scholarly journals Comparative profiling of small RNAs of pig seminal plasma and ejaculated and epididymal sperm

Reproduction ◽  
2017 ◽  
Vol 153 (6) ◽  
pp. 785-796 ◽  
Author(s):  
Cai Chen ◽  
Han Wu ◽  
Dan Shen ◽  
Saisai Wang ◽  
Li Zhang ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Small Rna ◽  
Small Rnas ◽  
Target Genes ◽  
Large Population ◽  
Small Nuclear Rna ◽  
Epididymal Sperm ◽  
Zygote Development ◽  
Similarities And Differences ◽  
Small Rna Species

The similarities and differences of small RNAs in seminal plasma, epididymal sperm and ejaculated sperm remain largely undefined. We conducted a systematic comparative analysis of small RNA profiles in pig ejaculated sperm, epididymal sperm and seminal plasma and found that the diversity distribution of small RNA species was generally similar, whereas the abundance of small RNAs is dramatically different across the three libraries; miRNAs and small RNAs derived from rRNA, tRNA, small nuclear RNA, 7SK RNA, NRON RNA and cis-regulatory RNA were enriched in the three libraries, but piRNA was absent. A large population of small RNAs from ejaculated sperm are ejaculated sperm specific, and only 8–30% of small RNAs overlapped with those of epididymal sperm or seminal plasma and a small proportion (5–18%) of small RNAs were shared in the three libraries, suggesting that, in addition to the testes, sperm RNAs may also originate from seminal plasma, epididymis as well as other resources. Most miRNAs were co-distributed but differentially expressed across the three libraries, with epididymal sperm exhibiting the highest abundance, followed by ejaculated sperm and seminal plasma. The prediction of target genes of the top 10 highly expressed miRNAs across the three libraries revealed that these miRNAs may be involved in spermatogenesis, zygote development and the interaction between the environment and animals. Our study provides the first description of the similarities and differences of small RNA profiles in ejaculated sperm, epididymal sperm and seminal plasma and indicates that sperm RNA may have origins other than the testes.

scholarly journals Analysis of miRNAs in the Heads of Different Castes of the Bumblebee Bombus lantschouensis (Hymenoptera: Apidae)

Insects ◽  
2019 ◽  
Vol 10 (10) ◽  
pp. 349 ◽  
Author(s):  
Liu ◽  
Huang ◽  
Zhang ◽  
Liu ◽  
An
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Target Genes ◽  
Solexa Sequencing ◽  
Biological Functions ◽  
Putative Target ◽  
Differentially Expressed Mirnas ◽  
Rna Deep Sequencing ◽  
Small Rna Deep Sequencing ◽  
Go Terms

Bumblebees are important insect pollinators for many wildflowers and crops. MicroRNAs (miRNAs) are endogenous non-coding small RNAs that regulate different biological functions in insects. In this study, the miRNAs in the heads of the three castes of the bumblebee Bombus lantschouensis were identified and characterized by small RNA deep sequencing. The significant differences in the expression of miRNAs and their target genes were analyzed. The results showed that the length of the small RNA reads from males, queens, and workers was distributed between 18 and 30 nt, with a peak at 22 nt. A total of 364 known and 89 novel miRNAs were identified from the heads of the three castes. The eight miRNAs with the highest expressed levels in males, queens, and workers were identical, although the order of these miRNAs based on expression differed. The male vs. queen, male vs. worker, and worker vs. queen comparisons identified nine, fourteen, and four miRNAs with significant differences in expression, respectively. The different castes were clustered based on the differentially expressed miRNAs (DE miRNAs), and the expression levels of the DE miRNAs obtained by RT-qPCR were consistent with the read counts obtained through Solexa sequencing. The putative target genes of these DE miRNAs were enriched in 29 Gene Ontology (GO) terms, and catalytic activity was the most enriched GO term, as demonstrated by its association with 2837 target genes in the male vs. queen comparison, 3535 target genes in the male vs. worker comparison, and 2185 target genes in the worker vs. queen comparison. This study highlights the characteristics of the miRNAs in the three B. lantschouensis castes and will aid further studies on the functions of miRNAs in bumblebees.

