RNA polymerase II (Pol II) plays an essential role in gene expression. We used plant native elongating transcript sequencing and global run-on sequencing to profile nascent RNAs genome wide in Arabidopsis. Researchers at Guangzhou University found that Pol II tends to accumulate downstream of the transcription start site (TSS). Moreover, Pol II with an unphosphorylated carboxyl-terminal domain (CTD) mainly accumulates downstream of the TSS, while Pol II with a Ser 5P CTD associates with spliceosomes, and Pol II with a Ser 2P CTD presents a sharp peak within 250 base pairs downstream of the polyadenylation site (PAS). Pol II pausing both at promoter-proximal regions and after PAS affects the transcription rate. Interestingly, active genes can be classified into three clusters based on the different modes of transcription. The researchers demonstrate that these two methods are suitable to study Pol II dynamics in planta. Although transcription is conserved overall within eukaryotes, there is plant-specific regulation.
Establishment of GRO-seq and pNET-seq in Arabidopsis
a, A diagram of the experimental approach. For GRO-seq, nuclei were isolated from frozen tissue and used in a run-on reaction supplemented with α32P-CTP and Br-UTP. The nascent RNA was purified, adapted and reverse transcribed to complementary DNA and sequenced from the 5′ end. For pNET-seq, the Pol II RNP was released from the nucleus via MNase treatment and immunoprecipitated using a Pol II CTD specific antibody. The Pol II-associated nascent RNA was end-labelled with γ32P-ATP and sequenced from the 3′ end using a small-RNA-seq protocol. b,c, Autoradiographs show 32P-labelled nascent RNA from the GRO-seq and pNET-seq procedures, respectively. Experiments were repeated twice independently with similar results.