scholarly journals Cis-directed cleavage and nonstoichiometric abundances of 21-nt reproductive phasiRNAs in grasses

2018 ◽  
Author(s):  
Saleh Tamim ◽  
Zhaoxia Cai ◽  
Sandra Mathioni ◽  
Jixian Zhai ◽  
Chong Teng ◽  
...  
Keyword(s):  
Computational Analysis ◽  
Developmental Stages ◽  
Transcriptional Gene Silencing ◽  
List Type ◽  
Small Interfering Rnas ◽  
Regulatory Activity ◽  
Sequence Composition ◽  
Post Transcriptional Gene Silencing ◽  
Non Coding Rnas ◽  
Weak Accumulation

SummaryPost-transcriptional gene silencing in plants results from independent activities of diverse small RNA types. In anthers of grasses, hundreds of loci yield non-coding RNAs that are processed into 21- and 24-nt phased small interfering RNAs (phasiRNAs); these are triggered by miR2118 and miR2275.We characterized these “reproductive phasiRNAs” from rice panicles and anthers across seven developmental stages. Our computational analysis identified characteristics of the 21-nt reproductive phasiRNAs that impact their biogenesis, stability, and potential functions.We demonstrate that 21-nt reproductive phasiRNAs can function in cis to target their own precursors. We observed evidence of this cis regulatory activity in both rice (Oryza sativa) and maize (Zea mays). We validated this activity with evidence of cleavage and a resulting shift in the pattern of phasiRNA production.We characterize biases in phasiRNA biogenesis, demonstrating that the Pol II-derived “top” strand phasiRNAs are consistently higher abundance than the bottom strand. The first phasiRNA from each precursor overlaps the miR2118 target site, and this impacts phasiRNA accumulation or stability, evident in the weak accumulation of this phasiRNA position. Additional influences on this first phasiRNA duplex include the sequence composition and length, and we show that these factors impact Argonaute loading.

scholarly journals An atypical class of non-coding small RNAs produced in rice leaves upon bacterial infection

2021 ◽  
Author(s):  
Ganna Reshetnyak ◽  
Jonathan M. Jacobs ◽  
Florence Auguy ◽  
Coline Sciallano ◽  
Lisa Claude ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Stress Responses ◽  
Small Rnas ◽  
Early Stage ◽  
Kinase Domain ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Virulent Strains ◽  
Microbe Interactions

ABSTRACTNon-coding small RNAs (sRNA) act as mediators of gene silencing and regulate plant growth, development and stress responses. Early insights into plant sRNAs established a role in antiviral defense and they are now extensively studied across plant-microbe interactions. Here, sRNA sequencing discovered a class of sRNA in rice (Oryza sativa) specifically associated with foliar diseases caused by Xanthomonas oryzae bacteria. Xanthomonas-induced small RNAs (xisRNAs) loci were distinctively upregulated in response to diverse virulent strains at an early stage of infection producing a single duplex of 20-22nt sRNAs. xisRNAs production was dependent on the Type III secretion system, a major bacterial virulence factor for host colonization. xisRNA loci overlap with annotated transcripts sequences often encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and some xisRNA loci coincide with the coding sequence of a conserved kinase motif. xisRNAs exhibit features of small interfering RNAs and their biosynthesis depend on canonical components OsDCL1 and OsHEN1. xisRNA induction possibly mediates post-transcriptional gene silencing but they do not broadly suppress cis-genes expression on the basis of mRNA-seq data. Overall, our results identify a group of unusual sRNAs with a potential role in plant-microbe interactions.

scholarly journals FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Chenjiang You ◽  
Wenrong He ◽  
Runlai Hang ◽  
Cuiju Zhang ◽  
Xiaofeng Cao ◽  
...  
Keyword(s):  
Rna Degradation ◽  
Transcriptional Gene Silencing ◽  
Rrna Processing ◽  
Small Interfering Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Processing Defects ◽  
And Function ◽  
Mirna Abundance ◽  
Unknown Link

Abstract Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, it also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5′−3′ RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5′−3′ exonuclease genes XRN2 and XRN3, we find that a large number of 21-nt risiRNAs are generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlates with pre-rRNA processing defects in these mutants. We also show that these risiRNAs are loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation.

