#Gro seq windows#

GRO-seq and PRO-seq performed a run-on with isolated nuclei, which might compromise the termination machinery. Several short-read Illumina-based methods have been developed to analyze nascent RNAs, but their aims are primarily on the elongating fraction of RNAs before termination, rather than the readthrough and cleaved ones. The majority of the termination RNA intermediates are transient and rapidly degraded after cleavage, and are therefore difficult to capture and characterize. Thus, we set out to study transcription termination in the model plant Arabidopsis. To date, the pattern of Pol II termination in plants remains largely unknown. Since the Arabidopsis genome is highly compact, with ~ 38,000 genes concentrated in only ~ 135 Mb DNA sequence, termination is essential to prevent the transcriptional collisions between flanking genes. As a result, readthrough transcription can continue for up to a few thousand nucleotides (nt) downstream of the poly(A) site. The kinetic competition between Pol II and exonuclease promotes termination. The readthrough transcripts accumulate in mutants with a defective cleavage and polyadenylation complex or a loss-of-function exonuclease. The cleavage at poly(A) site provides an entry for 5′ → 3′ exonuclease (Xrn2 in human, Rat1 in yeast, AtXRN2/3 in Arabidopsis) to degrade the 3′ cleavage product that is still presumably being synthesized by Pol II. The 3′ end of mature mRNA is determined by the cleavage event, rather than the site where transcription terminates. In mammalian cells, after Pol II passed through the poly(A) site, cleavage and polyadenylation process is initiated, and transcription continues until Pol II disassociated from the DNA template and nascent RNA released. Decades of extensive research in this area has proposed the allosteric/antiterminator model, the torpedo model, and later a unified model that combines these two to explain the mechanism of polyadenylation signal (PAS)-dependent termination. Termination is essential to prevent uncontrolled readthrough transcription from invading downstream genes. Transcription termination is the critical final step in RNA synthesis, during which nascent RNA is released from the complex of RNA Polymerase II (Pol II) and DNA template. Our method is applicable to establish a comprehensive termination landscape in a broad range of species. Consistent with previous reports, we also observe long chimeric transcripts with cryptic splicing in fpa mutant but loss of CG DNA methylation has no obvious impact on termination in the met1 mutant. 5′ Cleaved readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. We also observe efficient termination before downstream tRNA genes, suggesting that chromatin structure around the promoter region of tRNA genes may block pol II elongation.

#Gro seq windows#

Our data reveal a wide range of termination windows among genes, ranging from ~ 50 nt to over 1000 nt. Here, we use single-molecule nascent RNA sequencing to characterize the various forms of transient RNAs during termination at genome-wide scale in wildtype Arabidopsis and in atxrn3, fpa, and met1 mutants. The dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods.