Ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates the production of secondary siRNAs in plants

Iwakawa, Hiro-oki; Lam, Andy Y.W.; Mine, Akira; Fujita, Taketoshi; Kiyokawa, Kaori; Yoshikawa, Manabu; Takeda, Atsushi; Iwasaki, Shigeo; Tomari, Yukihide

doi:10.1016/j.celrep.2021.109300

Cited by 37 publications

(31 citation statements)

References 56 publications

(98 reference statements)

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“…Translation as an initiator of PTGS and epigenetic silencing Protein synthesis is commonly merely seen as a target of PTGS by reducing the amount of available RNA and/or interfering with translation. Our study adds to a growing body of work identifying translation also as a trigger for PTGS (Sun et al, 2020;Iwakawa et al, 2021). This became evident after epigenetic reactivation of EVD, from which splicing-coupled PCPA generates separate RNA isoforms from a single transcription unit.…”

Section: Discussionmentioning

confidence: 70%

“…This interpretation is neither compatible with the highly discrete nature of the stalling events experimentally detected in our study nor with the noticeable absence, from polysomes, of the TE RNAs used by Kim et al to build their model,EVD excepted (Fig 4A). Possible causes of ribosome stalling, including mRNA-extrinsic ones, are further evoked in Appendix Discussion and Appendix Fig S13 . In particular, we argue why a recently proposed model of RISC-mediated stalling-coupled RDR6 recruitment (Iwakawa et al, 2021) during tasiRNA biogenesis unlikely applies to EVD, leading us to consider a potential causal role for stalling-coupled 5'-OH RNA fragment generation in stimulating RDR6 activity. Since we were unable, however, to genetically impede stalling (Appendix Fig S10), we can only speculate in the model described in the next section since, at this stage, 5'OH RNA fragments might be mere byproducts of ribosome stalling.…”

Section: Discussionmentioning

confidence: 90%

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Innate, translation‐dependent silencing of an invasive transposon in Arabidopsis

Oberlin

Rajeswaran

et al. 2021

EMBO Reports

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Co-evolution between hosts' and parasites' genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral-like silencing response. To investigate this response's activation, we studied the single-copy element EVAD E (EVD), one of few representatives of the large Ty1/ Copia family able to proliferate in Arabidopsis when epigenetically reactivated. In Ty1/Copia elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full-length genomic flGAG-POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly nuclear flGAG-POL. During this process, an unusually intense ribosomal stalling event coincides with mRNA breakage yielding unconventional 5'OH RNA fragments that evade RNA quality control. The starting point of sRNA production by RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6), exclusively on shGAG, occurs precisely at this breakage point. This hitherto-unrecognized "translationdependent silencing" (TdS) is independent of codon usage or GC content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against EVD de novo invasions that underlies its associated sRNA pattern.

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Section: Discussionmentioning

confidence: 70%

Section: Discussionmentioning

confidence: 90%

Innate, translation‐dependent silencing of an invasive transposon in Arabidopsis

Oberlin

Rajeswaran

et al. 2021

EMBO Reports

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“…Several observations indicate the importance of ribosome association for miRNA-triggered siRNA production from TAS precursors. Ribosome profiling experiments demonstrate ribosome association with TAS3 precursors ( 89 , 90 ), and the TAS2 precursor contains a short open reading frame important for tasiRNA biogenesis, and both the precursor and cleavage fragments associate with polyribosomes ( 91 ). Furthermore, detailed biochemical analyses reveal the importance of ribosome stalling in proximity to the 5′ miR390 site in TAS3 precursors ( 90 ), and ribosome stalling and collision at rare codons in transposable element mRNAs correlates with their production of 21-nt siRNAs ( 58 ) (observed in mutants defective in DNA methylation, and, therefore, often referred to as epigenetically activated siRNAs, or easiRNAs ( 28 )).…”

Section: Discussionmentioning

confidence: 99%

“…Ribosome profiling experiments demonstrate ribosome association with TAS3 precursors ( 89 , 90 ), and the TAS2 precursor contains a short open reading frame important for tasiRNA biogenesis, and both the precursor and cleavage fragments associate with polyribosomes ( 91 ). Furthermore, detailed biochemical analyses reveal the importance of ribosome stalling in proximity to the 5′ miR390 site in TAS3 precursors ( 90 ), and ribosome stalling and collision at rare codons in transposable element mRNAs correlates with their production of 21-nt siRNAs ( 58 ) (observed in mutants defective in DNA methylation, and, therefore, often referred to as epigenetically activated siRNAs, or easiRNAs ( 28 )). It appears, therefore, that stalled ribosomes, in particular in combination with RISC, act as a trigger of secondary siRNA production, perhaps by extending the time of RISC association with target mRNA by AGO interaction.…”