scholarly journals Degradome Sequencing-based Identification of PhasiRNAs Biogenesis Pathways in Oryza Sativa

2020 ◽  
Author(s):  
Lan Yu ◽  
Rongkai Guo ◽  
Yeqin Jiang ◽  
Xinghuo Ye ◽  
Zhihong Yang ◽  
...  
Keyword(s):  
Oryza Sativa ◽  
Regulatory Network ◽  
Growth And Development ◽  
Methylation Status ◽  
Regulatory Function ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Comprehensive Understanding ◽  
Degradome Sequencing ◽  
Post Transcriptional Gene Silencing

Abstract Background: The miRNA-derived secondary phased small interfering RNAs (phasiRNAs) participate in post-transcriptional gene silencing and play important roles in various bio-processes in plants. In rice, two miRNAs, miR2118 and miR2275, were mainly responsible for the triggering of 21-nt and 24-nt phasiRNAs biogenesis, respectively. However, compare to other plant species, relative fewer phasiRNA biogenesis pathways have been discovered in rice, which limits the comprehensive understanding of the mechanism of phasiRNA biogenesis and the miRNA derived regulatory network. Results: In this study, we performed a systematical searching for phasiRNA biogenesis pathways in rice. As a result, five novel 21-nt phasiRNA biogenesis pathways and five novel 24-nt phasiRNA biogenesis pathways were identified. Further exploration of the regulatory function of phasiRNAs revealed eleven novel phasiRNAs with 21-nt length targeting forty-one genes, most of which involving in the growth and development of rice. In addition, five novel phasiRNAs with 24-nt length were found targeting the promoter of an OsCKI1 gene and causing higher methylation status in panicle, implying their regulatory function of the transcription of OsCKI1, and subsequently affect the development of rice. Conclusions: These results substantially extended the information of phasiRNA biogenesis pathways and their regulatory function in rice.

scholarly journals Improved computational analysis of ribosome dynamics from 5’P degradome data using fivepeseq

2020 ◽  
Author(s):  
Lilit Nersisyan ◽  
Maria Ropat ◽  
Vicent Pelechano
Keyword(s):  
Computational Analysis ◽  
Developmental Stages ◽  
Biological Information ◽  
List Type ◽  
Computational Tools ◽  
Ribosome Stalling ◽  
Specific Analysis ◽  
Key Points ◽  
Reproducible Analysis

ABSTRACTIn eukaryotes, 5’-3’ co-translation degradation machinery follows the last translating ribosome providing an in vivo footprint of its position. Thus 5’P degradome sequencing, in addition to informing about RNA decay, also provides valuable information regarding ribosome dynamics. Multiple experimental methods have been developed to investigate the mRNA degradome, however computational tools for their reproducible analysis are lacking. Here we present fivepseq: an easy-to-use application for analysis and interactive visualization of 5’P degradome data. This tool performs both metagene and gene specific analysis, and allows to easily investigate codon specific ribosome pauses. To demonstrate its ability to provide new biological information, we investigate gene specific ribosome pauses in S. cerevisiae after eIF5A depletion. In addition to identifying pauses at expected codon motifs, we identify multiple genes with strain-specific frameshifts. To show its wide applicability, we investigate more complex 5’P degradome from A. thaliana and discover both motif-specific ribosome protection associated with particular developmental stages, as well as generally increased ribosome protection at termination level associated with age. Our work shows how the use of improved analysis tools for the study of 5’P degradome can significantly increase the biological information that can be derived from such datasets and facilitate its reproducible analysis.KEY POINTSAnalysis of 5’P degradome data with fivepseq informs about global and gene-specific translational features.Frameshifts in translation-related genes in S. cerevisiae may be linked to ribosome stalling.Ribosome protection at termination and codon motifs are linked to development in A. Thaliana.

scholarly journals Binding and processing of small dsRNA molecules by the class 1 RNase III protein encoded by sweet potato chlorotic stunt virus

2014 ◽  
Vol 95 (2) ◽  
pp. 486-495 ◽  
Author(s):  
Isabel Weinheimer ◽  
Kajohn Boonrod ◽  
Mirko Moser ◽  
Michael Wassenegger ◽  
Gabi Krczal ◽  
...  
Keyword(s):  
Sweet Potato ◽  
Transcriptional Gene Silencing ◽  
Specific Class ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Rnase Iii ◽  
Potato Plants ◽  
Post Transcriptional Gene Silencing ◽  
Class 1

