GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing

Сидоренко, Л. В.; Lee, Tzuu‐fen; Woosley, Aaron; Moskal, William A.; Bevan, Scott A.; Merlo, P. Ann Owens; Walsh, Terence A.; Wang, Xiujuan; Weaver, Staci A.; Glancy, Todd; Wang, Po-Hao; Yang, Xiaozeng; Sriram, Shreedharan; Meyers, Blake C.

doi:10.1038/s41477-017-0040-6

Cited by 30 publications

(16 citation statements)

References 44 publications

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“…The weak translation of TE RNAs contributes to both RNA truncation and localization to siRNA bodies which are both important for the selective easiRNA processing of transposon RNAs. Although RDR6 can target several endogenous genes to produce trans -acting siRNAs in normal condition 13,22,65 , the major RNA templates of RDR6 are those from foreign and invasive genetic elements such as virus, transgenes and transposons 11,33,66 . Given that non-self or alien RNAs are presumably less optimal to the host’s codon usage and commonly regulated by the similar siRNA-mediated pathway, the epigenetic silencing of viral RNAs and transgenes might be triggered by the translation-coupled pathway as was seen in transposons.…”

Section: Discussionmentioning

confidence: 99%

Translational inhibition and phase separation primes the epigenetic silencing of transposons

Kim

Lei

et al. 2020

Preprint

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19Transposons are mobile DNAs that can cause fatal mutations. To counteract these 20 genome invaders, the host genomes deploy small interfering (si) RNAs to initiate and 21 establish the epigenetic silencing. However, the regulatory mechanisms for the selective 22 recognition of transposons by the host genomes remain still elusive. Here we show that plant 23 transposon RNAs undergo frequent ribosome stalling caused by their inherently unfavourable 24 codon sequence usage. The ribosome stalling then causes the RNA truncation and the 25 localization to siRNA bodies, which are both critical prerequisites for the siRNA processing. 26 In addition, SGS3, the key protein in the siRNA biogenesis pathway, forms liquid droplets in 27 vitro through its prion-like domains implicating the role of liquid-liquid phase separation in 28 the formation of the siRNA bodies. Our study provides a novel insight into the regulatory 29 mechanisms for the recognition of invasive genetic elements which is essential for the 30 maintenance of genome integrity. 31 32 33 Keywords 34 transposon, codon optimality, ribosome stalling, easiRNA, RDR6-RdDM, liquid-liquid phase 35 separation, siRNA body, stress granule 36 37 38 45 of transposons 5 . While the canonical RdDM is mainly for reinforcing the silent state of 46 transposons, the recently proposed alternative RdDM pathway suppresses the active 47 transposons that are induced upon the epigenetic mutations and in several development 48 stages 3,6-10 . RNA-DEPENDENT RNA POLYMERASE 6 (RDR6)-SUPPRESSOR OF GENE 49 SILENCING 3 (SGS3) complex serves as a detector of transposon RNAs that selectively 50 processes them into 21 or 22-nt siRNAs (also referred to as epigenetically activated siRNAs, 51 easiRNAs) 11,12 . Transposon-derived siRNAs trigger both post-transcriptional repression by 52 cleaving the transposon RNAs (and then subsequently initiating the secondary siRNA 53 biogenesis) and establishment of the epigenetic silencing by recruiting DNA 54 methyltransferases to the target TE chromatin 5,7,8 . The biogenesis of 21 or 22-nt siRNAs in 55 plants is initiated by RDR6 which is an RNA-dependent RNA polymerase that forms double-56stranded RNAs by templating directly the target RNAs 13 . SGS3 is an RNA-binding protein 57 that interacts with RDR6 and is essential for its function 13 . The duplex RNAs are then sliced 58 to 21 or 22-nt siRNAs by DICER-LIKE 3 or 4 (DCL3/4) 8,14,15 . Since RDR6 templates are 59 directly derived from the RNAs of those mobile elements, RDR6 and the resulting easiRNAs 60 are deemed the first line of the host immune system against the invasive genetic elements. 61The RDR6-mediated easiRNA production pathway (RDR6-RdDM) is usually 62 prevented in the transcripts derived from genes by the RNA decay pathways [16][17][18] . This raises 63 4 an important question of how RDR6 specifically recognizes its TE targets and establish their 64 epigenetic silencing. Previous reports showed that the initial cleavage of target transcripts is a 65 critical prerequisite for RDR6 recog...

