Polyadenylation site–induced decay of upstream transcripts enforces promoter directionality

Abstract

Ntini et al. 3The non-protein-coding part of the human genome is pervasively transcribed into a large diversity of non-coding (nc) RNA 1 . A substantial fraction of this material derives from, or near, active gene promoters, that are producing a range of small-( 1-6 ) and long non-coding RNA (lncRNA) 7 . Indeed, it has been estimated that >60% of lncRNAs in human embryonic stem cells derive from promoters of active proteincoding genes 8 . Although some lncRNAs have reported functions, these species are generally kept at low abundance by cellular degradation activities 9,10 . For example, we previously coupled depletion of the major nuclear 3'-5' exonucleolytic activity, the RNA exosome, with tiling microarrays to reveal PROMPTs closely upstream of active human gene promoters 9 . PROMPTs are 5'capped, >100nt long and 3'end adenylated in the absence of exosome activity 11 . The mechanism underlying the efficient exosome-mediated suppression of these lncRNAs, while preserving the promoter-downstream mRNA, remains enigmatic.Here, we couple exosome-depletion to high-throughput 5'end-, 3'end-and regular RNA-sequencing (RNAseq) to create a genome-wide map of PROMPTs. Our results demonstrate that PROMPT transcription initiates antisense with respect to the downstream gene. We suggest that such initiating RNAPII, if stalled at a PROMPT-TSS proximal position, can elicit the production of previously reported TSSa-RNA.Sequence motifs around PROMPT 3'ends adhere to a pA site consensus and are significantly more abundant upstream than downstream of gene promoters. This provides a directional RNA output from human promoters by rapidly terminating antisense transcription and enforcing degradation of its RNA product. RESULTS PROMPTs initiate from bi-directional promoter activityTo obtain strand-specific and positional information of PROMPTs, we first subjected total RNA from HeLa cells, that had been treated with either a control (ctrl) eGFP siRNA or RRP40 siRNA ( Supplementary Fig. 1a), to regular RNA sequencing (RNAseq) as well as cap-selected RNA 5'end sequencing (Cap Analysis of Gene Expression (CAGE)). We focused our analysis on protein-coding genes and therefore considered reads mapping to the -3kb to +1kb regions of 2428 UCSC gene promoters, which were selected not to overlap any other annotated mRNAs. When aligned to the TSSs of these promoters, both RNAseq-and CAGE-data disclosed a strong presence of exosome-sensitive transcripts originating closely upstream of the gene TSS Ntini et al. 4(average peak CAGE position at -110bp) and commencing in the antisense direction relative to the neighboring mRNA TSS (Fig. 1a bottom panel, compare 'ctrl' and 'RRP40' plots). Only minor signal was detected in the sense direction of the same region. Whereas the abundance of antisense PROMPT (asPROMPT) CAGE tags increased by an average of 8-fold upon RRP40 depletion, the corresponding sense CAGE signals of the same region were largely unaffected (Fig. 1b, P<2e-16, twosided t-test). This predominant occurrence of asPROMPTs was also visi...

Suppressor of sable [Su(s)] and Wdr82 down-regulate RNA from heat-shock-inducible repetitive elements by a mechanism that involves transcription termination

“…However, some aspects of Su(s)-Wdr82-dependent regulation differ from these other processes. For example, it appears that cryptic, canonical pA signals can mediate promoter-proximal termination and degradation of PROMPTs (Ntini et al 2013). However, the unstable αβ RNAs acquire poly(A) tails by a mechanism that is independent of the canonical pA signal.…”

“…For example, short, polyadenylated, and unstable RNAs known as PROMPTs (promoter upstream transcripts) arise from divergent transcription at bidirectional promoters (Preker et al 2008(Preker et al , 2011Jensen et al 2013;Ntini et al 2013). Transcription in the upstream antisense direction is thought to be nonproductive because cryptic pA signals induce termination, and the resulting RNAs are degraded by the nuclear exosome.…”

A long non-coding RNA links calreticulin-mediated immunogenic cell removal to RB1 transcription

“…[12][13][14][15][16] In many cases, antisense transcripts upstream of promoters are unstable and their emergence might be explained by aberrant transcription initiation of RNA polymerase II due to the depletion of nucleosomes on active promoters. 14,[17][18][19][20] In evolutionary terms, divergent transcription initiation could fuel the emergence of new functional genes, by exposing the transcribed DNA strand to mutagenic alterations as has been reported for the acquisition of splice sites. 21 Several studies have addressed the regulation of bidirectional transcription, 13,16,18,19,[22][23][24][25][26][27] with three of these shedding light on the functionality of ncRNAs that are bidirectionally expressed from human PCG promoters: Hung et al 28 investigated human cell cycle-regulated genes and identified 58 functional long ncRNAs that are co-expressed with PCGs from bidirectional promoters.…”

An end in sight? Xrn2 and transcriptional termination by RNA polymerase II

“…Additionally, promoter upstream transcripts (PROMPTs) are largely unaffected by Xrn2 (our unpublished findings). This is despite observations that PROMPTs are relatively rich in PAS’s with evidence that these are sometimes functional [23]. Thus, RNA cleavage does not automatically trigger Xrn2-dependent RNA degradation and termination.…”