A protein complex regulates RNA processing of intronic heterochromatin-containing genes in Arabidopsis

Abstract

In several eukaryotic organisms, heterochromatin (HC) in the introns of genes can regulate RNA processing, including polyadenylation, but the mechanism underlying this regulation is poorly understood. By promoting distal polyadenylation, the bromo-adjacent homology (BAH) domain-containing and RNA recognition motif-containing protein ASI1 and the H3K9me2-binding protein EDM2 are required for the expression of functional full-length transcripts of intronic HC-containing genes in Arabidopsis Here we report that ASI1 and EDM2 form a protein complex in vivo via a bridge protein, ASI1-Immunoprecipitated Protein 1 (AIPP1), which is another RNA recognition motif-containing protein. The complex also may contain the Pol II CTD phosphatase CPL2, the plant homeodomain-containing protein AIPP2, and another BAH domain protein, AIPP3. As is the case with dysfunction of ASI1 and EDM2, dysfunction of AIPP1 impedes the use of distal polyadenylation sites at tested intronic HC-containing genes, such as the histone demethylase gene IBM1, resulting in a lack of functional full-length transcripts. A mutation in AIPP1 causes silencing of the 35S-SUC2 transgene and genome-wide CHG hypermethylation at gene body regions, consistent with the lack of full-length functional IBM1 transcripts in the mutant. Interestingly, compared with asi1, edm2, and aipp1 mutations, mutations in CPL2, AIPP2, and AIPP3 cause the opposite effects on the expression of intronic HC-containing genes and other genes, suggesting that CPL2, AIPP2, and AIPP3 may form a distinct subcomplex. These results advance our understanding of the interplay between heterochromatic epigenetic modifications and RNA processing in higher eukaryotes.

Keywords: DNA methylation; RNA processing; heterochromatin; polyadenylation; transposable element.

Fig. 1.

AIPP1 interacts with ASI1 and EDM2. (A) Y2H assay showing that ASI1 does not directly interact with EDM2, but both ASI1 and EDM2 directly interact with AIPP1. (B) IP-MS results showing that EDM2, AIPP1, AIPP2, AIPP3, and CPL2 copurified with 3×Flag-ASI1. Results from one of five independent IP-MS experiments are shown. The “score” was calculated as the sum of the negative algorithms of the posterior error probability values of the connected peptide-spectrum matches. “Coverage” indicates the percentage of amino acid residues covered by the identified peptides. “Peptide” refers to the total number of identified peptide sequences matching the protein.

Fig. S1.

Split luciferase assays showing protein interactions among the ASI1-copurified proteins. Protein interactions among the indicated proteins were examined by split luciferase assays. (A) Split luciferase assays showing protein interactions between AIPP1 and ASI1, AIPP1 and EMD2 but not ASI1 and EDM2. (B) Split luciferase assays showing that AIPP2 can interact with ASI1, AIPP3, and CPL2. Luciferase activity was determined at 3 d after infiltration. N and C indicate the N-terminal and C-terminal fragments of luciferase, respectively.

Fig. S2.

ASI1 immunoprecipitated proteins identified by IP-MS assays. Results from four of five independent IP-MS experiments are shown.

Fig. S3.

Domain structures of ASI1 immunoprecipitated proteins. (A) Domain structures of AIPP1, AIPP2, and AIPP3 proteins. Conserved domains are predicted by the National Center for Biotechnology Information’s online tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). (B) Sequence alignment between the RRM domains of AIPP1 and ASI1.

Fig. 2.

ASI1, EDM2, and AIPP1 may form a protein complex in vivo. (A) Diagram of the construction of the entry vectors harboring two expression units. (B) The ASI1–EDM2 interaction mediated by AIPP1 was examined by yeast growth on a stringent medium deficient in Met, Leu, Trp, His, and adenine (SD-M/L/T/H/A). GFP, which was used as an unrelated donor to replace AIPP1, could not mediate the interaction between EDM2 and ASI1. The plates were photographed after incubation at 28 °C for 3 d. (C) Western blot analysis showing that the epitope-tagged ASI1, EDM2, and AIPP1 proteins are present in the same eluted fractions in gel filtration assays. The arrows indicate the molecular mass corresponding to the indicated fractions.

Fig. S4.

Mutant alleles for ASI1 immunoprecipitated proteins. (A) The genomic structure of the AIPP1 gene and mutations detected by DNA sequencing in two aipp1 mutant alleles. The underscored bases represent single guide RNA sequences used for gene editing by CRISPR/Cas9. Gray and blue boxes represent untranslated regions and exons, respectively. Red columns indicate mutated regions. (B) Diagram of T-DNA insertion lines for the CPL2, AIPP2, and AIPP3 genes. Inverted triangles indicate the insertion sites of T-DNA.

Fig. 3.