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Section: Discussionmentioning

confidence: 99%

Translational inhibition and phase separation primes the epigenetic silencing of transposons

Kim

Lei

et al. 2020

Preprint

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“…The quality of total RNA was evaluated using LabChipGX (Caliper LifeSciences, Waltham, MA, USA) and total RNA was used for sRNA library preparation and sequencing. The sRNA library was prepared, and data analyzed as described previously (Sidorenko et al, 2017). Raw sequencing reads were first trimmed of the adapter sequences and then mapped to the transgene sequences.…”

Section: Small Rna Library Construction Sequencing and Bioinformatimentioning

confidence: 99%

Transcription Terminator-Mediated Enhancement in Transgene Expression in Maize: Preponderance of the AUGAAU Motif Overlapping With Poly(A) Signals

et al. 2020

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The selection of transcription terminators (TTs) for pairing with high expressing constitutive promoters in chimeric constructs is crucial to deliver optimal transgene expression in plants. In this study, the use of the native combinations of four polyubiquitin gene promoters and corresponding TTs resulted in up to >3-fold increase in transgene expression in maize. Of the eight polyubiquitin promoter and TT regulatory elements utilized, seven were novel and identified from the polyubiquitin genes of Brachypodium distachyon, Setaria italica, and Zea mays. Furthermore, gene expression driven by the Cassava mosaic virus promoter was studied by pairing the promoter with distinct TTs derived from the high expressing genes of Arabidopsis. Of the three TTs studied, the polyubiquitin10 gene TT produced the highest transgene expression in maize. Polyadenylation patterns and mRNA abundance from eight distinct TTs were analyzed using 3′-RACE and next-generation sequencing. The results exhibited one to three unique polyadenylation sites in the TTs. The poly(A) site patterns for the StPinII TT were consistent when the same TT was deployed in chimeric constructs irrespective of the reporter gene and promoter used. Distal to the poly(A) sites, putative polyadenylation signals were identified in the near-upstream regions of the TTs based on previously reported mutagenesis and bioinformatics studies in rice and Arabidopsis. The putative polyadenylation signals were 9 to 11 nucleotides in length. Six of the eight TTs contained the putative polyadenylation signals that were overlaps of either canonical AAUAAA or AAUAAA-like polyadenylation signals and AUGAAU, a top-ranking-hexamer of rice and Arabidopsis gene near-upstream regions. Three of the polyubiquitin gene TTs contained the identical 9-nucleotide overlap, AUGAAUAAG, underscoring the functional significance of such overlaps in mRNA 3′ end processing. In addition to identifying new combinations of regulatory elements for high constitutive trait gene expression in maize, this study demonstrated the importance of TTs for optimizing gene expression in plants. Learning from this study could be applied to other dicotyledonous and monocotyledonous plant species for transgene

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“…An increased GC content of a transgenic construct, above whole genome GC content, was shown to decrease transgene DNA methylation and siRNA production from the Agrobacterium -introduced transgene constructs [41]. In our study, all reporter genes had considerably higher GC levels ( bar 69%, nptII 60%, GUS 48%) compared to the A. thaliana genome (~36%), however the bar gene had the least siRNA and methylC-seq coverage, providing a possible explanation for silencing of GUS and nptII , but not bar .…”

Section: Cc-by-nc-nd 40 International License It Is Made Available Umentioning

confidence: 99%

The complex architecture of plant transgene insertions

Jupe

et al. 2018

Preprint

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GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing

Cited by 30 publications

References 44 publications

Translational inhibition and phase separation primes the epigenetic silencing of transposons

Translational inhibition and phase separation primes the epigenetic silencing of transposons

Transcription Terminator-Mediated Enhancement in Transgene Expression in Maize: Preponderance of the AUGAAU Motif Overlapping With Poly(A) Signals

The complex architecture of plant transgene insertions

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