Transcript isoform sequencing reveals widespread promoter-proximal transcriptional termination

Higher organisms achieve optimal gene expression by tightly regulating the transcriptional activity of RNA Polymerase II (RNAPII) along DNA sequences of genes1. RNAPII density across genomes is typically highest where two key choices for transcription occur: near transcription start sites (TSSs) and polyadenylation sites (PASs) at the beginning and end of genes, respectively2,3. Alternative TSSs and PASs amplify the number of transcript isoforms from genes4, but how alternative TSSs connect to variable PASs is unresolved from common transcriptomics methods. Here, we define TSS/PAS pairs for individual transcripts in Arabidopsis thaliana using an improved Transcript Isoform sequencing (TIF-seq) protocol and find on average over four different isoforms corresponding to variable TSS/PAS pairs per expressed gene. While intragenic initiation represents a large source of regulated isoform diversity, we discover 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 coincides with increased RNAPII density, indicating these large pools of promoter-stalled RNAPII across genomes are often engaged in transcriptional termination. RNAPII elongation factors progress transcription beyond sites of sppRNA formation, demonstrating RNAPII density near promoters represents a checkpoint for early transcriptional termination that governs full-length gene isoform expression.