Methods for detecting RNA degradation intermediates in plants

Ueno, Daishin; Yamasaki, Satoshi; Kato, Ko

doi:10.1016/j.plantsci.2022.111241

Cited by 4 publications

(4 citation statements)

References 54 publications

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“…We selected Si sRNA24 to study the cleavage pattern of its significantly down-regulated target transcripts AT1G65090 and AT4G15765 using RNA-ligase-mediated Rapid Amplification of cDNA Ends (5’-RLM-RACE) (Llave et al, 2002; Ueno et al, 2022). For the predicted target transcript AT1G65090, no band corresponding to an expected canonical cleavage product was detected (Supplemental Figure 6A) (Carbonell, 2017c; Carthew & Sontheimer, 2009).…”

Section: Resultsmentioning

confidence: 99%

“…We selected amir296 and its predicted target gene AT2G45240 to investigate the canonical PTGS cleavage site using RNA-ligase-mediated Rapid Amplification of cDNA Ends (5’-RLM-RACE) (Llave et al, 2002; Ueno et al, 2022). Samples were obtained from transformed protoplasts at 24 hptr, from control protoplasts (mock-treated without amiRNA construct) and from non-treated protoplasts.…”

Section: Resultsmentioning

confidence: 99%

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A pipeline for validation of sRNA effectors suggests cross-kingdom communication in the symbiosis ofArabidopsiswithSerendipita indica

Nasfi,

Shahbazi,

Bitterlich

et al. 2023

Preprint

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Cross-kingdom RNA interference (ckRNAi), the bidirectional communication between microbes and their host counterparts, is a key element in the outcome of host colonization. Whether mutualistic fungi of the Serendipitaceae family with their broad host range use small RNA effectors (sRNAs) to colonize plant roots is still under debate. To investigate if ckRNAi is a factor in the symbiosis ofSerendipita indica(Si) withArabidopsis thaliana(Ath), we established a pipeline to validate expression, translocation and post-transcriptional gene silencing of host genes bySi-derived sRNAs (SisRNAs). First, we confirmed the expression ofSisRNAs both in axenic fungal culture and duringAthroot colonization using stem-loop PCR. Then, to verify the translocation of putativeSisRNA effectors, an ARGONAUTE 1 immuno-purification assay (AtAGO1-IP) was employed, detecting fungalSisRNAs being loaded into the plant RNAi machinery inSi-colonised roots. Subsequently,SisRNAs and artificial sRNAs (amiRNAs), were transiently expressed inAthprotoplasts to test their gene silencing activity. Stem-loop PCR confirmed expression of sRNA effectors and qPCR validated post-transcriptional gene silencing of their predicted target genes involved in cell wall organization, hormonal signalling regulation, plant immunity and gene expression. Moreover, 5’-RLM-RACE analysis revealed amiRNA-mediated canonical cleavage inArabidopsistargets. In conclusion, this study provides a blueprint for rapid selection and analysis of sRNA effectors in plant-microbe interactions in general and suggests cross-kingdom communication in the Sebacinoid symbiosis.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A pipeline for validation of sRNA effectors suggests cross-kingdom communication in the symbiosis ofArabidopsiswithSerendipita indica

Nasfi,

Shahbazi,

Bitterlich

et al. 2023

Preprint

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“…Moreover, some of these 5 0 P protected peaks were as abundant and sharp as miRNA-directed slicing 5 0 P peaks (Hou et al, 2016;Lee et al, 2020). These findings conflict with a common view of RNA degradome composition that the most prominent 5 0 P ends arise from endonucleolytic cleavage (Bracken et al, 2011;Ueno et al, 2022). The contribution of EJC-protected fragments to the major decay intermediates in Arabidopsis WT might remain underestimated given that in human, half of the EJC binding sites map to non-canonical sites (Sauliere et al, 2012;Singh et al, 2012).…”