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) causes heavy yield losses in sweet potato plants co-infected with other viruses. The dsRNA-specific class 1 RNase III–like endoribonuclease (RNase3) encoded by SPCSV suppresses post-transcriptional gene silencing and eliminates antiviral defence in sweet potato plants in an endoribonuclease activity-dependent manner. RNase3 can cleave long dsRNA molecules, synthetic small interfering RNAs (siRNAs), and plant- and virus-derived siRNAs extracted from sweet potato plants. In this study, conditions for efficient expression and purification of enzymically active recombinant RNase3 were established. Similar to bacterial class 1 RNase III enzymes, RNase3-Ala (a dsRNA cleavage-deficient mutant) bound to and processed double-stranded siRNA (ds-siRNA) as a dimer. The results support the classification of SPCSV RNase3 as a class 1 RNase III enzyme. There is little information about the specificity of RNase III enzymes on small dsRNAs. In vitro assays indicated that ds-siRNAs and microRNAs (miRNAs) with a regular A-form conformation were cleaved by RNase3, but asymmetrical bulges, extensive mismatches and 2′-O-methylation of ds-siRNA and miRNA interfered with processing. Whereas Mg2+ was the cation that best supported the catalytic activity of RNase3, binding of 21 nt small dsRNA molecules was most efficient in the presence of Mn2+. Processing of long dsRNA by RNase3 was efficient at pH 7.5 and 8.5, whereas ds-siRNA was processed more efficiently at pH 8.5. The results revealed factors that influence binding and processing of small dsRNA substrates by class 1 RNase III in vitro or make them unsuitable for processing by the enzyme.

scholarly journals Comparison between the Repression Potency of siRNA Targeting the Coding Region and the 3′-Untranslated Region of mRNA

2013 ◽  
Vol 2013 ◽  
pp. 1-5 ◽  
Author(s):  
Ching-Fang Lai ◽  
Chih-Ying Chen ◽  
Lo-Chun Au
Keyword(s):  
Gene Silencing ◽  
Thermodynamic Stability ◽  
Untranslated Region ◽  
Stop Codon ◽  
Secondary Structures ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Coding Region ◽  
Post Transcriptional Gene Silencing ◽  
Flanking Sequences

Small interfering RNAs (siRNAs) are applied for post-transcriptional gene silencing by binding target mRNA. A target coding region is usually chosen, although the3′-untranslated region (3′-UTR) can also be a target. This study elucidates whether the coding region or3′-UTR elicits higher repression. pFLuc and pRLuc are two reporter plasmids. A segment ofFLucgene was PCR-amplified and inserted behind the stop codon of theRLucgene of the pRLuc. Similarly, a segment ofRLucgene was inserted behind the stop codon ofFLuc. Two siFLuc and two siRLuc were siRNAs designed to target the central portions of these segments. Therefore, the siRNA encountered the same targets and flanking sequences. Results showed that the two siFLuc elicited higher repression when theFLucsegment resided in the coding region. Conversely, the two siRLuc showed higher repression when theRLucsegment was in the3′-UTR. These results indicate that both the coding region and the3′-UTR can be more effective targets. The thermodynamic stability of the secondary structures was analyzed. The siRNA elicited higher repression in the coding region when the target configuration was stable, and needed to be solved by translation. A siRNA may otherwise favor the target at3′-UTR.

scholarly journals Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)

2009 ◽  
Vol 2009 ◽  
pp. 1-13 ◽  
Author(s):  
Eric J. Devor ◽  
Lingyan Huang ◽  
Abdusattor Abdukarimov ◽  
Ibrokhim Y. Abdurakhmonov
Keyword(s):  
Small Rna ◽  
High Throughput Sequencing ◽  
Rna World ◽  
Regulatory Elements ◽  
Plant Genome ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Sequencing Technologies ◽  
Post Transcriptional Gene Silencing