AIPP1 is required for the expression of full-length transcripts of selected ASI1 and EDM2 target intronic HC-TRE genes. (A–C) Three ASI1 and EDM2 target genes that contain intronic HC, including the H3K9me2 demethylase gene IBM1 (A), AT3G05410 (B), and AT1G58602 (RPP7) (C) were selected for expression analysis in aipp1 mutants. Col-0 served as the WT control, and asi1-2 and edm2-4 served as positive controls. Two representative forms of pre-mRNA transcripts with different polyadenylation sites are shown in the diagram. Black and green boxes represent exons and intronic HC elements, respectively. 3′- and 5′-specific primer pairs were used for detection of long (L) and 5′ (sum of distal and proximal transcripts) transcripts by RT-qPCR. The relative expression of different transcripts was first normalized to ACTIN2 and then to Col-0 plants. Error bars indicate SD; n = 3. (D–F) Snapshots of mRNA-seq and DNA methylation profiles from the IGV showing the read coverage (Upper) and DNA methylation levels (Lower) of the IBM1 (D), AT3G05410 (E), and RPP7 (F) genes. The genomic structure of each gene is shown at the top. Green boxes represent TREs.

Fig. S5.

DEXSeq results showing exon expression at the IBM1 (A), AT3G05410 (B), and RPP7 (C) genes. The plot indicates the estimated expression of each exon based on mRNA-seq (two replicates for each genotype). The estimates are from counts of mRNA-seq reads using each exon as a unit. The plots were generated with the R package “DEXSeq” function “plotDEXSeq” (21).

Fig. S6.

Intronic TE-containing genes affected in aipp1-1, asi1-2, and edm2-4 mutants. The Venn diagram shows the overlap of intronic TE-containing genes with reduced mRNA-seq 3′ reads in aipp1-1, asi1-2, and edm2-4 mutants.

Fig. S7.

Comparison of the effects of indicated single and double mutants on the accumulation of full-length transcripts of the tested loci. The expression of full-length transcripts (L) of the intronic HC-containing genes AT3G05410 and IBM1 was examined in aipp1-1, asi1-2, and edm2-4 single mutants and in their double mutants using 3′-specific primer pairs. Error bars indicate SD; n = 3.

Fig. S8.

AIPP1 prevents silencing of the 35S-SUC2 transgene locus. (A) The root growth phenotype of the aipp1-1 mutant in the 35S-SUC2 transgene background in half-strength Murashige and Skoog MS medium supplemented with 1% sucrose or 1% glucose. asi1-1 and edm2-6, which were already in the 35S-SUC2 transgene background, were grown as parallel controls. (B) RT-qPCR results showing that expression of the SUC2, HPTII, and NPTII transgenes was significantly reduced in aipp1-1, asi1-1, and edm2-6 mutant plants in comparison with the WT parental 35S-SUC2 plants. All plants were of the 35S-SUC2 background. Relative expression of the transgenes was first normalized to ACTIN2 plants and then to 35S-SUC2 plants. Error bars indicate SD; n = 3. **P < 0.01, Student’s t test.

Fig. 4.

AIPP1 controls gene body CHG methylation. (A) Boxplot diagram showing DNA methylation levels of each genotype at ibm1-4, aipp1-1, and aipp-2 hyper-DMRs. r1, replicate 1; r2, replicate 2. (B) Composition of the hyper-DMRs in aipp1-1, aipp1-2, and ibm1-4 mutants. (C) Distribution of DNA methylation along gene (Upper)/TE (Lower) bodies and their upstream/downstream 2-kb flanking sequences in different cytosine contexts.

Fig. S9.

AIPP1 dysfunction causes increased CHG methylation in gene body regions of long genes. (A and B) CHG methylation levels of body regions and their upstream and downstream 2-kb flanking sequences of different-sized genes (A) and TEs (B). (C) IGV snapshot showing increased CHG methylation at the body regions of genes in a representative genomic region in aipp1 and ibm1 mutants. Orange boxes represent TEs or TE genes.

Fig. 5.

AIPP2 can directly interact with ASI1, AIPP3, and CPL2 and regulate intronic HC-TRE genes. (A) Y2H assay results showing interaction of AIPP2 with AIPP3, ASI1, and CPL2. (B) Effect of the different mutations on the accumulation of full-length transcripts from the indicated intronic HC-TRE genes. 3′-specific primer pairs were used for detection of full-length transcripts (L) of IBM1, AT3G05410, and RPP7 by RT-qPCR. Relative expression of different transcripts was first normalized to ACTIN2 and then to Col-0 plants. Error bars indicate SD; n = 3. **P < 0.01; *P < 0.05, Student’s t test.

Fig. 6.

Dysfunction of ASI1 and its associated proteins affects the expression of a nonintroinc HC gene. RT-qPCR shows that the ASI1-AIPP1-EDM2 subcomplex is required for the expression of AT4G16870, a TE gene with heavy DNA methylation. A snapshot of DNA methylation in this locus is shown. Relative expression in different mutants was first normalized to ACTIN2 and then to Col-0 plants. Error bars indicate SD; n = 3. **P < 0.01, Student’s t test.