RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

Tirumalai, Varsha; Prasad, M. Rajendra; Shivaprasad, P. V.

doi:10.3791/61394-v

Cited by 3 publications

(4 citation statements)

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“…Isolated RNA was quantified using a Qubit Fluorimeter with RNA BR dye (Thermo Fisher Scientific). sRNA Northern hybridization was performed as described previously (Shivaprasad et al., 2012; Tirumalai et al., 2020). Roughly 10 μg of total RNA was electrophoresed at 80 V for 3 h in a 15% acrylamide gel (with 8 m urea and acrylamide:bisacrylamide [19:1]).…”

Section: Methodsmentioning

confidence: 99%

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Indian Himalayan natural Arabidopsis thaliana accessions with abolished miR158 levels exhibit robust miR173‐initiated trans‐acting cascade silencing

et al. 2023

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Small RNAs (sRNAs) such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) are short 20-24nucleotide non-coding RNAs. They are key regulators of gene expression in plants and other organisms. Several 22-nucleotide miRNAs trigger biogenesis cascades of trans-acting secondary siRNAs, which are involved in various developmental and stress responses. Here we show that Himalayan Arabidopsis thaliana accessions having natural mutations in the miR158 locus exhibit robust cascade silencing of the pentatricopeptide repeat (PPR)-like locus. Furthermore, we show that these cascade sRNAs trigger tertiary silencing of a gene involved in transpiration and stomatal opening. The natural deletions or insertions in MIR158 led to improper processing of miR158 precursors, thereby blocking synthesis of mature miR158. Reduced miR158 levels led to increased levels of its target, a pseudo-PPR gene that is targeted by tasiRNAs generated by the miR173 cascade in other accessions. Using sRNA datasets derived from Indian Himalayan accessions, as well as overexpression and knockout lines of miR158, we show that absence of miR158 led to buildup of pseudo-PPR-derived tertiary sRNAs. These tertiary sRNAs mediated robust silencing of a gene involved in stomatal closure in Himalayan accessions lacking miR158 expression. We functionally validated the tertiary phasiRNA that targets NHX2, which encodes a Na + -K + /H + antiporter protein, thereby regulating transpiration and stomatal conductance. Overall, we report the role of the miRNA-TAS-siRNA-pseudogene-tertiary phasiRNA-NHX2 pathway in plant adaptation.

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Section: Methodsmentioning

confidence: 99%

“…The blots were exposed to phosphor screen (GE Healthcare) and imaged on a Typhoon molecular imager (GE Healthcare). The membranes were stripped at 80°C, followed by repeated hybridization with other probes as described before (Tirumalai et al., 2020).…”

Section: Methodsmentioning

confidence: 99%

Indian Himalayan natural Arabidopsis thaliana accessions with abolished miR158 levels exhibit robust miR173‐initiated trans‐acting cascade silencing

et al. 2023

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“…About 8-15 µg of total RNA was used for sRNA northern as described earlier (Shivaprasad et al, 2012; Tirumalai et al, 2020). Membranes were stripped and used for multiple hybridizations.…”

Section: Methodsmentioning

confidence: 99%

Upstream regulator of genomic imprinting in rice endosperm is a small RNA-associated chromatin remodeler

Pal,

Sundar G,

et al. 2023

Preprint

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Genomic imprinting is observed in endosperm, a placenta-like seed tissue, where transposable elements (TEs) and repeat-derived small(s)RNAs mediate epigenetic changes in plants. In imprinting, uniparental gene expression arises due to parent-specific epigenetic marks on one allele but not on the other. The importance of sRNAs and their regulation in endosperm development or in imprinting is poorly understood in crops. Here we show that a previously uncharacterized CLASSY (CLSY)-family chromatin remodeler named OsCLSY3 is essential for rice endosperm development and imprinting, acting as an upstream player in sRNA pathway. Comparative transcriptome and genetic analysis indicated its endosperm-preferred expression and its paternally imprinted nature. These important features were modulated by RNA-directed DNA methylation (RdDM) of tandemly arranged TEs in its promoter. Upon perturbation of OsCLSY3 in transgenic lines we observed defects in endosperm development and loss of around 70% of all sRNAs. Interestingly, well-conserved endosperm-specific sRNAs (siren) that are vital for reproductive fitness in angiosperms were dependent on OsCLSY3. We also observed many imprinted genes and seed development-associated genes under the control of CLSY3-dependent RdDM. These results support an essential role of OsCLSY3 in rice endosperm development and imprinting, and propose similar regulatory strategies involving CLSY3 homologs among other cereals.

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“…sRNA northern hybridisation sRNA northern hybridisation was performed as described earlier (Shivaprasad et al, 2012;Tirumalai et al, 2020). Around 8 µg of total RNA was electrophoresed on a denaturing gel, blotted onto Hybond N+ membrane (Cytiva) and UV crosslinked.…”

Section: Multiple Sequence Alignment and Phylogenymentioning

confidence: 99%

Oryzagenera-specific novel Histone H4 variant predisposes H4 Lysine5 Acetylation marks to modulate salt stress responses

Sotelo-Parrilla

Raju

et al. 2023

Preprint

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Paralogous variants of canonical histones guide accessibility to DNA and function as additional layers of genome regulation. Across eukaryotes, the occurrence, mechanism of action and functional significance of several variants of core histones are well known except that of histone H4. Here we show that a novel variant of H4 (H4.V), expressing tissue-specifically among members of Oryza genera, mediates specific epigenetic changes contributing majorly to salt tolerance. H4.V was incorporated to specific chromosomal locations where it blocked deposition of active histone marks. Under salt stress, large scale re-distribution of H4.V enabled incorporation of stress dependent histone H4 Lysine5 Acetylation (H4K5Ac) marks. Mis-expression of H4.V led to defects at morphological level especially in reproductive tissues, and in mounting stress responses. H4.V mediated these alterations by condensing chromatin at specific genomic regions as seen with cryo-EM structure of reconstituted H4.V containing nucleosomes. These results not only uncovered the presence of a H4 variant in plants, but also a novel chromatin regulation of stress responses that might have contributed to success of semi-aquatic Oryza members under variable water-limiting conditions.

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RNA Blot Analysis for the Detection and Quantification of Plant MicroRNAs

Cited by 3 publications

References 0 publications

Indian Himalayan natural Arabidopsis thaliana accessions with abolished miR158 levels exhibit robust miR173‐initiated trans‐acting cascade silencing

Indian Himalayan natural Arabidopsis thaliana accessions with abolished miR158 levels exhibit robust miR173‐initiated trans‐acting cascade silencing

Upstream regulator of genomic imprinting in rice endosperm is a small RNA-associated chromatin remodeler

Oryzagenera-specific novel Histone H4 variant predisposes H4 Lysine5 Acetylation marks to modulate salt stress responses

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