Characterization of Arabidopsis thaliana promoter bidirectionality and antisense RNAs by depletion of nuclear RNA decay enzymes

Abstract

In animals, transcription by RNA polymerase II initiates bidirectionally from gene promoters to produce pre-mRNAs on the forward strand and promoter upstream transcripts (PROMPTs) on the reverse strand. PROMPTs are rapidly degraded by the nuclear exosome. Similarly, active enhancer regions in animals initiate transcription of exosome-sensitive enhancer RNAs (eRNAs). Previous studies based on nascent RNA approaches concluded that Arabidopsis thaliana does not produce PROMPTs . Here, we used steady-state RNA sequencing methods in mutants defective in nuclear RNA decay, including by the exosome, to reassess the existence of PROMPTs and eRNAs in A. thaliana . While PROMPTs are overall rare in A. thaliana , about 100 clear cases of exosome-sensitive PROMPTs and 113 loci producing eRNA-like transcripts were identified. In addition, we found~200 transcription start sites within 3'-UTR-encoding regions that produce unspliced exosome-sensitive antisense RNAs covering much of the cognate pre-mRNA. A typical representative of this class of RNAs is the previously characterized non-coding RNA controlling the expression of the key seed dormancy regulator, DELAY OF GERMINATION1 . Exosome-sensitive antisense RNAs are overrepresented in transcription factor genes, suggesting a potential for widespread control of gene expression.Lastly, we assess the use of alternative promoters in A. thaliana and compare the accuracy of existing TSS annotations.

Long non-coding RNAs in plants: emerging modulators of gene activity in development and stress responses

“…These eRNAs often lack polyA tails and are degraded by the exosome when they are released from RNA polymerase II (RNA pol II, Shlyueva et al 2014 ). Bidirectional transcripts are not typically detected in enhancers or promoters of Arabidopsis and other plants, most likely due to rapid degradation (Thieffry et al 2020 and references therein). Most eRNAs are functionally uncharacterized.…”

“…Furthermore, the directionality of these divergent lncRNAs is determined by the asymmetry of U1 snRNP and polyadenylation signals (Quinn and Chang 2016 ). However, divergent transcription does not appear to occur in the majority of genes in Arabidopsis thaliana (Hetzel et al 2016 ; Thieffry et al 2020 ). In addition to the RNA polymerase machinery, transcription factors (TFs) and chromatin environment (e.g., histone modification and DNA methylation) also contribute to the regulation of lncRNA expression (Quinn and Chang 2016 ).…”

“…Like mRNAs, plant lncRNAs can be degraded by both 3′–5′ exonucleolysis via the nuclear exosome and 5′–3′ exonucleolysis via exonucleases such as XRN2 and XRN3 (Kurihara et al 2012 ). In mutants of exosome subunits, a set of specialized lncRNAs similar to CUTs (Cryptic unstable transcripts) and PROMPTs (Promoter upstream transcripts) emerged from TSSs of mRNAs (Chekanova et al 2007 ; Chekanova 2015 ; Thieffry et al 2020 ). Data from humans suggest that exosome-regulated lncRNAs modulate the activity of enhancers, resolving deleterious R-loop structures by the exosome (Pefanis et al 2015 ; Nair et al 2020 ).…”

Araport Lives: An Updated Framework for Arabidopsis Bioinformatics

“…One is a set of 41 tracks representing a multipronged gene expression experiment to track the response to various abiotic stresses from Lee and Bailey-Serres (2019). The other is a set of 4 tracks based on Cap Analysis of Gene Expression (CAGE) experiments to determine promoter bidirectionality performed by Thieffry et al (2019). The CAGE data are visualized using the Stranded View plugin (Hofmeister and Schmitz, 2018), which allows separation of the display of expression values into plus and minus strands in a single track.…”

PAMP-triggered Genetic Reprogramming Involves Widespread Alternative Transcription Initiation and an Immediate Transcription Factor Wave

“…Because the study of enhancers in plants is still rudimentary, it is not yet clear whether eRNA-like-producing loci correspond to active enhancers. Nonetheless, intergenic and intronic loci producing eRNA-like transcripts have been observed in cassava using nascent RNA sequencing techniques (Lozano et al, n.d.), and we previously identified around 100 similar loci in unchallenged Arabidopsis seedlings using CAGE (Thieffry et al 2020). In this latter case, detection of the short-lived eRNAs required inactivation of components of the nuclear exosome-mediated RNA decay pathway, achieved either by knockout mutation of the DEAD box helicase HUA ENHANCER2 (HEN2), a requisite, nucleoplasmic exosome cofactor (Lange et al 2014), or by partial loss of function of the core exosome subunit RRP4 (Hématy et al 2016; Thieffry et al 2020).…”