Section: Discussionmentioning

confidence: 99%

Nuclear and cytoplasmic RNA exosomes and PELOTA1 prevent miRNA-induced secondary siRNA production in Arabidopsis

Vigh

Bressendorff

Thieffry

et al. 2022

Nucleic Acids Research

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Amplification of short interfering RNA (siRNAs) via RNA-dependent RNA polymerases (RdRPs) is of fundamental importance in RNA silencing. Plant microRNA (miRNA) action generally does not involve engagement of RdRPs, in part thanks to a poorly understood activity of the cytoplasmic exosome adaptor SKI2. Here, we show that inactivation of the exosome subunit RRP45B and SKI2 results in similar patterns of miRNA-induced siRNA production. Furthermore, loss of the nuclear exosome adaptor HEN2 leads to secondary siRNA production from miRNA targets largely distinct from those producing siRNAs in ski2. Importantly, mutation of the Release Factor paralogue PELOTA1 required for subunit dissociation of stalled ribosomes causes siRNA production from miRNA targets overlapping with, but distinct from, those affected in ski2 and rrp45b mutants. We also show that in exosome mutants, miRNA targets can be sorted into producers and non-producers of illicit secondary siRNAs based on trigger miRNA levels and miRNA:target affinity rather than on presence of 5′-cleavage fragments. We propose that stalled RNA-Induced Silencing Complex (RISC) and ribosomes, but not mRNA cleavage fragments released from RISC, trigger siRNA production, and that the exosome limits siRNA amplification by reducing RISC dwell time on miRNA target mRNAs while PELOTA1 does so by reducing ribosome stalling.

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“…In addition, the level of translation was inversely correlated to 22G-RNA abundance, suggesting that elongating ribosomes antagonize the production of 22G-RNAs 51 . In plants ( Arabidopsis thaliana ), ribosome stalling occurs 12–13 nt upstream of several miRNA binding sites, mediated by the binding of the plant-specific dsRNA-binding protein SGS3, which triggers the production of downstream secondary phased siRNAs 52 . Although the mechanisms differ in each case, the stop codon enrichment at piRNA 5′ ends suggests that also piRNA biogenesis may be connected to the translational machinery.…”

Section: Discussionmentioning

confidence: 99%

An evolutionarily conserved stop codon enrichment at the 5′ ends of mammalian piRNAs

2022

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PIWI-interacting RNAs (piRNAs) are small RNAs required to recognize and silence transposable elements. The 5’ ends of mature piRNAs are defined through cleavage of long precursor transcripts, primarily by Zucchini (Zuc). Zuc-dependent cleavage typically occurs immediately upstream of a uridine. However, Zuc lacks sequence preference in vitro, pointing towards additional unknown specificity factors. Here, we examine murine piRNAs and reveal a strong and specific enrichment of three sequences (UAA, UAG, UGA)—corresponding to stop codons—at piRNA 5’ ends. Stop codon sequences are also enriched immediately after piRNA processing intermediates, reflecting their Zuc-dependent tail-to-head arrangement. Further analyses reveal that a Zuc in vivo cleavage preference at four sequences (UAA, UAG, UGA, UAC) promotes 5’ end stop codons. This observation is conserved across mammals and possibly further. Our work provides new insights into Zuc-dependent cleavage and may point to a previously unrecognized connection between piRNA biogenesis and the translational machinery.

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Ribosome stalling caused by the Argonaute-microRNA-SGS3 complex regulates the production of secondary siRNAs in plants

Cited by 37 publications

References 56 publications

Innate, translation‐dependent silencing of an invasive transposon in Arabidopsis

Innate, translation‐dependent silencing of an invasive transposon in Arabidopsis

Nuclear and cytoplasmic RNA exosomes and PELOTA1 prevent miRNA-induced secondary siRNA production in Arabidopsis

An evolutionarily conserved stop codon enrichment at the 5′ ends of mammalian piRNAs

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