Section: Xrn4 Products In the Rna Degradomementioning

confidence: 91%

Arabidopsis mRNA decay landscape shaped by XRN 5′‐3′ exoribonucleases

et al. 2023

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5 0 -3 0 exoribonucleases (XRNs) play crucial roles in the control of RNA processing, quality, and quantity in eukaryotes. Although genome-wide profiling of RNA decay fragments is now feasible, how XRNs shape the plant mRNA degradome remains elusive. Here, we profiled and analyzed the RNA degradomes of Arabidopsis wild-type and mutant plants with defects in XRN activity. Deficiency of nuclear XRN3 or cytoplasmic XRN4 activity but not nuclear XRN2 activity greatly altered Arabidopsis mRNA decay profiles. Short excised linear introns and cleaved pre-mRNA fragments downstream of polyadenylation sites were polyadenylated and stabilized in the xrn3 mutant, demonstrating the unique function of XRN3 in the removal of cleavage remnants from pre-mRNA processing. Further analysis of stabilized XRN3 substrates confirmed that pre-mRNA 3 0 end cleavage frequently occurs after adenosine. The most abundant decay intermediates in wildtype plants include not only the primary substrates of XRN4 but also the products of XRN4-mediated cytoplasmic decay. An increase in decay intermediates with 5 0 ends upstream of a consensus motif in the xrn4 mutant suggests that there is an endonucleolytic cleavage mechanism targeting the 3 0 untranslated regions of many Arabidopsis mRNAs. However, analysis of decay fragments in the xrn4 mutant indicated that, except for microRNA-directed slicing, endonucleolytic cleavage events in the coding sequence rarely result in major decay intermediates. Together, these findings reveal the major substrates and products of nuclear and cytoplasmic XRNs along Arabidopsis transcripts and provide a basis for precise interpretation of RNA degradome data.

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“…Additionally, certain degradation of RNA in samples is inevitable, due to internal regulatory processes, especially the cell's mechanism in preventing RNA accumulation in the cells. This lead to the inevitable degradation of some RNA by the endonucleases produced in the cells [31]. Hence, prevention measures can be taken to minimise the effect of endonucleases towards RNA in the samples.…”

Section: Total Rna Quantification and Quality Assessmentmentioning

confidence: 99%

Identification of MiR398 and Its Regulatory Roles in Terpenoid Biosynthesis of Persicaria odorata

Mahadi,

Abd Samad,

A. Samad

2024

Mal. J. Fund. Appl. Sci.

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Persicaria odorata is an herbaceous plant with antifungal and antibacterial properties. The plant produces secondary metabolites, including phenols, sulphur-containing compounds and terpenoids, in response to biotic and abiotic stresses. Terpenoids in P. odorata are synthesized through the mevalonate (MVA) and the methylerythritol phosphate (MEP) pathways. 1-deoxy-D-xylulose-5phosphate isomerase (DXR) is a rate-limiting enzyme in the MEP pathway and may be regulated by microRNA (miRNA) miR398. There is a lack of evidence showing miR398 regulation in terpenoid biosynthesis in P. odorata through DXR-targeting. The study aimed to verify the stem-loop structure of miR398 and analyse its expression towards the target gene, DXR, in treated and control P. odorata. RNA was quantified and qualitatively analysed using a Nanodrop spectrophotometer and agarose gel electrophoresis. The stem loop of miR398 was verified using reverse transcriptase PCR (RT-PCR) and agarose gel electrophoresis. The expression of miR398 and DXR was compared between control and treated samples. Treated leaves were punctured with needles and left for 48h before harvest. Gene expressions were quantified and normalised using reference genes. The stem-loop structure of miR398 was confirmed, despite possible primer mismatches and unspecific binding. This step was essential before comparing and assessing the gene expression of miR398 and DXR. A decrease in abundance of miR398 whereas in increase in abundance was in the treated sample compared to the control sample indicating that miR398 negatively regulated DXR under stress conditions, suggesting an increase in terpenoid synthesis as a defence mechanism. DXR acts as a rate-limiting enzyme in the MEP metabolic pathway. Further studies are needed to quantify the effects of miR398 on terpenoid biosynthesis after wounding in P. odorata.

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Methods for detecting RNA degradation intermediates in plants

Cited by 4 publications

References 54 publications

A pipeline for validation of sRNA effectors suggests cross-kingdom communication in the symbiosis ofArabidopsiswithSerendipita indica

A pipeline for validation of sRNA effectors suggests cross-kingdom communication in the symbiosis ofArabidopsiswithSerendipita indica

Arabidopsis mRNA decay landscape shaped by XRN 5′‐3′ exoribonucleases

Identification of MiR398 and Its Regulatory Roles in Terpenoid Biosynthesis of Persicaria odorata

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