed genes in the drh-3(ne4253) mutant, which correlates with their reduced transcription. Our study shows that when abundant RdRP-produced siRNAs are depleted, there is ectopic elevation of noncoding RNAs linked to increase in silencing chromatin marks. Moreover, our results suggest that enhancer small RNAs may guide local H3K9 methylation.

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Comparative Analysis of Biochemical Biases by Ligation- and Template-Switch-Based Small RNA Library Preparation Protocols

Clinical Chemistry ◽

10.1373/clinchem.2019.305045 ◽

2019 ◽

Vol 65 (12) ◽

pp. 1581-1591 ◽

Cited By ~ 2

Author(s):

Morgane Meistertzheim ◽

Tobias Fehlmann ◽

Franziska Drews ◽

Marcello Pirritano ◽

Gilles Gasparoni ◽

...

Keyword(s):

Small Rna ◽

Small Rnas ◽

Regulation Of Gene Expression ◽

Biochemical Properties ◽

Library Preparation ◽

Key Players ◽

Rna Fragments ◽

Template Switch ◽

Small Rna Species ◽

Small Rna Library

Abstract BACKGROUND Small RNAs are key players in the regulation of gene expression and differentiation. However, many different classes of small RNAs (sRNAs) have been described with distinct biogenesis pathways and, as a result, with different biochemical properties. To analyze sRNAs by deep sequencing, complementary DNA synthesis requires manipulation of the RNA molecule itself. Thus, enzymatic activities during library preparation bias the library content owing to biochemical criteria. METHODS We compared 4 different manipulations of RNA for library preparation: (a) a ligation-based procedure allowing only 5′-mono-phosphorylated RNA to enter the library, (b) a ligation-based procedure allowing additional 5′-triphosphates and Cap structures, (c) a ligation-independent, template-switch-based library preparation, and (d) a template-switch-based library preparation allowing 3′-phosphorylated RNAs to enter the library. RESULTS Our data show large differences between ligation-dependent and ligation-independent libraries in terms of their preference for individual sRNA classes such as microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and transfer RNA fragments. Moreover, the miRNA composition is different between both procedures, and more microRNA isoforms (isomiRs) can be identified after pyrophosphatase treatment. piRNAs are enriched in template-switch libraries, and this procedure apparently includes more different RNA species. CONCLUSIONS Our data indicate that miRNAomics from both methods will hardly be comparable. Ligation-based libraries enrich for canonical miRNAs, which thus may be suitable methods for miRNAomics. Template-switch libraries contain increased numbers and different compositions of fragments and long RNAs. Following different interests for other small RNA species, ligation-independent libraries appear to show a more realistic sRNA landscape with lower bias against biochemical modifications.

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A low-bias and sensitive small RNA library preparation method using randomized splint ligation

Nucleic Acids Research ◽

10.1093/nar/gkaa480 ◽

2020 ◽

Vol 48 (14) ◽

pp. e80-e80 ◽

Cited By ~ 3

Author(s):

Sean Maguire ◽

Gregory J S Lohman ◽

Shengxi Guan

Keyword(s):

Rna Sequencing ◽

Small Rna ◽

Small Rnas ◽

Small Rna Sequencing ◽

Preparation Process ◽

Library Preparation ◽

Normal Tissues ◽

Levels Of Detail ◽

Low Sensitivity ◽

Next Generation Sequencing Ngs

Abstract Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2′-O-methyl modifications (2′OMe) on the 3′ terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2′OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2′OMe modified RNA with new levels of detail.

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GTSF-1 is required for the formation of a functional RNA-dependent RNA Polymerase complex in C. elegans

10.1101/271270 ◽

2018 ◽

Cited By ~ 1

Author(s):

Miguel Vasconcelos Almeida ◽

Sabrina Dietz ◽

Stefan Redl ◽

Emil Karaulanov ◽

Andrea Hildebrandt ◽

...

