Simple and accurate transcriptional start site identification using Smar2C2 and examination of conserved promoter features

Abstract

SUMMARY The precise and accurate identification and quantification of transcriptional start sites (TSSs) is key to understanding the control of transcription. The core promoter consists of the TSS and proximal non‐coding sequences, which are critical in transcriptional regulation. Therefore, the accurate identification of TSSs is important for understanding the molecular regulation of transcription. Existing protocols for TSS identification are challenging and expensive, leaving high‐quality data available for a small subset of organisms. This sparsity of data impairs study of TSS usage across tissues or in an evolutionary context. To address these shortcomings, we developed Smart‐Seq2 Rolling Circle to Concatemeric Consensus (Smar2C2), which identifies and quantifies TSSs and transcription termination sites. Smar2C2 incorporates unique molecular identifiers that allowed for the identification of as many as 70 million sites, with no known upper limit. We have also generated TSS data sets from as little as 40 pg of total RNA, which was the smallest input tested. In this study, we used Smar2C2 to identify TSSs in Glycine max (soybean), Oryza sativa (rice), Sorghum bicolor (sorghum), Triticum aestivum (wheat) and Zea mays (maize) across multiple tissues. This wide panel of plant TSSs facilitated the identification of evolutionarily conserved features, such as novel patterns in the dinucleotides that compose the initiator element (Inr), that correlated with promoter expression levels across all species examined. We also discovered sequence variations in known promoter motifs that are positioned reliably close to the TSS, such as differences in the TATA box and in the Inr that may prove significant to our understanding and control of transcription initiation. Smar2C2 allows for the easy study of these critical sequences, providing a tool to facilitate discovery.

Enhancers associated with unstable RNAs are rare in plants

“…In eukaryotes, most genes display dispersed transcription initiation from multiple TSSs within 20-100 bp in the same promoter or enhancer region (Haberle and Stark 2018; Murray et al 2022). Therefore, we will hereafter jointly refer to all strand-specific individual or clusters of TSSs as transcription start regions (TSRs, Fig.…”

“…In eukaryotes, most genes display dispersed transcription initiation from multiple TSSs within 20-100 bp in the same promoter or enhancer region (Haberle and Stark 2018;Murray et al 2022).…”

Abundance of Transgene Transcript Variants Associated with Somatically Active Transgenic Helitrons from Multiple T-DNA Integration Sites in Maize

“…Recently, the identification of evolutionarily conserved features correlated with promoter expression levels in TSSs has become an important area in understanding the control of transcription initiation and regulation in a plant, including the identification of novel patterns in the dinucleotides composing the initiator element [ 39 ]. To gain a glimpse into the alteration of general sequence patterns surrounding the TSS resulting from Helitron insertions, 5′ RACE assay showed that Helitron insertions generally did not affect the patterns in the dinucleotides composing the initiator element since highly expressed transgenes were enriched most commonly for CA, followed by CG, TG, which perfectly agrees with recent research [ 39 ]. This occurred regardless of the presence of the TATA box and the varied content of CG nucleotide between the TATA box and TSS, the initiator element ( Figure 1 B, Table S6 ).…”

“…Although, occasionally, the dHel functions as a novel promoter to drive the transcription of the transgene in the constructs, such as in the case of S3-HelA2 [ 45 ]. Furthermore, the Helitron insertion in different construct has no substantial effects on the patterns in the dinucleotides composing the initiator element, which is important and highly conserved to the control of transcription initiation and regulation in plant [ 39 ], but on the alternative splicing and aberrant splicing ( Table S6 ).…”

Smar2C2: A Simple and Efficient Protocol for the Identification of Transcription Start Sites

“…The number of TSSs that can be measured using this technique is largely dependent on the sequencing depth used. We were able to measure 70 million unique TSS reads from a single input (Murray, Mendieta, Vollmers, & Schmitz, 2022), and we have not reached or been able to determine a sequencing saturation point. If extremely low levels of RNA input are used then sequencing saturation may be reached earlier, but currently we do not know what these levels are.…”