scholarly journals Isolation and detection of small RNA molecules

2008 ◽  
Vol 53 (No. 10) ◽  
pp. 451-455 ◽  
Author(s):  
J. Fulneček
Keyword(s):  
Small Rna ◽  
Northern Blotting ◽  
Rna Molecules

The article describes in detail methodical procedures for isolation of small RNA molecules, their electrophoresis, northern blotting and finally detection by hybridization with radioactively labeled riboprobes. The presented protocols were successfully introduced and used in our laboratory.

scholarly journals Small RNA-Sequencing: Approaches and Considerations for miRNA Analysis

Diagnostics ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 964
Author(s):  
Sarka Benesova ◽  
Mikael Kubista ◽  
Lukas Valihrach
Keyword(s):  
Rna Sequencing ◽  
Small Rna ◽  
High Sensitivity ◽  
Small Rna Sequencing ◽  
Rna Seq ◽  
Liquid Biopsies ◽  
Comprehensive Overview ◽  
Rna Molecules ◽  
Novel Mirna ◽  
The Many

MicroRNAs (miRNAs) are a class of small RNA molecules that have an important regulatory role in multiple physiological and pathological processes. Their disease-specific profiles and presence in biofluids are properties that enable miRNAs to be employed as non-invasive biomarkers. In the past decades, several methods have been developed for miRNA analysis, including small RNA sequencing (RNA-seq). Small RNA-seq enables genome-wide profiling and analysis of known, as well as novel, miRNA variants. Moreover, its high sensitivity allows for profiling of low input samples such as liquid biopsies, which have now found applications in diagnostics and prognostics. Still, due to technical bias and the limited ability to capture the true miRNA representation, its potential remains unfulfilled. The introduction of many new small RNA-seq approaches that tried to minimize this bias, has led to the existence of the many small RNA-seq protocols seen today. Here, we review all current approaches to cDNA library construction used during the small RNA-seq workflow, with particular focus on their implementation in commercially available protocols. We provide an overview of each protocol and discuss their applicability. We also review recent benchmarking studies comparing each protocol’s performance and summarize the major conclusions that can be gathered from their usage. The result documents variable performance of the protocols and highlights their different applications in miRNA research. Taken together, our review provides a comprehensive overview of all the current small RNA-seq approaches, summarizes their strengths and weaknesses, and provides guidelines for their applications in miRNA research.

scholarly journals Sequencing of small RNAs of the fern Pleopeltis minima (Polypodiaceae) offers insight into the evolution of the microRNA repertoire in land plants

2016 ◽  
Author(s):  
Florencia Berruezo ◽  
Flavio S. J. de Souza ◽  
Pablo I. Picca ◽  
Sergio I. Nemirovsky ◽  
Leandro Martinez-Tosar ◽  
...  
Keyword(s):  
Small Rna ◽  
Land Plant ◽  
Land Plants ◽  
Phylogenetic Position ◽  
Seed Plants ◽  
Messenger Rnas ◽  
Rna Molecules ◽  
Functional Studies ◽  
Mirna Genes ◽  
Ecological Importance

AbstractMicroRNAs (miRNAs) are short, single stranded RNA molecules that regulate the stability and translation of messenger RNAs in diverse eukaryotic groups. Several miRNA genes are of ancient origin and have been maintained in the genomes of animal and plant taxa for hundreds of millions of years, and functional studies indicate that ancient miRNAs play key roles in development and physiology. In the last decade, genome and small RNA (sRNA) sequencing of several plant species have helped unveil the evolutionary history of land plant miRNAs. Land plants are divided into bryophytes (liverworts, mosses), lycopods (clubmosses and spikemosses), monilophytes (ferns and horsetails), gymnosperms (cycads, conifers and allies) and angiosperms (flowering plants). Among these, the fern group occupies a key phylogenetic position, since it represents the closest extant cousin taxon of seed plants, i.e. gymno- and angiosperms. However, in spite of their evolutionary, economic and ecological importance, no fern genome has been sequenced yet and few genomic resources are available for this group. Here, we sequenced the small RNA fraction of an epiphytic South American fern, Pleopeltis minima (Polypodiaceae), and compared it to plant miRNA databases, allowing for the identification of miRNA families that are shared by all land plants, shared by all vascular plants (tracheophytes) or shared by euphyllophytes (ferns and seed plants) only. Using the recently described transcriptome of another fern, Lygodium japonicum, we also estimated the degree of conservation of fern miRNA targets in relation to other plant groups. Our results pinpoint the origin of several miRNA families in the land plant evolutionary tree with more precision and are a resource for future genomic and functional studies of fern miRNAs.

scholarly journals Histone H1 prevents non-CG methylation-mediated small RNA biogenesis in Arabidopsis heterochromatin

2021 ◽  
Author(s):  
Jaemyung Choi ◽  
David Bruce Lyons ◽  
Daniel Zilberman
Keyword(s):  
Dna Methylation ◽  
Transposable Elements ◽  
Small Rna ◽  
Histone H3 ◽  
Histone H1 ◽  
Dna Methyltransferases ◽  
Flowering Plants ◽  
Genomic Sequences ◽  
Rna Molecules ◽  
Rna Biogenesis

Flowering plants utilize small RNA molecules to guide DNA methyltransferases to genomic sequences. This RNA-directed DNA methylation (RdDM) pathway preferentially targets euchromatic transposable elements. However, RdDM is thought to be recruited by methylation of histone H3 at lysine 9 (H3K9me), a hallmark of heterochromatin. How RdDM is targeted to euchromatin despite an affinity for H3K9me is unclear. Here we show that loss of histone H1 enhances heterochromatic RdDM, preferentially at nucleosome linker DNA. Surprisingly, this does not require SHH1, the RdDM component that binds H3K9me. Furthermore, H3K9me is dispensable for RdDM, as is CG DNA methylation. Instead, we find that non-CG methylation is specifically required for small RNA biogenesis, and without H1 small RNA production quantitatively expands to non-CG methylated loci. Our results demonstrate that H1 enforces the separation of euchromatic and heterochromatic DNA methylation pathways by excluding the small RNA-generating branch of RdDM from non-CG methylated heterochromatin.