Keyword(s):

Rna Polymerase ◽

Gene Silencing ◽

Small Rna ◽

Small Rnas ◽

Zinc Fingers ◽

Specific Factor ◽

Rna Dependent Rna Polymerase ◽

C Elegans ◽

Argonaute Proteins ◽

Rna Pathways

AbstractIn every domain of life, Argonaute proteins and their associated small RNAs regulate gene expression. Despite great conservation of Argonaute proteins throughout evolution, many proteins acting in small RNA pathways are not widely conserved. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured, acidic C-terminal tail, are conserved in animals and act in small RNA pathways. In fly and mouse, they are required for fertility and have been shown to interact with Piwi clade Argonautes. We identified T06A10.3 as the Caenorhabditis elegans Gtsf1 homolog and named it gtsf-1. Given its conserved nature and roles in Piwi-mediated gene silencing, we sought out to characterize GTSF-1 in the context of the small RNA pathways of C. elegans. Like its homologs, GTSF-1 is required for normal fertility. Surprisingly, we report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, gtsf-1 mutants show strong depletion of a class of endogenous small RNAs, known as 26G-RNAs, and fully phenocopy mutants lacking RRF-3, the RNA-dependent RNA Polymerase that synthesizes 26G-RNAs. We show, both in vivo and in vitro, that GTSF-1 specifically and robustly interacts with RRF-3 via its tandem CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex, also known as ERIC, thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may similarly act to drive the assembly of larger complexes that subsequently act in small RNA production and/or in imposing small RNA-mediated silencing activities.

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The RNA phosphatase PIR-1 regulates endogenous small RNA pathways in C. elegans

10.1101/2020.08.03.235143 ◽

2020 ◽

Cited By ~ 1

Author(s):

Daniel A Chaves ◽

Hui Dai ◽

Lichao Li ◽

James J Moresco ◽

Myung Eun Oh ◽

...

Keyword(s):

Small Rna ◽

Small Rnas ◽

Cellular Functions ◽

Germline Development ◽

C Elegans ◽

Rna Sensors ◽

Rna Pathways ◽

Rna Capping ◽

Capping Enzymes ◽

Insight Into

SUMMARYEukaryotic cells regulate 5’ triphosphorylated (ppp-) RNAs to promote cellular functions and prevent recognition by antiviral RNA sensors. For example, RNA capping enzymes possess triphosphatase domains that remove the γ phosphates of ppp-RNAs during RNA capping. Members of the closely related PIR1 family of RNA polyphosphatases remove both the β and γ phosphates from ppp-RNAs. Here we show that C. elegans PIR-1 dephosphorylates ppp-RNAs made by cellular RdRPs and is required for the maturation of 26G-RNAs, Dicer-dependent small RNAs that regulate thousands of genes during spermatogenesis and embryogenesis. PIR-1 also regulates the CSR-1 22G-RNA pathway and has critical functions in both somatic and germline development. Our findings suggest that PIR-1 modulates both Dicer-dependent and - independent Argonaute pathways, and provide insight into how cells and viruses use a conserved RNA phosphatase to regulate and respond to ppp-RNA species.

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Multigenerational Regulation of the Caenorhabditis elegans Chromatin Landscape by Germline Small RNAs

Annual Review of Genetics ◽

10.1146/annurev-genet-112618-043505 ◽

2019 ◽

Vol 53 (1) ◽

pp. 289-311 ◽

Cited By ~ 10

Author(s):

Natasha E. Weiser ◽

John K. Kim

Keyword(s):

Gene Expression ◽

Caenorhabditis Elegans ◽

Small Rnas ◽

Model Organism ◽

Epigenetic Inheritance ◽

Small Interfering Rnas ◽

Transgenerational Epigenetic Inheritance ◽

C Elegans ◽

Small Noncoding Rnas ◽

Rna Pathways

In animals, small noncoding RNAs that are expressed in the germline and transmitted to progeny control gene expression to promote fertility. Germline-expressed small RNAs, including endogenous small interfering RNAs (endo-siRNAs) and Piwi-interacting RNAs (piRNAs), drive the repression of deleterious transcripts such as transposons, repetitive elements, and pseudogenes. Recent studies have highlighted an important role for small RNAs in transgenerational epigenetic inheritance via regulation of heritable chromatin marks; therefore, small RNAs are thought to convey an epigenetic memory of genomic self and nonself elements. Small RNA pathways are highly conserved in metazoans and have been best described for the model organism Caenorhabditis elegans. In this review, we describe the biogenesis, regulation, and function of C. elegans endo-siRNAs and piRNAs, along with recent insights into how these distinct pathways are integrated to collectively regulate germline gene expression, transgenerational epigenetic inheritance, and ultimately, animal fertility.

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