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

Quentin Angelo Thomas,Bingnan Li,Sebastian Marquardt,Vicent Pelechano,Jingwen Wang,Jinghan Liu,Ryan Ard

doi:10.1038/s41467-020-16390-7

Quentin Angelo Thomas, Bingnan Li + Show 5 more

Open Access

https://doi.org/10.1038/s41467-020-16390-7

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Abstract

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.

Highlights

RNA polymerase II (RNAPII) transcription converts the DNA sequence of a single gene into multiple transcript isoforms that may carry alternative functions
We compared transcript boundaries detected by transcription isoform sequencing (TIF-seq) to boundaries determined by methods identifying TSSs35 and polyadenylation sites (PASs) by Direct RNA sequencing (DRS)[46]
TIF-seq validated previously characterised alternative gene isoforms in Arabidopsis that result from alterative transcription start sites (TSSs) or PASs (Supplementary Fig. 3e, f), as well as antisense lncRNA variants (Supplementary Fig. 3g)

Summary

Introduction

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 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. Chromatin-based mechanisms counter-acting intragenic TSSs during RNAPII elongation could affect the diversity of proteins derived from a gene[10]. Transcriptional termination at intragenic polyadenylation sites (PASs) results in mRNA isoforms lacking C-

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