Stress-induced endogenous siRNAs targeting regulatory intron sequences in Brachypodium.

Exposure to abiotic stresses triggers global changes in the expression of thousands of eukaryotic genes at the transcriptional and post-transcriptional levels. Small RNA (smRNA) pathways and splicing both function as crucial mechanisms regulating stress-responsive gene expression. However, examples of smRNAs regulating gene expression remain largely limited to effects on mRNA stability, translation, and epigenetic regulation. Also, our understanding of the networks controlling plant gene expression in response to environmental changes, and examples of these regulatory pathways intersecting, remains limited. Here, to investigate the role of smRNAs in stress responses we examined smRNA transcriptomes of Brachypodium distachyon plants subjected to various abiotic stresses. We found that exposure to different abiotic stresses specifically induced a group of novel, endogenous small interfering RNAs (stress-induced, UTR-derived siRNAs, or sutr-siRNAs) that originate from the 3' UTRs of a subset of coding genes. Our bioinformatics analyses predicted that sutr-siRNAs have potential regulatory functions and that over 90% of sutr-siRNAs target intronic regions of many mRNAs in trans. Importantly, a subgroup of these sutr-siRNAs target the important intron regulatory regions, such as branch point sequences, that could affect splicing. Our study indicates that in Brachypodium, sutr-siRNAs may affect splicing by masking or changing accessibility of specific cis-elements through base-pairing interactions to mediate gene expression in response to stresses. We hypothesize that this mode of regulation of gene expression may also serve as a general mechanism for regulation of gene expression in plants and potentially in other eukaryotes.

The role of the Arabidopsis Exosome in siRNA-independent silencing of heterochromatic loci.

The exosome functions throughout eukaryotic RNA metabolism and has a prominent role in gene silencing in yeast. In Arabidopsis, exosome regulates expression of a "hidden" transcriptome layer from centromeric, pericentromeric, and other heterochromatic loci that are also controlled by small (sm)RNA-based de novo DNA methylation (RdDM). However, the relationship between exosome and smRNAs in gene silencing in Arabidopsis remains unexplored. To investigate whether exosome interacts with RdDM, we profiled Arabidopsis smRNAs by deep sequencing in exosome and RdDM mutants and also analyzed RdDM-controlled loci. We found that exosome loss had a very minor effect on global smRNA populations, suggesting that, in contrast to fission yeast, in Arabidopsis the exosome does not control the spurious entry of RNAs into smRNA pathways. Exosome defects resulted in decreased histone H3K9 dimethylation at RdDM-controlled loci, without affecting smRNAs or DNA methylation. Exosome also exhibits a strong genetic interaction with RNA Pol V, but not Pol IV, and physically associates with transcripts produced from the scaffold RNAs generating region. We also show that two Arabidopsis rrp6 homologues act in gene silencing. Our data suggest that Arabidopsis exosome may act in parallel with RdDM in gene silencing, by epigenetic effects on chromatin structure, not through siRNAs or DNA methylation.