The “RNA revolution” that started at the end of the 20th century with the discovery of post-transcriptional gene silencing and its mechanism via RNA interference (RNAi) placed tiny 21-24 nucleotide long noncoding RNAs (ncRNAs) in the forefront of biology as one of the most important regulatory elements in a host of physiologic processes. The discovery of new classes of ncRNAs including endogenous small interfering RNAs, microRNAs, and PIWI-interacting RNAs is a hallmark in the understanding of RNA-dependent gene regulation. New generation high-throughput sequencing technologies further accelerated the studies of this “tiny world” and provided their global characterization and validation in many biological systems with sequenced genomes. Nevertheless, for the many “yet-unsequenced” plant genomes, the discovery of small RNA world requires in vitro cloning from purified cellular RNAs. Thus, reproducible methods for in vitro small RNA cloning are of paramount importance and will remain so into the foreseeable future. In this paper, we present a description of existing small RNA cloning methods as well as next-generation sequencing methods that have accelerated this research along with a description of the application of one in vitro cloning method in an initial small RNA survey in the “still unsequenced” allotetraploid cotton genome.

scholarly journals MicroRNAs in the skin: role in development, homoeostasis and regeneration

2017 ◽  
Vol 131 (15) ◽  
pp. 1923-1940 ◽  
Author(s):  
Steven Horsburgh ◽  
Nicola Fullard ◽  
Mathilde Roger ◽  
Abbie Degnan ◽  
Stephen Todryk ◽  
...  
Keyword(s):  
Therapeutic Interventions ◽  
Transcriptional Gene Silencing ◽  
Biological Functions ◽  
Vast Number ◽  
Regulate Gene Expression ◽  
Post Transcriptional Gene Silencing ◽  
Non Coding Rnas ◽  
And Function ◽  
Molecular Signals

The skin is the largest organ of the integumentary system and possesses a vast number of functions. Due to the distinct layers of the skin and the variety of cells which populate each, a tightly regulated network of molecular signals control development and regeneration, whether due to programmed cell termination or injury. MicroRNAs (miRs) are a relatively recent discovery; they are a class of small non-coding RNAs which possess a multitude of biological functions due to their ability to regulate gene expression via post-transcriptional gene silencing. Of interest, is that a plethora of data demonstrates that a number of miRs are highly expressed within the skin, and are evidently key regulators of numerous vital processes to maintain non-aberrant functioning. Recently, miRs have been targeted as therapeutic interventions due to the ability of synthetic ‘antagomiRs’ to down-regulate abnormal miR expression, thereby potentiating wound healing and attenuating fibrotic processes which can contribute to disease such as systemic sclerosis (SSc). This review will provide an introduction to the structure and function of the skin and miR biogenesis, before summarizing the literature pertaining to the role of miRs. Finally, miR therapies will also be discussed, highlighting important future areas of research.

Therapeutic targeting of non-coding RNAs

2013 ◽  
Vol 54 ◽  
pp. 127-145 ◽  
Author(s):  
Thomas C. Roberts ◽  
Matthew J.A. Wood
Keyword(s):  
Gene Expression ◽  
Antisense Oligonucleotides ◽  
Regulation Of Gene Expression ◽  
Gene Activation ◽  
Mature Mirnas ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Promising Therapeutic Approach ◽  
Non Coding Rnas ◽  
Mirna Sponges

ncRNAs (non-coding RNAs) are implicated in a wide variety of cellular processes, including the regulation of gene expression. In the present chapter we consider two classes of ncRNA: miRNAs (microRNAs) which are post-transcriptional regulators of gene expression and lncRNAs (long ncRNAs) which mediate interactions between epigenetic remodelling complexes and chromatin. Mutation and misexpression of ncRNAs have been implicated in many disease conditions and, as such, pharmacological modulation of ncRNAs is a promising therapeutic approach. miRNA activity can be antagonized with antisense oligonucleotides which sequester or degrade mature miRNAs, and expressed miRNA sponges which compete with target transcripts for miRNA binding. Conversely, synthetic or expressed miRNA mimics can be used to treat a deficiency in miRNA expression. Similarly, conventional antisense technologies can be used to silence lncRNAs. Targeting promoter-associated RNAs with siRNAs (small interfering RNAs) results in recruitment of chromatin-modifying activities and induces transcriptional gene silencing. Alternatively, targeting natural antisense transcripts with siRNAs or antisense oligonucleotides can abrogate endogenous epigenetic silencing leading to transcriptional gene activation. The ability to modulate gene expression at the epigenetic level presents exciting new opportunities for the treatment of human disease.

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