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RNA polymerase II activity revealed by GRO-seq and pNET-seq in Arabidopsis

ABSTRACT: RNA polymerase II (Pol II) play an essential role in gene expression. Here, we adapted mammalian Native Elongation Transcript sequencing and Global Run On sequencing to profile nascent RNA genome wide in Arabidopsis. We found Pol II tends to accumulate downstream of transcription start site and pausing at proximal promoter is an important regulatory step for Pol II transcription although loosely controlled. Furthermore, the Pol II with unphosphorylated carboxyl-terminal domain (CTD) mainly accumulates downstream the TSS, while the Ser5P CTD Pol II associates with spliceosome, and the Ser2P CTD Pol II presents a sharp peak 250 base pair downstream of polyadenylation site indicating a stringent control of termination for protein coding genes; whilst the termination of noncoding genes is not. Active expressed genes can be classified into three clusters according to the distribution patterns of different Pol II isoforms. In summary, we demonstrated the modified plant GRO-seq and mNET-seq are suitable to study RNA Pol II dynamics in planta. Although transcription is conserved among high eukaryotes, Pol II has its feature in Arabidopsis.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE109974 | GEO | 2018/09/30

REPOSITORIES: GEO

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Project description:Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is directed by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex and requires CTD-Ser2 phosphorylation. Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar RNAs (snoRNAs) is preformed by the Nrd1 complex, and requires CTD-Ser5 phosphorylation. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1 are more similar to loss of a subunit of the Ser5P binding Nrd1-complex, than to loss of Ser2P binding factors. Tiling array analysis of ctk1Δ reveals readthrough at several hundred sites, including snoRNAs, as reported previously, but also many cryptic unstable transcripts, stable untranslated transcripts (SUTs) and other transcripts. Surprisingly, neither loss of CTK1 nor a Pol II CTD-Ser2 substitution mutant results in a global defect in termination of mRNAs, indicating that Ser2P is not essential for proper termination of most mRNAs. At snoRNA, Nrd1 location is shifted downstream in ctk1∆, indicating defective release rather than recruitment of Nrd1. Weakening the interaction between Nrd1 and Pol II rescues the readthrough in ctk1∆, likely by facilitating Nrd1 release. The termination defect is kinase activity dependent, but cannot be completely explained by loss of CTD-Ser2 phosphorylation , a major substrate of Ctk1, suggesting the involvement of an additional substrate. Mutant alleles of the elongation factor Spt5 rescue ctk1∆-dependent readthrough, indicating a role for Spt5 in this process, perhaps as a substrate of Ctk1. The results show that Ctk1 is more intimately involved in termination of small non-coding RNAs than was previously assumed and lead to a model in which Ctk1 influences Spt5 activity to achieve this.

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RNA polymerase II activity revealed by GRO-seq and pNET-seq in Arabidopsis

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