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 Functional analysis of virus and host components that mediate potyvirus-induced diseases

2008 ◽  
Author(s):  
Steven A. Whitham ◽  
Amit Gal-On ◽  
Tzahi Arazi
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Binding Activity ◽  
Symptom Expression ◽  
Wild Type ◽  
Disease Symptoms ◽  
Virus Accumulation ◽  
Plant Genes ◽  
Small Rna Species ◽  
Host Genes

The mechanisms underlying the development of symptoms in response to virus infection remain to be discovered in plants. Insight into symptoms induced by potyviruses comes from evidence implicating the potyviral HC-Pro protein in symptom development. In particular, recent studies link the development of symptoms in infected plants to HC-Pro's ability to interfere with small RNA metabolism and function in plant hosts. Moreover, mutation of the highly conserved FRNK amino acid motif to FINK in the HC-Pro of Zucchini yellow mosaic virus (ZYMV) converts a severe strain into an asymptomatic strain, but does not affect virus accumulation in cucurbit hosts. The ability of this FINK mutation to uncouple symptoms from virus accumulation creates a unique opportunity to study symptom etiology, which is usually confounded by simultaneous attenuation of both symptoms and virus accumulation. Our goal was to determine how mutations in the conserved FRNK motif affect host responses to potyvirus infection in cucurbits and Arabidopsis thaliana. Our first objective was to define those amino acids in the FRNK motif that are required for symptoms by mutating the FRNK motif in ZYMV and Turnip mosaic virus (TuMV). Symptom expression and accumulation of resulting mutant viruses in cucurbits and Arabidopsis was determined. Our second objective was to identify plant genes associated with virus disease symptoms by profiling gene expression in cucurbits and Arabidopsis in response to mutant and wild type ZYMV and TuMV, respectively. Genes from the two host species that are differentially expressed led us to focus on a subset of genes that are expected to be involved in symptom expression. Our third objective was to determine the functions of small RNA species in response to mutant and wild type HC-Pro protein expression by monitoring the accumulation of small RNAs and their targets in Arabidopsis and cucurbit plants infected with wild type and mutant TuMV and ZYMV, respectively. We have found that the maintenance of the charge of the amino acids in the FRNK motif of HC-Pro is required for symptom expression. Reduced charge (FRNA, FRNL) lessen virus symptoms, and maintain the suppression of RNA silencing. The FRNK motif is involved in binding of small RNA species including microRNAs (miRNA) and short interfering RNAs (siRNA). This binding activity mediated by the FRNK motif has a role in protecting the viral genome from degradation by the host RNA silencing system. However, it also provides a mechanism by which the FRNK motif participates in inducing the symptoms of viral infection. Small RNA species, such as miRNA and siRNA, can regulate the functions of plant genes that affect plant growth and development. Thus, this binding activity suggests a mechanism by which ZYMVHC-Pro can interfere with plant development resulting in disease symptoms. Because the host genes regulated by small RNAs are known, we have identified candidate host genes that are expected to play a role in symptoms when their regulation is disrupted during viral infections. As a result of this work, we have a better understanding of the FRNK amino acid motif of HC-Pro and its contribution to the functions of HC-Pro, and we have identified plant genes that potentially contribute to symptoms of virus infected plants when their expression becomes misregulated during potyviral infections. The results set the stage to establish the roles of specific host genes in viral pathogenicity. The potential benefits include the development of novel strategies for controlling diseases caused by viruses, methods to ensure stable expression of transgenes in genetically improved crops, and improved potyvirus vectors for expression of proteins or peptides in plants.   

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

2019 ◽  
Vol 65 (12) ◽  
pp. 1581-1591 ◽  
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.

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 TEsmall identifies small RNAs associated with targeted inhibitor resistance in melanoma

2018 ◽  
Author(s):  
Kathryn O’Neill ◽  
Wen-Wei Liao ◽  
Ami Patel ◽  
Molly Gale Hammell
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Target Genes ◽  
Mapk Pathway ◽  
Single Agent ◽  
Resistance Mechanisms ◽  
Melanoma Cells ◽  
Cancer Therapies ◽  
Mrna Targets ◽  
Inhibitor Resistance

