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 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 Sequences throughout the basic β-1,3-glucanase mRNA coding region are targets for homology dependent post-transcriptional gene silencing

1999 ◽  
Vol 20 (2) ◽  
pp. 143-152 ◽  
Author(s):  
John J. M. R. Jacobs ◽  
Matthew Sanders ◽  
Marc Bots ◽  
Marco Andriessen ◽  
Gerben J. van Eldik ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Transcriptional Gene Silencing ◽  
Coding Region ◽  
Post Transcriptional Gene Silencing

scholarly journals RNA Silencing against Geminivirus: Complementary Action of Posttranscriptional Gene Silencing and Transcriptional Gene Silencing in Host Recovery

2008 ◽  
Vol 83 (3) ◽  
pp. 1332-1340 ◽  
Author(s):  
Edgar A. Rodríguez-Negrete ◽  
Jimena Carrillo-Tripp ◽  
Rafael F. Rivera-Bustamante
Keyword(s):  
Gene Silencing ◽  
Intergenic Region ◽  
Rna Silencing ◽  
Methylation Status ◽  
Inverse Correlation ◽  
Plant Viruses ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Coding Region ◽  
Natural Defense

ABSTRACT RNA silencing in plants is a natural defense system mechanism against invading nucleic acids such as viruses. Geminiviruses, a family of plant viruses characterized by a circular, single-stranded DNA genome, are thought to be both inducers and targets of RNA silencing. Some natural geminivirus-host interactions lead to symptom remission or host recovery, a process commonly associated with RNA silencing-mediated defense. Pepper golden mosaic virus (PepGMV)-infected pepper plants show a recovery phenotype, which has been associated with the presence of virus-derived small RNAs. The results presented here suggest that PepGMV is targeted by both posttranscriptional and transcriptional gene silencing mechanisms. Two types of virus-related small interfering RNAs (siRNAs) were detected: siRNAs of 21 to 22 nucleotides (nt) in size that are related to the coding regions (Rep, TrAP, REn, and movement protein genes) and a 24-nt population primarily associated to the intergenic regions. Methylation levels of the PepGMV A intergenic and coat protein (CP) coding region were measured by a bisulfite sequencing approach. An inverse correlation was observed between the methylation status of the intergenic region and the concentration of viral DNA and symptom severity. The intergenic region also showed a methylation profile conserved in all times analyzed. The CP region, on the other hand, did not show a defined profile, and its methylation density was significantly lower than the one found on the intergenic region. The participation of both PTGS and TGS mechanisms in host recovery is discussed.

scholarly journals In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs

2015 ◽  
Author(s):  
Angel Emilio Martínez de Alba ◽  
Ana Moreno ◽  
Marc Gabriel ◽  
Allison C Mallory ◽  
Aurélie Christ ◽  
...  
Keyword(s):  
Quality Control ◽  
Rna Polymerase ◽  
Gene Silencing ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Rna Dependent Rna Polymerase ◽  
P Bodies ◽  
New Class ◽  
Post Transcriptional Gene Silencing ◽  
Rna Quality Control

Cytoplasmic degradation of endogenous RNAs is an integral part of RNA quality control (RQC) and often relies on the removal of the 5’ cap structure and their subsequent 5’ to 3’ degradation in cytoplasmic processing (P-)bodies. In parallel, many eukaryotes degrade exogenous and selected endogenous RNAs through post-transcriptional gene silencing (PTGS). In plants, PTGS depends on small interfering (si)RNAs produced after the conversion of single-stranded RNAs to doublestranded RNAs by the cellular RNA DEPENDENT RNA POLYMERASE 6 (RDR6) in cytoplasmic siRNA-bodies. PTGS and RQC compete for transgene-derived RNAs, but it is unknown whether this competition also occurs for endogenous transcripts. We show that the lethality of decapping mutants is suppressed by impairing RDR6 activity. We establish that upon decapping impairment hundreds of endogenous mRNAs give rise to a new class of rqc-siRNAs, that over-accumulate when RQC processes are impaired, a subset of which depending on RDR6 for their production. We observe that P- and siRNA-bodies often are dynamically juxtaposed, potentially allowing for crosstalk of the two machineries. Our results suggest that the decapping of endogenous RNA limits their entry into the PTGS pathway. We anticipate that the rqc-siRNAs identified in decapping mutants represent a subset of a larger ensemble of endogenous siRNAs.

scholarly journals Small circular interfering RNAs (sciRNAs) as a potent therapeutic platform for gene-silencing

2021 ◽  
Vol 49 (18) ◽  
pp. 10250-10264
Author(s):  
Hartmut Jahns ◽  
Rohan Degaonkar ◽  
Peter Podbevsek ◽  
Swati Gupta ◽  
Anna Bisbe ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Circular Rna ◽  
Antisense Strand ◽  
Rnai Pathway ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Sense Strand

Abstract In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using ‘click’ chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5′-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.

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

2021 ◽  
Vol 11 (1) ◽  
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 20–22 nt 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, with about half of them encoding protein kinase domain proteins. A number of the corresponding rice cis-genes have documented functions in immune signaling and xisRNA loci predominantly 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 Viroids as a Tool to Study RNA-Directed DNA Methylation in Plants

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1187
Author(s):  
Michael Wassenegger ◽  
Athanasios Dalakouras
Keyword(s):  
Dna Methylation ◽  
Rna Interference ◽  
Gene Silencing ◽  
Plant Cells ◽  
Transcriptional Gene Silencing ◽  
Rnai Machinery ◽  
Double Stranded Rna ◽  
Post Transcriptional Gene Silencing ◽  
Cumulative Amount

Viroids are plant pathogenic, circular, non-coding, single-stranded RNAs (ssRNAs). Members of the Pospiviroidae family replicate in the nucleus of plant cells through double-stranded RNA (dsRNA) intermediates, thus triggering the host’s RNA interference (RNAi) machinery. In plants, the two RNAi pillars are Post-Transcriptional Gene Silencing (PTGS) and RNA-directed DNA Methylation (RdDM), and the latter has the potential to trigger Transcriptional Gene Silencing (TGS). Over the last three decades, the employment of viroid-based systems has immensely contributed to our understanding of both of these RNAi facets. In this review, we highlight the role of Pospiviroidae in the discovery of RdDM, expound the gradual elucidation through the years of the diverse array of RdDM’s mechanistic details and propose a revised RdDM model based on the cumulative amount of evidence from viroid and non-viroid systems.

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