Novel Stress-Inducible Antisense RNAs of Protein-Coding Loci Are Synthesized by RNA-Dependent RNA Polymerase

Matsui, Atsushi; Iida, Kei; Tanaka, Maho; Yamaguchi, Katsushi; Mizuhashi, Kayoko; Kim, Jong-Myong; Takahashi, Satoshi; Kobayashi, Norio; Shigenobu, Shuji; Shinozaki, Kazuo; Seki, Motoaki

doi:10.1104/pp.17.00787

Cited by 18 publications

(9 citation statements)

References 66 publications

(118 reference statements)

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“…Since only two out of 9,445 (0.02%) reads mapped antisense to the ERCC RNA Spike-In collection 11 and 0 of 19,665 reads mapped antisense to the highly expressed gene RUBISCO ACTIVASE ( RCA ) ( AT2G39730 ), we conclude that spurious antisense is rare or absent from nanopore DRS data. This simplifies the interpretation of authentic antisense RNAs, which is important in Arabidopsis because the distinction between RT-dependent template switching and authentic antisense RNAs produced by RNA-dependent RNA polymerases that copy mRNA is not straightforward 17 . For example, by nanopore DRS, we could identify Arabidopsis long non-coding antisense RNAs, such as those at the auxin efflux carriers PIN4 and PIN7 (Figure 1C, Supplementary figure 1G).…”

Section: Resultsmentioning

confidence: 99%

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Parker

Knop

Sherwood

et al. 2019

Preprint

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21Understanding genome organization and gene regulation requires insight into RNA transcription, 22 processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a 23 wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA 24 methylation (m 6 A). Here we show that m 6 A can be mapped in full-length mRNAs transcriptome-wide 25 and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, 26 poly(A) site choice and poly(A) tail length. Loss of m 6 A from 3' untranslated regions is associated 27 with decreased relative transcript abundance and defective RNA 3′ end formation. A functional 28 consequence of disrupted m 6 A is a lengthening of the circadian period. We conclude that nanopore 29 direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length 30 single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying 31 this approach to less well-studied species could transform our understanding of what their genomes 32 encode. 33 34 misidentification of 3′ ends through internal priming 3 , spurious antisense and splicing events 46 produced by RT template switching 4,5 , and the inability to detect all base modifications in the 47 copying process 6 . The fragmentation of RNA prior to short-read sequencing makes it difficult to 48 interpret the combination of authentic RNA processing events and remains an unsolved problem 7 . 49We investigated whether long-read direct RNA sequencing (DRS) with nanopores 8 could 50 reveal the complexity of Arabidopsis mRNA processing and modifications. In nanopore DRS, the 51 protein pore (nanopore) sits in a membrane through which an electrical current is passed, and intact 52 RNA is fed through the nanopore by a motor protein 8 . Each RNA sequence within the nanopore 53 (5 bases) can be identified by the magnitude of signal it produces. Arabidopsis is a pathfinder model 54 in plant biology, and its genome annotation strongly influences the annotation and our 55 understanding of what other plant genomes encode. We applied nanopore DRS and Illumina RNAseq 56 to wild-type Arabidopsis (Col-0) and mutants defective in m 6 A 9 and exosome-mediated RNA decay 10 . 57We reveal m 6 A and combinations of RNA processing events (alternative patterns of 5′ capped 58 transcription start sites, splicing, 3′ polyadenylation and poly(A) tail length) in full-length Arabidopsis 59 mRNAs transcriptome-wide. 60 61 Results 62Nanopore DRS detects long, complex mRNAs and short, structured non-coding RNAs 63We purified poly (A)+ RNA from four biological replicates of 14-day-old Arabidopsis Col-0 seedlings. 64We incorporated synthetic External RNA Controls Consortium (ERCC) RNA Spike-In mixes into all 65 replicates 11,12 and carried out nanopore DRS. Illumina RNAseq was performed in parallel on similar 66 material. Using Guppy base-calling (Oxford Nanopore Technologies) and minimap2 alignment 67 software 13 , we identified around 1 mi...

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Section: Resultsmentioning

confidence: 99%

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Parker

Knop

Sherwood

et al. 2019

Preprint

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“…The finding that 58 annotated MIRNAs are downregulated in rdr1/2/6 mutant plants suggests that RDR1, RDR2, and RDR6 may have redundant roles in regulating processes that were not previously uncovered by studying RDR1, RDR2, or RDR6 alone. Similarly, another study found that loss of RDR1/2/6 reduces the expression of antisense RNAs during plant response to abiotic stresses, and TAS2 antisense RNAs were lower in rdr1/6 , rdr2/6 , or rdr1/2/6 mutant plants when compared with rdr6 single-mutant plants [ 62 ]. These findings reveal that RDR1/2/6 regulate the abundance of stress-related antisense RNAs cooperatively, but this functionality is separate from RDR6 functionality in siRNA biogenesis due to the fact that TAS2 was still found to accumulate in the absence of RDR6 function ( rdr6 mutation).…”

