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...

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Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within~500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels.

Abstract ARS2 is a conserved protein centrally involved in both nuclear RNA productive and destructive processes. To map features of ARS2 promoting RNA decay, we utilized two different RNA reporters, one of which depends on direct ARS2 tethering for its degradation. In both cases, ARS2 triggers a degradation phenotype aided by its interaction with the poly(A) tail exosome targeting (PAXT) connection. Interestingly, C-terminal amino acids of ARS2, responsible for binding the RNA 5′cap binding complex (CBC), become dispensable when ARS2 is directly tethered to the reporter RNA. In contrast, the Zinc-finger (ZnF) domain of ARS2 is essential for the decay of both reporters and consistently co-immunoprecipitation analyses reveal a necessity of this domain for the interaction of ARS2 with the PAXT-associated RNA helicase MTR4. Taken together, our results map the domains of ARS2 underlying two essential properties of the protein: its RNP targeting ability and its capacity to recruit the RNA decay machinery.

RNA polymerase II (RNAPII)-mediated gene transcription initiates at promoters and ends at terminators. Transcription termination is intimately connected to 3'-end processing of the produced RNA and already when loaded at the promoter, RNAPII starts to become configured for this downstream event. Conversely, RNAPII is 'reset' as part of the 3'-end processing/termination event, thus preparing the enzyme for its next round of transcription--possibly on the same gene. There is both direct and circumstantial evidence for preferential recycling of RNAPII from the gene terminator back to its own promoter, which supposedly increases the efficiency of the transcription process under conditions where RNAPII levels are rate limiting. Here, we review differences and commonalities between initiation and 3'-end processing/termination processes on various types of RNAPII transcribed genes. In doing so, we discuss the requirements for efficient 3'-end processing/termination and how these may relate to proper recycling of RNAPII.