Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters

Mammalian transcriptomes are complex and formed by extensive promoter activity. Moreover, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by early polyadenylation (pA) sites, promoters often cluster so that the divergent activity of one might impact another. Here, we find that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but due to pA site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provides a framework with which evolution shapes transcriptomes. Overall design: Mapping of paired 5' and 3'ends of capped and polyadenylated RNAs from RRP40-depleted and eGFP control HeLa cells and using transcript isoform sequencing (TIF-Seq, see Pelechano et al. Nat Protoc 2014 (PMID: 24967623) and Pelechano et al. Nature 2013 (PMID: 23615609)).