A simple and robust method for isolating and analyzing chromatin-bound RNAs in Arabidopsis

Abstract

Background: Chromatin-bound RNAs are the primary product of transcription that undergo on-chromatin processing such as capping, splicing, and polyadenylation. These processing steps then determine the fate of RNAs. Albeit its vital importance, a simple and robust method for isolating different fractions of chromatin-bound RNAs is missing in plants.

Result: Here, we describe our updated method and the associated step-by-step protocol for chromatin-bound RNAs isolation in A. thaliana. The chromatin-bound RNAs isolation is based on the 1 M UREA wash that removes the majority of non-chromatin-associated proteins from the nucleus, as previously developed in mammalian cells. On-demand, the isolated chromatin-bound RNAs can be either used directly for gene-specific analysis or subject to further rRNA removal and also the optional polyadenylated RNA removal, followed by high-throughput sequencing. Detailed protocols for these procedures are also provided. Comparison of sequencing results of chromatin-bound RNAs with and without polyadenylated RNA removal revealed that a small fraction of CB-RNAs is polyadenylated but not yet fully spliced, representing RNA-processing intermediate on-chromatin.

Conclusion: This optimized chromatin-bound RNAs purification method is simple and robust and can be used to study transcription and its-coupled RNA processing in plants.

Keywords: Arabidopsis thaliana; Chromatin-bound RNAs (CB-RNAs); Co-transcriptional splicing (CTS); Transcription regulation.

Fig. 1

A brief workflow of chromatin-bound RNA extraction in plants. A The cartoon demonstrates the workflow of chromatin-bound RNA extraction in Arabidopsis. B Western-blot detection of different proteins in different fractions that are obtained by chromatin-bound RNA extraction. Note that the UBC1 is absent from the nucleoplasm and chromatin fraction, while H3 and Pol II are highly enriched in the chromatin fraction. C Gel-electrophoresis detection of RNAs in different fractions. Note that 28S and 18S rRNA bands can be observed clearly in different fractions, indicating RNA degradation is minimal during extraction

Fig. 2

Q-PCR analysis to evaluate the enrichment of nascent RNA in different fractions.The qPCR results showed 5 specific introns’ enrichment at different RNA fractions. The Y-axis indicates the values of unspliced RNA normalized to spliced RNA. The primer locations are shown at the top of each gene structure. Note that the unspliced RNA is mostly enriched in the CB-RNA fraction. Data are presented as mean ± SEM (n = 2). Asterisk indicates significant difference among various RNA fractions based on two-tailed Student’s test

Fig. 3

Library construction workflow of mRNA seq and CB-RNA-seq.The workflow of library construction steps is summarized. The different RNA fractions (RNAe, RNAf, RNAe + RNAf) subjected to the library construction are indicated in red. Instructions on how to isolate each RNA fraction are provided in the method section

Fig. 4

The overview of CB-RNA-seq and mRNA-seq. A Three examples showed CB-RNA-seq (red) and mRNA-seq (blue) results. A 5ʹ to 3ʹ declining slope is observed in the CB-RNA-seq. The gene structure is indicated at the top of each track, and the length of each gene is indicated at the bottom. B Meta profile showed the reads distribution of CB-RNA-seq and mRNA-seq along the gene. TSS: transcription start site. PAS: Polyadenylation site. C Comparison of gene’s intron/exon ratio between CB-RNA-seq and mRNA-seq. ***p < 0.001, Wilcoxon test. D Diagram showed the calculation method of 5ʹ SS ratio and 3ʹ SS ratio. E Boxplot showed splicing ratio of 5ʹ SS and 3ʹ SS ratio of CB-RNA-seq and mRNA-seq in Arabidopsis. The boxplot showed the median and the 25th and 75th percentiles

Fig. 5

The comparison of intron splicing status in different CB-RNAs. A The cartoons show different fractions of the chromatin-bound RNAs. RNAe indicates the nascent being transcribed at the locus. RNAf indicates the full-length RNA, which is polyadenylated yet still attached to the chromatin. B Boxplot showed splicing ratio of 5ʹ SS and 3ʹ SS ratio of RNAe, RNAf, and RNAe + RNAf. ***p < 0.001, Wilcoxon test. The boxplot showed the median and the 25th and 75th percentiles. C The relationship between co-transcriptional splicing (CTS) and intron order in RNAe, RNAf, and RNAe + RNAf. The genes containing 9 introns were selected as representatives. X-axis indicated the intron order from 1st to 9th. The analyzed gene number was indicated at the top of each box. D The relationship between co-transcriptional splicing (CTS) and intron number in RNAe, RNAf, and RNAe + RNAf. The genes containing 1 to 10 introns were selected as representatives. X-axis indicated the intron number from 1 to 10. The analyzed gene number was indicated at the top of each boxplot