scholarly journals Enhanced RNAi stability through imperfect inverted repeats: nucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes in plants

2021 ◽  
Author(s):  
Daai Zhang ◽  
Chengcheng Zhong ◽  
Neil Smith ◽  
Rob Defeyter ◽  
Ian Greaves ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Full Potential ◽  
Nucleotide Substitutions ◽  
Hairpin Rna ◽  
Developmental Progression ◽  
Target Dna ◽  
Transgenic Lines ◽  
Rna Biogenesis ◽  
And Function ◽  
Interfering Rna

Abstract Hairpin RNA (hpRNA) transgenes, with a perfect inverted-repeat (IR) DNA, have been the most successful RNA interference (RNAi) method in plants. Here we show that hpRNA transgenes were invariably methylated in the IR DNA and the adjacent promoter, causing transcriptional self-silencing and preventing the full potential of RNAi. Nucleotide substitutions in the sense sequence, which disrupts the perfect IR DNA structure, were sufficient to prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi. Substituting all cytosine (C) with thymine (T) nucleotides, in a G:U hpRNA design, prevented DNA methylation and self-silencing but still allowed for the formation of perfect hpRNA due to G:U wobble base-pairing. The G:U design induces effective RNAi in 90–96% of transgenic lines, compared to 57–65% for the traditional hpRNA design. Furthermore, while a traditional hpRNA transgene showed increasing DNA methylation and self-silencing from cotyledons to true leaves, the G:U transgenes avoided this developmental progression of self-silencing and induced RNAi throughout plant growth. The G:U and traditional hpRNA transgenes generated small interfering RNA (siRNA) with different 5’ phosphorylation, which resembled the endogenous tasiRNA and miRNA, respectively. Furthermore, our results suggest that siRNAs from the two transgene designs function differently to induce target DNA methylation, one (from traditional hpRNA) through the canonical RdDM pathway and the other (G:U hpRNA) a non-canonical pathway. Our study not only revealed a methylation-resistant RNAi transgene design but also provided new mechanistic insights into small RNA biogenesis and function in plants.

scholarly journals Large-Scale, High-Throughput Validation of Short Hairpin RNA Sequences for RNA Interference

2006 ◽  
Vol 11 (3) ◽  
pp. 236-246 ◽  
Author(s):  
Laurence H. Lamarcq ◽  
Bradley J. Scherer ◽  
Michael L. Phelan ◽  
Nikolai N. Kalnine ◽  
Yen H. Nguyen ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Strong Support ◽  
Gc Content ◽  
Rapid Identification ◽  
Hairpin Rna ◽  
Rna Sequences ◽  
Short Hairpin ◽  
Short Hairpin Rnas ◽  
Interfering Rna

A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for positionspecific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNAinterference (RNAi).

scholarly journals Impact of DNA methylation on 3D genome structure

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Diana Buitrago ◽  
Mireia Labrador ◽  
Juan Pablo Arcon ◽  
Rafael Lema ◽  
Oscar Flores ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Chromatin Structure ◽  
Chromatin Condensation ◽  
Genome Structure ◽  
Dna Methyltransferases ◽  
Model System ◽  
Structure And Function ◽  
3D Genome ◽  
Cellular Machinery ◽  
And Function

AbstractDetermining the effect of DNA methylation on chromatin structure and function in higher organisms is challenging due to the extreme complexity of epigenetic regulation. We studied a simpler model system, budding yeast, that lacks DNA methylation machinery making it a perfect model system to study the intrinsic role of DNA methylation in chromatin structure and function. We expressed the murine DNA methyltransferases in Saccharomyces cerevisiae and analyzed the correlation between DNA methylation, nucleosome positioning, gene expression and 3D genome organization. Despite lacking the machinery for positioning and reading methylation marks, induced DNA methylation follows a conserved pattern with low methylation levels at the 5’ end of the gene increasing gradually toward the 3’ end, with concentration of methylated DNA in linkers and nucleosome free regions, and with actively expressed genes showing low and high levels of methylation at transcription start and terminating sites respectively, mimicking the patterns seen in mammals. We also see that DNA methylation increases chromatin condensation in peri-centromeric regions, decreases overall DNA flexibility, and favors the heterochromatin state. Taken together, these results demonstrate that methylation intrinsically modulates chromatin structure and function even in the absence of cellular machinery evolved to recognize and process the methylation signal.

scholarly journals DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα

2013 ◽  
Vol 451 (3) ◽  
pp. 453-461 ◽  
Author(s):  
Claudia C. S. Chini ◽  
Carlos Escande ◽  
Veronica Nin ◽  
Eduardo N. Chini
Keyword(s):  
Breast Cancer ◽  
Nuclear Receptor ◽  
Clock Genes ◽  
Mrna Levels ◽  
Protein Levels ◽  
The Stability ◽  
And Function ◽  
Interfering Rna ◽  
In Cells

The nuclear receptor Rev-erbα has been implicated as a major regulator of the circadian clock and integrates circadian rhythm and metabolism. Rev-erbα controls circadian oscillations of several clock genes and Rev-erbα protein degradation is important for maintenance of the circadian oscillations and also for adipocyte differentiation. Elucidating the mechanisms that regulate Rev-erbα stability is essential for our understanding of these processes. In the present paper, we report that the protein DBC1 (Deleted in Breast Cancer 1) is a novel regulator of Rev-erbα. Rev-erbα and DBC1 interact in cells and in vivo, and DBC1 modulates the Rev-erbα repressor function. Depletion of DBC1 by siRNA (small interfering RNA) in cells or in DBC1-KO (knockout) mice produced a marked decrease in Rev-erbα protein levels, but not in mRNA levels. In contrast, DBC1 overexpression significantly enhanced Rev-erbα protein stability by preventing its ubiquitination and degradation. The regulation of Rev-erbα protein levels and function by DBC1 depends on both the N-terminal and C-terminal domains of DBC1. More importantly, in cells depleted of DBC1, there was a dramatic decrease in circadian oscillations of both Rev-erbα and BMAL1. In summary, our data identify DBC1 as an important regulator of the circadian receptor Rev-erbα and proposes that Rev-erbα could be involved in mediating some of the physiological effects of DBC1.

