Transcript isoform sequencing reveals widespread promoter-proximal transcriptional termination in Arabidopsis

RNA polymerase II (RNAPII) transcription converts the DNA sequence of a single gene into multiple transcript isoforms that may carry alternative functions. Gene isoforms result from variable transcription start sites (TSSs) at the beginning and polyadenylation sites (PASs) at the end of transcripts. How alternative TSSs relate to variable PASs is poorly understood. Here, we identify both ends of RNA molecules in Arabidopsis thaliana by transcription isoform sequencing (TIF-seq) and report four transcript isoforms per expressed gene. While intragenic initiation represents a large source of regulated isoform diversity, we observe that ~14% of expressed genes generate relatively unstable short promoter-proximal RNAs (sppRNAs) from nascent transcript cleavage and polyadenylation shortly after initiation. The location of sppRNAs correlates with the position of promoter-proximal RNAPII stalling, indicating that large pools of promoter-stalled RNAPII may engage in transcriptional termination. We propose that promoter-proximal RNAPII stalling-linked to premature transcriptional termination may represent a checkpoint that governs plant gene expression. TIF-Seq was used to simultaneously map the transcription initiation sites and termination sites on A.thaliana seedlings (Col-0,Col-0 cold treated, HEN2-deficient genotype, HEN2-deficient cold treated and FACT-deficient genotypes). We also used TSS-Seq in these genotypes to validate the TIF-Seq analaysis and quantify initiation of transcription