Regulatory aspects of RNAi in plant production.

2021 ◽  
pp. 154-158
Author(s):  
Werner Schenkel ◽  
Achim Gathmann
Keyword(s):  
Small Rna ◽  
Plant Protection ◽  
Plant Protection Products ◽  
Plant Production ◽  
The European Union ◽  
Relevant Aspect ◽  
Rna Molecules ◽  
Rnai Technology ◽  
Living Organisms ◽  
Intended Use

Abstract Technologies based on RNA interference (RNAi) may be used in plant production in different contexts. With respect to applicable regulations, a major distinction is to be made between plants producing small RNA molecules due to modifications of the genome and topically applied plant protection products (PPPs) based on double-stranded RNA (dsRNA). The first group may be further divided into those using RNAi technology to achieve changes in the plant's metabolism and those where plant-produced RNA molecules are intended to impact other organisms that interact with the plant. For PPPs, relevant aspects are whether the product contains living organisms or only purified molecules. The intended use of the product is another relevant aspect with respect to regulation. It is expected that PPPs will be among the first products utilizing the RNAi mechanism in the European Union. This chapter discusses the regulation of modified RNAi plants and the regulation of PPPs utilizing RNAi mechanisms.

scholarly journals Degradation of Fungal MicroRNAs Triggered by Short Tandem Target Mimics Is via the Small-RNA-Degrading Nuclease

2019 ◽  
Vol 85 (9) ◽  
Author(s):  
Yulong Wang ◽  
Zhangxun Wang ◽  
Wenjing Yang ◽  
Xiangyun Xie ◽  
Haiyan Cheng ◽  
...  
Keyword(s):  
Small Rna ◽  
Target Genes ◽  
Rna Binding ◽  
Fungal Species ◽  
Metarhizium Robertsii ◽  
Content Type ◽  
Rna Molecules ◽  
Significant Difference ◽  
Pol Iii ◽  
Short Tandem

ABSTRACT MicroRNAs (miRNAs) have been recognized as sequence-specific regulators of the genome, transcriptome, and proteome in eukaryotes. However, the functions and working mechanisms of hundreds of fungal miRNA-like (miR-like) RNAs are obscure. Here, we report that a short tandem target mimic (STTM) triggered the degradation of several fungal miR-like RNAs in two different fungal species, Metarhizium robertsii and Aspergillus flavus, and that small-RNA-degrading nucleases (SDNs) were indispensable for such degradation. STTMs were most effective when the fungal polymerase II (Pol II) promoter was used for their expression, while the Pol III promoter was less effective. The length of the STTM spacer, approximately 48 to 96 nucleotides, and the number of miR-like RNA binding sites, from 2 to 4 copies, showed no significant difference in the degradation of miR-like RNAs. STTMs modulated the miR-like RNA expression levels in at least two different fungal species, which further impacted fungal asexual growth and sporulation. Further analysis showed that the degraded miR-like RNAs in STTM mutants led to the upregulation of potential target genes involved in fungal development and conidial production, which result in different phenotypes in these mutants. The STTM technology developed in this study is an effective and powerful tool for the functional dissection of fungal miR-like RNAs. IMPORTANCE The development and application of STTM technology to block miR-like RNAs in M. robertsii and A. flavus may allow for efficient generation of miR-like RNA mutants in various fungi, providing a powerful tool for functional genomics of small RNA molecules in fungi.

scholarly journals Selective Uptake of Small RNA Molecules in the Virion of Murine Gammaherpesvirus 68

2008 ◽  
Vol 83 (5) ◽  
pp. 2321-2326 ◽  
Author(s):  
Anna R. Cliffe ◽  
Anthony A. Nash ◽  
Bernadette M. Dutia
Keyword(s):  
Small Rna ◽  
Nuclear Staining ◽  
Rna Molecules ◽  
Murine Gammaherpesvirus ◽  
Barr Virus ◽  
Genes Encoding ◽  
Infected Cells ◽  
Epstein Barr ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Noncoding RNAs are a feature of many herpesvirus genomes. They include microRNAs, whose function is the subject of intense investigation, in addition to longer RNA molecules such as the Epstein-Barr virus-encoded RNAs and herpesvirus saimiri U RNAs, which have been known for some time but whose function is still not well defined. Murine gammaherpesvirus 68 (MHV-68) encodes eight viral tRNA-like molecules (vtRNAs) of unknown function. Investigating the kinetics of expression of the vtRNAs, we observed that they were present directly after infection with the virus. This strongly suggested that vtRNAs were part of the virion structure, which was confirmed by their detection within various purified, RNase-treated preparations. Although both viral and cellular mRNAs were also detected within the MHV-68 virion, the major RNA species present were small RNAs of around 70 nucleotides in length. Interestingly, incorporation of viral mRNA was not related to the relative abundance in infected cells, as M11 mRNA, which is present at low abundance, was found in virions. MHV-76, which lacks the genes encoding the vtRNAs, also incorporated small RNA molecules within the virion, suggesting a requirement for these molecules for virion maturation. In productively infected cells the vtRNAs localized predominantly within the cytoplasm, although they also exhibited a globular pattern of nuclear staining. Their presence in the cytoplasm is consistent with interaction with virion components prior to maturation of virus particles. The significance of these findings for virion architecture and function is discussed.

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