MicroRNAs (miRNAs) are small 21-22nt RNAs that act to regulate the expression of mRNA target genes through direct binding to mRNA targets. While miRNAs typically dominate small RNA transcriptomes, many other classes are present including tRNAs, snoRNAs, snRNAs, Y-RNAs, piRNAs, and siRNAs. Interactions between processing machinery and targeting networks of these various small RNA classes remains unclear, largely because these small RNAs are typically analyzed separately. Here we present TEsmall, a tool that allows for the simultaneous processing and analysis of small RNAs from each annotated class in a single integrated workflow. The pipeline begins with raw fastq reads and proceeds all the way to producing count tables formatted for differential expression. Several interactive charts are also produced to look at overall distributions in length and annotation classes. We next applied the TEsmall pipeline to small RNA libraries generated from melanoma cells responding to targeted inhibitors of the MAPK pathway. Targeted oncogene inhibitors have emerged as way to tailor cancer therapies to the particular mutations present in a given tumor. While these targeted strategies are typically effective for short intervals, the emergence of resistance is extremely common, limiting the effectiveness of single-agent therapeutics and driving the need for a better understanding of resistance mechanisms. Using TEsmall, we identified several microRNAs and other small RNA classes that are enriched in inhibitor resistant melanoma cells in multiple melanoma cell lines and may be able to serve as markers of resistant populations more generally.

scholarly journals Non-Templated Oligo-Adenylation of Small RNAs in the Parasite Entamoeba: Potential Roles for Small RNA Control in Single Celled Eukaryotic Pathogen

2020 ◽  
Author(s):  
Hanbang Zhang ◽  
Gretchen M Ehrenkaufer ◽  
Neil Hall ◽  
Upinder Singh
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Cellular Localization ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Protozoan Parasite ◽  
Rnai Pathway ◽  
Regulation Mechanism ◽  
Protozoan Parasites ◽  
Modification Process

Abstract Background: The RNA interference (RNAi) pathway is a gene regulation mechanism that uitilizes small RNA (sRNA) and Argonaute (Ago) proteins to silence target genes. Our previous work identified a functional RNAi pathway in the protozoan parasite Entamoeba histolytica, including abundant 27nt antisense sRNA populations derived from the secondary RNAi pathway which associate with EhAgo2-2 protein. However, there is lack of understanding about sRNAs that are bound to two other EhAgos (EhAgo2-1 and 2-3), and the mechanism of sRNA regulation itself is unclear in this parasite. Results: In the present study, we sequenced sRNA libraries from both total RNAs and EhAgo bound RNAs. We identified a new population of 31nt sRNAs that results from the addition of a non-templated 3-4 adenosine nucleotides at the 3´-end of the 27nt sRNA populations, indicating a non-templated RNA-tailing event in the parasite. We found that both sRNA populations (27nt and 31nt) are unchanged during the development of E. invadens. However, we detected an alteration in their relative abundance for the targeted gene in parasites transfected with a trigger-gene silencing construct, indicating that non-templated RNA-tailing is likely a pathway for sRNA turnover when the targeted gene is unable to be silenced in this parasite. In sequencing the sRNAs associating with the three EhAgo proteins, we observed that despite distinct cellular localization, all three EhAgo sRNA libraries contain 27nt sRNAs with 5´-polyphosphate (5´-polyP) structure and a largely overlapping sRNA repertoire, mainly targeting retrotransposons and a subset of ~226 genes that are endogenously silenced. Furthermore, our data show that 31nt sRNA populations paritally associate with wildtype EhAgo2-2 but not with its mutant protein (EhAgo2-2 C-terminal deletion), indicating an intact RISC is essential for the sRNA modification process.Conclusion: High-throughput sequencing of sRNA in Entamoeba has identified a new population of sRNA with non-templated adenylation modification, which is the first such observation amongst single cell protozoan parasites. Our sRNA sequencing libraries provide the first comprehensive sRNA dataset for all three Entamoeba Ago proteins, which can serve as a useful database for the amoeba community.

scholarly journals Effect of RNA silencing suppression activity of chrysanthemum virus B p12 protein on small RNA species

