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Global analysis of RNA-dependent RNA Polymerase-dependent small RNAs reveals new substrates and functions for these proteins and SGS3 in Arabidopsis

ABSTRACT: In plants, smRNAs are often derived from long double-stranded RNA (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 co-factors 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 co-factors 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 RDR clade proteins (RDR3 - 5), which had not been well-characterized previously. We also identified new substrates for the other three RDRs (RDR1, 2, and 6) as well as the RDR2 co-factor RNA-DIRECTED DNA METHYLATION 12 (RDM12) and the RDR6 co-factor SUPRESSOR 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 co-factor for the RDR family 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 higher survival rate under salt treatment in comparison to wild-type (Col-0). 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.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE169270 | GEO | 2021/04/22

REPOSITORIES: GEO

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Project description:Whole genome small RNA sequencing (wild-type Col-0, abh1-1, ein5-6, ein5-6 abh1-1) and 5'-RACE (GMUCT) (wild-type Col-0 and ein5-6) sequencing of parenthetically indicated genotypes of Arabidopsis using the Illumina Genetic Analyzer. Whole-genome oligonucleotide tiling microarrays were used for gene expression studies of the entire transcriptome for wildtype Col-0, abh1-1, ein5-6, and ein5-6 abh1-1 plants. Micro (mi)RNAs and small-interfering (si)RNAs are abundant endogenous small (sm)RNAs that control transcript expression by post-transcriptional gene silencing. Here, we show that concomitant loss of XRN4/EIN5, a 5’-3’ exoribonuclease, and CBP80/ABH1, the largest subunit of the mRNA cap binding complex, results in Arabidopsis plants manifesting myriad developmental defects. Through the analysis of ein5 abh1 double mutant plants, we find that CBP80/ABH1 regulates the levels of mature miRNAs, which suggests this protein is a novel component of the miRNA-mediated RNA silencing pathway. Additionally, we show that a novel class of smRNAs are processed from both sense and anti-sense strands of ~130 endogenous transcripts that apparently are converted to double-stranded RNA and subsequently processed into smRNAs, and accumulate in the absence of XRN4/EIN5. Moreover, we demonstrate that accumulation of these smRNAs is often synergistically increased in ein5 abh1 double mutant plants, which suggests that these proteins act coordinately to regulate the substrates from which they are processed. Finally, we find that the parent transcripts of these novel smRNAs accumulate in an uncapped form upon loss of XRN4/EIN5. These results suggest that uncapped endogenous transcripts can shuttle into an RNA silencing pathway where they become smRNA biogenesis substrates. Overall, our results reveal unexpected connections between RNA metabolism and silencing. Keywords: transcriptome analysis using tiling array; whole genome small RNA sequencing; 5'-RACE sequencing

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Global analysis of RNA-dependent RNA Polymerase-dependent small RNAs reveals new substrates and functions for these proteins and SGS3 in Arabidopsis

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