RIP-seq of the Arabidopsis mRNA export mutant aly1 and associated controls.

RNA-directed DNA methylation (RdDM) is a set of mechanisms by which transcriptionally-repressive DNA methylation and histone modifications are targeted de novo to transposable element and transgene regions of the genome. We identified a mutant in which all forms of RdDM are deficient, leading to the transcriptional activation of some transposable elements and the complete inability to initiate transgene silencing. The mutated gene, ALY1, encodes an RNA-binding nuclear export protein belonging to a family of four ALY proteins in Arabidopsis. ALY proteins function together to export a large number of mRNAs from the nucleus for translation, but we found that ALY1 is unique in its ability to enable RdDM. We sequenced methylomes (via MethylC-seq) to identify the genome-wide loss of CHH methylation targeted by RdDM in aly1 mutants. We sequenced small RNAs and determined that ALY1 functions in RdDM downstream of small RNA production and export. We sequenced total, nuclear and cytoplasmic mRNAs to determine that ALY1 functions in the nuclear export directly or indirectly on 2,612 mRNAs. Additionally, we sequenced RNAs bound to the ALY1 protein using RNA-immunoprecipitation followed by high throughput sequencing. Examination of ALY1-bound RNA population in two replicates of wild-type Columbia and four replicates of the ALY1pro::ALY1-GFP transgene complementing the aly1-1 mutation, using a GFP antibody.