2020 ◽  
Vol 165 (12) ◽  
pp. 2953-2959
Author(s):  
Ramesh R. Vetukuri ◽  
Pruthvi B. Kalyandurg ◽  
Ganapathi Varma Saripella ◽  
Diya Sen ◽  
Jose Fernando Gil ◽  
...  
Keyword(s):  
Size Distribution ◽  
Small Rna ◽  
Small Rnas ◽  
Rna Silencing ◽  
Transcriptional Activator ◽  
Lower Abundance ◽  
Silencing Suppression ◽  
The Impact ◽  
Small Rna Species ◽  
Suppressor Of Rna Silencing

Abstract Chrysanthemum virus B encodes a multifunctional p12 protein that acts as a transcriptional activator in the nucleus and as a suppressor of RNA silencing in the cytoplasm. Here, we investigated the impact of p12 on accumulation of major classes of small RNAs (sRNAs). The results show dramatic changes in the sRNA profiles characterised by an overall reduction in sRNA accumulation, changes in the pattern of size distribution of canonical siRNAs and in the ratio between sense and antisense strands, lower abundance of siRNAs with a U residue at the 5′-terminus, and changes in the expression of certain miRNAs, most of which were downregulated.

scholarly journals Heterologous Expression of Salvia miltiorrhiza MicroRNA408 Enhances Tolerance to Salt Stress in Nicotiana benthamiana

2018 ◽  
Vol 19 (12) ◽  
pp. 3985 ◽  
Author(s):  
Xiaorong Guo ◽  
Junfeng Niu ◽  
Xiaoyan Cao
Keyword(s):  
Salt Stress ◽  
Abiotic Stress ◽  
Transgenic Tobacco ◽  
Small Rna ◽  
Small Rnas ◽  
Target Genes ◽  
Salvia Miltiorrhiza ◽  
Promoter Sequence ◽  
Oxygen Species ◽  
Gus Staining

MicroRNAs (miRNAs) are a class of endogenous small RNAs that regulate the expression of target genes post-transcriptionally; they are known to play major roles in development and responses to abiotic stress. MicroRNA408 (miR408) is a conserved small RNA in plants; it was reported that miR408 genes were involved in abiotic stress in Arabidopsis. However, miR408 in Salvia miltiorrhiza has been rarely investigated. In this study, we cloned Sm-MIR408, the miR408 precursor sequence, and its promoter sequence from S. miltiorrhiza and the role in tolerance to salt stress is described. The effects of salt stress on miR408 expression were studied by using β-glucuronidase (GUS) staining. Our data indicated that transgenic tobacco overexpressing Sm-MIR408 promoted seed germination and reduced the accumulation of reactive oxygen species under salt stress. Transcript levels of antioxidative genes, i.e., NbSOD, NbPOD, and NbCAT, and their enzyme activities increased in salinity-stressed transgenic tobacco plants, suggesting a better antioxidant system to cope the oxidative damage caused by salinity stress. Taken together, these findings indicated that miR408 functions in positive responses to salt tolerance in tobacco.

scholarly journals Contribution of Small RNA Pathway to Interactions of Rice with Pathogens and Insect Pests

Rice ◽  
2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Qin Feng ◽  
Yan Li ◽  
Zhi-Xue Zhao ◽  
Wen-Ming Wang
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Recent Progress ◽  
Target Genes ◽  
Insect Pests ◽  
Circular Rnas ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Argonaute Proteins ◽  
Non Coding Rnas

AbstractSmall RNAs (sRNAs) are mainly classified into microRNAs (miRNAs) and small interfering RNAs (siRNAs) according to their origin. miRNAs originate from single-stranded RNA precursors, whereas siRNAs originate from double-stranded RNA precursors that are synthesized by RNA-dependent RNA polymerases. Both of single-stranded and double-stranded RNA precursors are processed into sRNAs by Dicer-like proteins. Then, the sRNAs are loaded into ARGONAUTE proteins, forming RNA-induced silencing complexes (RISCs). The RISCs repress the expression of target genes with sequences complementary to the sRNAs through the cleavage of transcripts, the inhibition of translation or DNA methylation. Here, we summarize the recent progress of sRNA pathway in the interactions of rice with various parasitic organisms, including fungi, viruses, bacteria, as well as insects. Besides, we also discuss the hormone signal in sRNA pathway, and the emerging roles of circular RNAs and long non-coding RNAs in rice immunity. Obviously, small RNA pathway may act as a part of rice innate immunity to coordinate with growth and development.

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