Genome-Wide Profiling and Analysis of Arabidopsis siRNAs

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Abstract
Eukaryotes contain a diversified set of small RNA-guided pathways that control genes, repeated sequences, and viruses at the transcriptional and posttranscriptional levels. Genome-wide profiles and analyses of small RNAs, particularly the large class of 24-nucleotide (nt) short interfering RNAs (siRNAs), were done for wild-type Arabidopsis thaliana and silencing pathway mutants with defects in three RNA-dependent RNA polymerase (RDR) and four Dicer-like (DCL) genes. The profiling involved direct analysis using a multiplexed, parallel-sequencing strategy. Small RNA-generating loci, especially those producing predominantly 24-nt siRNAs, were found to be highly correlated with repetitive elements across the genome. These were found to be largely RDR2- and DCL3-dependent, although alternative DCL activities were detected on a widespread level in the absence of DCL3. In contrast, no evidence for RDR2-alternative activities was detected. Analysis of RDR2- and DCL3-dependent small RNA accumulation patterns in and around protein-coding genes revealed that upstream gene regulatory sequences systematically lack siRNA-generating activities. Further, expression profiling suggested that relatively few genes, proximal to abundant 24-nt siRNAs, are regulated directly by RDR2- and DCL3-dependent silencing. We conclude that the widespread accumulation patterns for RDR2- and DCL3-dependent siRNAs throughout the Arabidopsis genome largely reflect mechanisms to silence highly repeated sequences. The discovery of small RNA molecules that inactivate genes during normal growth and development or on invasion by viruses and other mobile genetic elements has profoundly altered our views about how genes are regulated. We set out to investigate the global patterns of small RNA formation and activity across the entire genome of the plant, Arabidopsis thaliana. We used new technology and bioinformatics to characterize and compare these patterns in normal plants and in plants with defects in various genes involved in RNA silencing. The results show vast populations of small RNAs in plants and reveal how specialized branches of the RNA-silencing pathway have been adopted for inactivation of different types of genome sequences.