Simultaneous mapping of transcript ends at single-nucleotide resolution and identification of widespread promoter-associated non-coding RNA governed by TATA elements

Abstract

Understanding the relationships between regulatory factor binding, chromatin structure, cis-regulatory elements and RNA-regulation mechanisms relies on accurate information about transcription start sites (TSS) and polyadenylation sites (PAS). Although several approaches have identified transcript ends in yeast, limitations of resolution and coverage have remained, and definitive identification of TSS and PAS with single-nucleotide resolution has not yet been achieved. We developed SMORE-seq (simultaneous mapping of RNA ends by sequencing) and used it to simultaneously identify the strongest TSS for 5207 (90%) genes and PAS for 5277 (91%) genes. The new transcript annotations identified by SMORE-seq showed improved distance relationships with TATA-like regulatory elements, nucleosome positions and active RNA polymerase. We found 150 genes whose TSS were downstream of the annotated start codon, and additional analysis of evolutionary conservation and ribosome footprinting suggests that these protein-coding sequences are likely to be mis-annotated. SMORE-seq detected short non-coding RNAs transcribed divergently from more than a thousand promoters in wild-type cells under normal conditions. These divergent non-coding RNAs were less evident at promoters containing canonical TATA boxes, suggesting a model where transcription initiation at promoters by RNAPII is bidirectional, with TATA elements serving to constrain the directionality of initiation.

Snf1-Dependent Transcription Confers Glucose-Induced Decay upon the mRNA Product

“…Sequences of all yeast mRNAs were retrieved using an R script as described above for the qPAR-CLIP data analysis but this time using the transcription start site (TSS) and polyadenylation site (PAS) coordinates obtained from Park et al (49). Sequences without a TSS coordinate were retrieved beginning 100 bp upstream from the start codon, while sequences without a PAS coordinate were retrieved ending 150 bp downstream from the stop codon.…”

Volatile evolution of long noncoding RNA repertoires: mechanisms and biological implications

“…This is far more than predicted under random expectation, and many bidirectional gene pairs have been stably associated over long periods of evolution [86-89]. The key feature underlying this organization is the inherent property of many RNAPII promoters (primarily TATA-less and CG-rich) to drive divergent transcription, which has been documented in diverse eukaryotes [90-92]. Typically, transcription initiation at such promoters leads to the production of upstream short, capped and polyadenylated noncoding RNAs (often termed promoter upstream transcripts, or PROMPTs) that have no known function and are rapidly degraded by the nuclear exosome [93-95] (Figure 3A).…”

Histone H3K4 and H3K36 Methylation Independently Recruit the NuA3 Histone Acetyltransferase in Saccharomyces cerevisiae

“…For average plots showing the immunoprecipitation (IP) divided by input, a coverage value of one was added to both the IP and the input to avoid division by zero. Values past the polyadenylation sequence (Park et al 2014) were excluded from the average calculation, and the fraction of genes included in the average calculation at any given distance from the transcription start site (TSS) was shown.…”