GSE11070
A link between RNA metabolism and silencing affecting Arabidopsis development
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 Overall design: Two biological replicates were done for each of the four genotypes (wild-type Col-0, abh1-1, ein5-6, and ein5-6 abh1-1) for the gene expression studies of the entire transcriptome employing tiling microarrays for the reverse (Crick) and forward (Watson) chromosomal strands, which meant a total of 16 whole-genome oligonucleotide tiling microarrays were used for these studies. The small RNA component of the transcriptome was sequenced from the four genotypes (wild-type Col-0, abh1-1, ein5-6, and ein5-6 abh1-1) using the Illumina Genetic Analyzer. A genome-wide survey of 5'-RACE products was sequenced (GMUCT) from two genotypes (wild-type Col-0 and ein5-6) using the Illumina Genetic Analyzer.