scholarly journals On the origin and evolutionary consequences of gene body DNA methylation

2016 ◽  
Vol 113 (32) ◽  
pp. 9111-9116 ◽  
Author(s):  
Adam J. Bewick ◽  
Lexiang Ji ◽  
Chad E. Niederhuth ◽  
Eva-Maria Willing ◽  
Brigitte T. Hofmeister ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Recombinant Inbred Lines ◽  
Dna Methyltransferases ◽  
Inbred Lines ◽  
Gene Body ◽  
Gene Body Methylation ◽  
Eutrema Salsugineum ◽  
And Function ◽  
Evolutionary Consequences

In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum. Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales.

A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

2005 ◽  
Vol 32 (8) ◽  
pp. 671 ◽  
Author(s):  
Song Chen ◽  
Christopher A. Helliwell ◽  
Li-Min Wu ◽  
Elizabeth S. Dennis ◽  
Narayana M. Upadhyaya ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Marker Gene ◽  
Direct Repeat ◽  
Transgene Silencing ◽  
Selective Marker ◽  
Hairpin Rna ◽  
Transgenic Lines ◽  
Flanking Sequences ◽  
Dna Vector ◽  
Selection Of

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d / r) or inverted-repeat (i / r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i / r T-DNA insertions is a primary inducer of transgene silencing in plants.

scholarly journals Reinforcement of transcriptional silencing by a positive feedback between DNA methylation and non-coding transcription

2021 ◽  
Author(s):  
M Hafiz Rothi ◽  
Masayuki Tsuzuki ◽  
Shriya Sethuraman ◽  
Andrzej T Wierzbicki
Keyword(s):  
Dna Methylation ◽  
Small Interfering Rna ◽  
Important Determinant ◽  
Dna Methyltransferases ◽  
Heterochromatin Formation ◽  
Pol V ◽  
Non Coding Rnas ◽  
Silencing Pathways ◽  
Interfering Rna ◽  
The Impact

Abstract Non-coding transcription is an important determinant of heterochromatin formation. In Arabidopsis thaliana a specialized RNA polymerase V (Pol V) transcribes pervasively and produces long non-coding RNAs. These transcripts work with small interfering RNA to facilitate locus-specific establishment of RNA-directed DNA methylation (RdDM). Subsequent maintenance of RdDM is associated with elevated levels of Pol V transcription. However, the impact of DNA methylation on Pol V transcription remained unresolved. We found that DNA methylation strongly enhances Pol V transcription. The level of Pol V transcription is reduced in mutants defective in RdDM components working downstream of Pol V, indicating that RdDM is maintained by a mutual reinforcement of DNA methylation and Pol V transcription. Pol V transcription is affected only on loci that lose DNA methylation in all sequence contexts in a particular mutant, including mutants lacking maintenance DNA methyltransferases, which suggests that RdDM works in a complex crosstalk with other silencing pathways.

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.

scholarly journals Developmental Patterning and Neurogenetic Gradients of Nurr1 Positive Neurons in the Rat Claustrum and Lateral Cortex

2021 ◽  
Vol 15 ◽  
Author(s):  
Chao Fang ◽  
Hong Wang ◽  
Robert Konrad Naumann
Keyword(s):  
Expression Patterns ◽  
Prenatal Development ◽  
Superficial Layer ◽  
Lateral Cortex ◽  
Developmental Progression ◽  
Previous Birth ◽  
Developmental Patterning ◽  
And Function ◽  
Anterior Posterior

The claustrum is an enigmatic brain structure thought to be important for conscious sensations. Recent studies have focused on gene expression patterns, connectivity, and function of the claustrum, but relatively little is known about its development. Interestingly, claustrum-enriched genes, including the previously identified marker Nurr1, are not only expressed in the classical claustrum complex, but also embedded within lateral neocortical regions in rodents. Recent studies suggest that Nurr1 positive neurons in the lateral cortex share a highly conserved genetic expression pattern with claustrum neurons. Thus, we focus on the developmental progression and birth dating pattern of the claustrum and Nurr1 positive neurons in the lateral cortex. We comprehensively investigate the expression of Nurr1 at various stages of development in the rat and find that Nurr1 expression first appears as an elongated line along the anterior-posterior axis on embryonic day 13.5 (E13.5) and then gradually differentiates into multiple sub-regions during prenatal development. Previous birth dating studies of the claustrum have led to conflicting results, therefore, we combine 5-ethynyl-2′-deoxyuridine (EdU) labeling with in situ hybridization for Nurr1 to study birth dating patterns. We find that most dorsal endopiriform (DEn) neurons are born on E13.5 to E14.5. Ventral claustrum (vCL) and dorsal claustrum (dCL) are mainly born on E14.5 to E15.5. Nurr1 positive cortical deep layer neurons (dLn) and superficial layer neurons (sLn) are mainly born on E14.5 to E15.5 and E15.5 to E17.5, respectively. Finally, we identify ventral to dorsal and posterior to anterior neurogenetic gradients within vCL and DEn. Thus, our findings suggest that claustrum and Nurr1 positive neurons in the lateral cortex are born sequentially over several days of embryonic development and contribute toward charting the complex developmental pattern of the claustrum in rodents.

Sign in / Sign up

Export Citation Format

Share Document