Specific gene silencing using small interfering RNAs in fish embryos

2003 ◽  
Vol 310 (4) ◽  
pp. 1089-1095 ◽  
Author(s):  
Surintorn Boonanuntanasarn ◽  
Goro Yoshizaki ◽  
Toshio Takeuchi
Keyword(s):  
Gene Silencing ◽  
Specific Gene ◽  
Small Interfering Rnas ◽  
Fish Embryos

scholarly journals Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA

2020 ◽  
Author(s):  
Mi Seul Park ◽  
GeunYoung Sim ◽  
Audrey C. Kehling ◽  
Kotaro Nakanishi
Keyword(s):  
Gene Silencing ◽  
Fold Increase ◽  
Specific Gene ◽  
Small Interfering Rnas ◽  
Guide Rna ◽  
Guide Rnas ◽  
Argonaute Proteins ◽  
Complementary Rnas ◽  
Standing Question

AbstractRNA interfering is a eukaryote-specific gene silencing by 20∼23 nucleotide (nt) microRNAs and small interfering RNAs that recruit Argonaute proteins to complementary RNAs for degradation. In humans, Argonaute2 (AGO2) has been known as the only slicer while Argonaute3 (AGO3) barely cleaves RNAs. Therefore, the intrinsic slicing activity of AGO3 remains controversial and a long-standing question. Here, we report 14-nt 3′ end-shortened variants of let-7a, miR-27a, and specific miR-17-92 families that make AGO3 an extremely competent slicer by an ∼ 82-fold increase in target cleavage. These RNAs, named cleavage-inducing tiny guide RNAs (cityRNAs), conversely lower the activity of AGO2, demonstrating that AGO2 and AGO3 have different optimum guide lengths for target cleavage. Our study sheds light on the role of tiny guide RNAs.

scholarly journals Human Argonaute2 and Argonaute3 are catalytically activated by different lengths of guide RNA

2020 ◽  
Vol 117 (46) ◽  
pp. 28576-28578
Author(s):  
Mi Seul Park ◽  
GeunYoung Sim ◽  
Audrey C. Kehling ◽  
Kotaro Nakanishi
Keyword(s):  
Gene Silencing ◽  
Specific Gene ◽  
Small Interfering Rnas ◽  
Guide Rna ◽  
Guide Rnas ◽  
Argonaute Proteins ◽  
Rna Interfering ◽  
Complementary Rnas ◽  
Standing Question

RNA interfering is a eukaryote-specific gene silencing by 20∼23-nucleotide (nt) microRNAs and small interfering RNAs that recruit Argonaute proteins to complementary RNAs for degradation. In humans, Argonaute2 (AGO2) has been known as the only slicer while Argonaute3 (AGO3) barely cleaves RNAs. Therefore, the intrinsic slicing activity of AGO3 remains controversial and a long-standing question. Here, we report 14-nt 3′ end-shortened variants of let-7a, miR-27a, and specific miR-17–92 families that make AGO3 an extremely competent slicer, increasing target cleavage up to ∼82-fold in some instances. These RNAs, named cleavage-inducing tiny guide RNAs (cityRNAs), conversely lower the activity of AGO2, demonstrating that AGO2 and AGO3 have different optimum guide lengths for target cleavage. Our study sheds light on the role of tiny guide RNAs.

scholarly journals RNA Interference: Biology, Mechanism, and Applications

2003 ◽  
Vol 67 (4) ◽  
pp. 657-685 ◽  
Author(s):  
Neema Agrawal ◽  
P. V. N. Dasaradhi ◽  
Asif Mohmmed ◽  
Pawan Malhotra ◽  
Raj K. Bhatnagar ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Defense Mechanisms ◽  
Specific Gene ◽  
Small Interfering Rnas ◽  
Allelic Series ◽  
Rnase Iii ◽  
Heterochromatin Formation ◽  
Regulatory Processes ◽  
Micro Rnas ◽  
And Function

SUMMARY Double-stranded RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. The silencing of a gene is a consequence of degradation of RNA into short RNAs that activate ribonucleases to target homologous mRNA. The resulting phenotypes either are identical to those of genetic null mutants or resemble an allelic series of mutants. Specific gene silencing has been shown to be related to two ancient processes, cosuppression in plants and quelling in fungi, and has also been associated with regulatory processes such as transposon silencing, antiviral defense mechanisms, gene regulation, and chromosomal modification. Extensive genetic and biochemical analysis revealed a two-step mechanism of RNAi-induced gene silencing. The first step involves degradation of dsRNA into small interfering RNAs (siRNAs), 21 to 25 nucleotides long, by an RNase III-like activity. In the second step, the siRNAs join an RNase complex, RISC (RNA-induced silencing complex), which acts on the cognate mRNA and degrades it. Several key components such as Dicer, RNA-dependent RNA polymerase, helicases, and dsRNA endonucleases have been identified in different organisms for their roles in RNAi. Some of these components also control the development of many organisms by processing many noncoding RNAs, called micro-RNAs. The biogenesis and function of micro-RNAs resemble RNAi activities to a large extent. Recent studies indicate that in the context of RNAi, the genome also undergoes alterations in the form of DNA methylation, heterochromatin formation, and programmed DNA elimination. As a result of these changes, the silencing effect of gene functions is exercised as tightly as possible. Because of its exquisite specificity and efficiency, RNAi is being considered as an important tool not only for functional genomics, but also for gene-specific therapeutic activities that target the mRNAs of disease-related genes.

scholarly journals Chemical strategies for strand selection in short-interfering RNAs

RSC Advances ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 2415-2426
Author(s):  
Andrew J. Varley ◽  
Jean-Paul Desaulniers
Keyword(s):  
Rna Interference ◽  
Gene Silencing ◽  
Rnai Pathway ◽  
Specific Gene ◽  
Small Interfering Rnas ◽  
Chemical Strategies