Section: Discussionmentioning

confidence: 99%

Global Analysis of RNA-Dependent RNA Polymerase-Dependent Small RNAs Reveals New Substrates and Functions for These Proteins and SGS3 in Arabidopsis

Hua

Berkowitz

Willmann

et al. 2021

ncRNA

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RNA silencing pathways control eukaryotic gene expression transcriptionally or posttranscriptionally in a sequence-specific manner. In RNA silencing, the production of double-stranded RNA (dsRNA) gives rise to various classes of 20–24 nucleotide (nt) small RNAs (smRNAs). In Arabidopsis thaliana, smRNAs are often derived from long dsRNA molecules synthesized by one of the six genomically encoded RNA-dependent RNA Polymerase (RDR) proteins. However, the full complement of the RDR-dependent smRNAs and functions that these proteins and their RNA-binding cofactors play in plant RNA silencing has not been fully uncovered. To address this gap, we performed a global genomic analysis of all six RDRs and two of their cofactors to find new substrates for RDRs and targets of the resulting RDR-derived siRNAs to uncover new functions for these proteins in plants. Based on these analyses, we identified substrates for the three RDRγ clade proteins (RDR3–5), which had not been well-characterized previously. We also identified new substrates for the other three RDRs (RDR1, RDR2, and RDR6) as well as the RDR2 cofactor RNA-directed DNA methylation 12 (RDM12) and the RDR6 cofactor suppressor of gene silencing 3 (SGS3). These findings revealed that the target substrates of SGS3 are not limited to those solely utilized by RDR6, but that this protein seems to be a more general cofactor for the RDR family of proteins. Additionally, we found that RDR6 and SGS3 are involved in the production of smRNAs that target transcripts related to abiotic stresses, including water deprivation, salt stress, and ABA response, and as expected the levels of these mRNAs are increased in rdr6 and sgs3 mutant plants. Correspondingly, plants that lack these proteins (rdr6 and sgs3 mutants) are hypersensitive to ABA treatment, tolerant to high levels of PEG8000, and have a higher survival rate under salt treatment in comparison to wild-type plants. In total, our analyses have provided an extremely data-rich resource for uncovering new functions of RDR-dependent RNA silencing in plants, while also revealing a previously unexplored link between the RDR6/SGS3-dependent pathway and plant abiotic stress responses.

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“…On the other hand, OsRDR4 should be involved in RNA interference and silencing: RNA-dependent RNA polymerases (RDRs), in fact, are the core proteins mediating RNA interference as they can amplify microRNAs and small temporal RNAs and can also produce double-stranded RNA using small interfering RNAs as primers [ 158 ]. In addition, RDRs have antisense RNA synthesis activity independent of the endogenous small RNA pathways [ 159 ]. Quite interestingly, OsRDR4 expression appears to be quite specific, as it does not show detectable levels in vegetative tissues except the shoot apical meristem [ 158 ].…”

Section: Discussionmentioning

confidence: 99%

Seed Dormancy Involves a Transcriptional Program That Supports Early Plastid Functionality during Imbibition

Gianinetti¹,

Finocchiaro²,

Bagnaresi³

et al. 2018

Plants

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Red rice fully dormant seeds do not germinate even under favorable germination conditions. In several species, including rice, seed dormancy can be removed by dry-afterripening (warm storage); thus, dormant and non-dormant seeds can be compared for the same genotype. A weedy (red) rice genotype with strong dormancy was used for mRNA expression profiling, by RNA-Seq, of dormant and non-dormant dehulled caryopses (here addressed as seeds) at two temperatures (30 °C and 10 °C) and two durations of incubation in water (8 h and 8 days). Aim of the study was to highlight the differences in the transcriptome of dormant and non-dormant imbibed seeds. Transcript data suggested important differences between these seeds (at least, as inferred by expression-based metabolism reconstruction): dry-afterripening seems to impose a respiratory impairment onto non-dormant seeds, thus glycolysis is deduced to be preferentially directed to alcoholic fermentation in non-dormant seeds but to alanine production in dormant ones; phosphoenolpyruvate carboxykinase, pyruvate phosphate dikinase and alanine aminotransferase pathways appear to have an important gluconeogenetic role associated with the restoration of plastid functions in the dormant seed following imbibition; correspondingly, co-expression analysis pointed out a commitment to guarantee plastid functionality in dormant seeds. At 8 h of imbibition, as inferred by gene expression, dormant seeds appear to preferentially use carbon and nitrogen resources for biosynthetic processes in the plastid, including starch and proanthocyanidins accumulation. Chromatin modification appears to be a possible mechanism involved in the transition from dormancy to germination. Non-dormant seeds show higher expression of genes related to cell wall modification, suggesting they prepare for acrospire/radicle elongation.

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Novel Stress-Inducible Antisense RNAs of Protein-Coding Loci Are Synthesized by RNA-Dependent RNA Polymerase

Cited by 18 publications

References 66 publications

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Global Analysis of RNA-Dependent RNA Polymerase-Dependent Small RNAs Reveals New Substrates and Functions for These Proteins and SGS3 in Arabidopsis

Seed Dormancy Involves a Transcriptional Program That Supports Early Plastid Functionality during Imbibition

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