RNA 3' processing functions of Arabidopsis FCA and FPA limit intergenic transcription

Abstract

The RNA-binding proteins FCA and FPA were identified based on their repression of the flowering time regulator FLC but have since been shown to have widespread roles in the Arabidopsis thaliana genome. Here, we use whole-genome tiling arrays to show that a wide spectrum of genes and transposable elements are misexpressed in the fca-9 fpa-7 (fcafpa) double mutant at two stages of seedling development. There was a significant bias for misregulated genomic segments mapping to the 3' region of genes. In addition, the double mutant misexpressed a large number of previously unannotated genomic segments corresponding to intergenic regions. We characterized a subset of these misexpressed unannotated segments and established that they resulted from extensive transcriptional read-through, use of downstream polyadenylation sites, and alternative splicing. In some cases, the transcriptional read-through significantly reduced expression of the associated genes. FCA/FPA-dependent changes in DNA methylation were found at several loci, supporting previous associations of FCA/FPA function with chromatin modifications. Our data suggest that FCA and FPA play important roles in the A. thaliana genome in RNA 3' processing and transcription termination, thus limiting intergenic transcription.

Fig. 1.

qRT-PCR analysis of UA segments in (A) fca-9 fpa-7, (B) fpa-7, and (C) fca-9 mutants. Histograms show mean values ± SEM for three independent PCR amplifications on at least three biological replicates. The y axis shows the fold change in C and fold change on a logarithmic (base 10) scale in A and B relative to WT Columbia (Col = 1) after normalization to UBC gene expression.

Fig. 2.

Schematic representation of a selection of verified UA segments and their locations in the A. thaliana genome (A–F). The schemes are adapted from http://neomorph.salk.edu/epigenome/epigenome.html with minor alterations. The blue arrows designate the UA segments, with the arrowheads pointing to the 3′ end of the transcript, green boxes are exons, red boxes are 5′ and 3′ UTRs, thin gray lines are introns, and gray boxes are nongenic annotations (i.e., transposons and pseudogenes). A′–F′ are plotted based on sequencing either the RT-PCR products (B′, C′, E′, and F′) amplified with primer pairs indicated in Fig. S2 or 5′ and 3′ RACE products (A′ and D′). The dashed lines and asterisk in A′ indicate the ∼3-kb intron splicing event; two asterisks in F′ designate the predicted outcome of loss of fcafpa on the affected genes. The TAIR9 annotation numbers are designated above or below each annotation. Different scales are used for the different gene representations.

Fig. 3.

ChIP analysis of genomic regions around At1g28140 and At2g23780. (A) A schematic of the genomic regions, (B) enrichment of FCA in Col-0 compared with fca-9, and (C) FPA::YFP compared with YFP after immunoprecipitation with an FCA or GFP antibody, respectively, are shown. Histograms show mean values ± SEM for enrichment calculated by percent input normalized against actin for three qPCR amplifications and two biological replicates. qPCR primers for UA2, At1g28140, UA10B, and At2g23780 are designated by solid bars on the gene models in A. Table S3 lists the primer sequences.

Fig. 4.

qRT-PCR analysis of the segment UA228 in various RNA silencing mutants. Histograms show mean values ± SEM for three independent PCR amplifications on two biological replicates. The y axis shows the relative fold change in logarithmic (base 10) scale normalized to UBC gene expression. Table S3 lists the mutant alleles.

Fig. 5.

DNA methylation analysis at the UA228 segment in Col vs. fcafpa by (A) McrBC digestion. Equal amounts of McrBC-digested (+) and undigested (−) DNA from two biological replicates of Col and fcafpa were amplified by PCR. (B) Bisulfite sequencing.