Therapeutic small interfering RNAs (siRNAs) are double stranded RNAs capable of potent and specific gene silencing through activation of the RNA interference (RNAi) pathway.

scholarly journals Widespread occurrence of microRNA-mediated target cleavage on membrane-bound polysomes

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Xiaoyu Yang ◽  
Chenjiang You ◽  
Xufeng Wang ◽  
Lei Gao ◽  
Beixin Mo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Gene Silencing ◽  
High Frequency ◽  
Small Rnas ◽  
High Throughput Sequencing ◽  
Noncoding Rnas ◽  
Small Interfering Rnas ◽  
Widespread Occurrence ◽  
Membrane Bound ◽  
Different Tissues

Abstract Background Small RNAs (sRNAs) including microRNAs (miRNAs) and small interfering RNAs (siRNAs) serve as core players in gene silencing at transcriptional and post-transcriptional levels in plants, but their subcellular localization has not yet been well studied, thus limiting our mechanistic understanding of sRNA action. Results We investigate the cytoplasmic partitioning of sRNAs and their targets globally in maize (Zea mays, inbred line “B73”) and rice (Oryza sativa, cv. “Nipponbare”) by high-throughput sequencing of polysome-associated sRNAs and 3′ cleavage fragments, and find that both miRNAs and a subset of 21-nucleotide (nt)/22-nt siRNAs are enriched on membrane-bound polysomes (MBPs) relative to total polysomes (TPs) across different tissues. Most of the siRNAs are generated from transposable elements (TEs), and retrotransposons positively contributed to MBP overaccumulation of 22-nt TE-derived siRNAs (TE-siRNAs) as opposed to DNA transposons. Widespread occurrence of miRNA-mediated target cleavage is observed on MBPs, and a large proportion of these cleavage events are MBP-unique. Reproductive 21PHAS (21-nt phasiRNA-generating) and 24PHAS (24-nt phasiRNA-generating) precursors, which were commonly considered as noncoding RNAs, are bound by polysomes, and high-frequency cleavage of 21PHAS precursors by miR2118 and 24PHAS precursors by miR2275 is further detected on MBPs. Reproductive 21-nt phasiRNAs are enriched on MBPs as opposed to TPs, whereas 24-nt phasiRNAs are nearly completely devoid of polysome occupancy. Conclusions MBP overaccumulation is a conserved pattern for cytoplasmic partitioning of sRNAs, and endoplasmic reticulum (ER)-bound ribosomes function as an independent regulatory layer for miRNA-induced gene silencing and reproductive phasiRNA biosynthesis in maize and rice.

High-throughput vectors for efficient gene silencing in plants

2002 ◽  
Vol 29 (10) ◽  
pp. 1217 ◽  
Author(s):  
Chris A. Helliwell ◽  
S. Varsha Wesley ◽  
Anna J. Wielopolska ◽  
Peter M. Waterhouse
Keyword(s):  
Gene Silencing ◽  
Insertional Mutagenesis ◽  
Single Step ◽  
Plant Genome ◽  
Specific Gene ◽  
Single Polymerase Chain Reaction ◽  
Efficient Gene ◽  
Gene Redundancy ◽  
Endogenous Genes ◽  
Rapid Generation

A major challenge in the post-genome era of plant biology is to determine the functions of all genes in the plant genome. A straightforward approach to this problem is to reduce or knockout expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study but is limited by gene redundancy, lethal knockouts, non-tagged mutants, and the inability to target the inserted element to a specific gene. The efficacy of gene silencing in plants using inverted-repeat transgene constructs that encode a hairpin RNA (hpRNA) has been demonstrated by a number of groups, and has several advantages over insertional mutagenesis. In this paper we describe two improved pHellsgate vectors that facilitate rapid generation of hpRNA-encoding constructs. pHellsgate 4 allows the production of an hpRNA construct in a single step from a single polymerase chain reaction product, while pHellsgate 8 requires a two-step process via an intermediate vector. We show that these vectors are effective at silencing three endogenous genes in Arabidopsis, FLOWERING LOCUS C, PHYTOENE DESATURASE and ETHYLENE INSENSITIVE 2. We also show that a construct of sequences from two genes silences both genes.

Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis

Science ◽  
2015 ◽  
Vol 348 (6230) ◽  
pp. 120-123 ◽  
Author(s):  
Xinyan Zhang ◽  
Ying Zhu ◽  
Xiaodan Liu ◽  
Xinyu Hong ◽  
Yang Xu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Plant Immunity ◽  
Rna Decay ◽  
Foreign Gene ◽  
Small Interfering Rnas ◽  
Developmental Defects ◽  
Posttranscriptional Gene Silencing ◽  
Endogenous Gene ◽  
Cytoplasmic Rna ◽  
Cognate Gene

Plant immunity against foreign gene invasion takes advantage of posttranscriptional gene silencing (PTGS). How plants elaborately avert inappropriate PTGS of endogenous coding genes remains unclear. We demonstrate in Arabidopsis that both 5′-3′ and 3′-5′ cytoplasmic RNA decay pathways act as repressors of transgene and endogenous PTGS. Disruption of bidirectional cytoplasmic RNA decay leads to pleiotropic developmental defects and drastic transcriptomic alterations, which are substantially rescued by PTGS mutants. Upon dysfunction of bidirectional RNA decay, a large number of 21- to 22-nucleotide endogenous small interfering RNAs are produced from coding transcripts, including multiple microRNA targets, which could interfere with their cognate gene expression and functions. This study highlights the risk of unwanted PTGS and identifies cytoplasmic RNA decay pathways as safeguards of plant transcriptome and development.

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