that the sense complex may promote antisense preinitiation complex formation in the upstream region (16). Second, as RNAPII elongates the sense transcript, negative supercoiling of the DNA will occur upstream, perhaps promoting the antisense initiation process (17). This divergent transcription could structure chromatin and nascent RNA at the TSS for subsequent regulation.References and Notes 1. G. Orphanides, D. Reinberg, Cell 108, 439 (2002 fig. S1B), demonstrating diminished exosome function. Oligo dT-primed, double-stranded cDNA from cells that had been treated with either a control [enhanced green fluorescent protein (eGFP)] or hRrp40 small interfering RNA (siRNA) was hybridized to an encyclopedia of DNA elements (ENCODE) tiling array, which covers a representative~1% of the human genome (1). Comparison of array data to public gene annotations revealed overall stabilization of mRNAs (exons in Fig. 1A), as expected. RNA from intronic and intergenic regions were largely unaffected, with the exception of a 1.5-kb region immediately upstream of transcription start sites (TSSs) that was stabilized~1.5-fold on average (Fig. 1A). The relative stabilization of

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...

ABSTRACT

Most eukaryotic pre-messenger RNAs are processed at their 3' ends by endonucleolytic cleavage and polyadenylation. In yeast, this processing requires polyadenylate [poly(A)] polymerase (PAP) and other proteins that have not yet been characterized. Here, mutations in the PAP1 gene were shown to be synergistically lethal with previously identified mutations in the RNA14 and RNA15 genes, which suggests that their encoded proteins participate in 3'-end processing. Indeed, extracts from ma14 and rna15 mutants were shown to be deficient in both steps of processing. Biochemical complementation experiments and reconstitution of both activities with partially purified cleavage factor I (CF I) validated the genetic prediction.

We have identified an essential gene, called FIP1, encoding a 327 amino acid protein interacting with yeast poly(A) polymerase (PAP1) in the two-hybrid assay. Recombinant FIP1 protein forms a 1:1 complex with PAP1 in vitro. At 37 degrees C, a thermosensitive allele of FIP1 shows a shortening of poly(A) tails and a decrease in the steady-state level of actin transcripts. When assayed for 3'-end processing in vitro, fip1 mutant extracts exhibit normal cleavage activity, but fail to polyadenylate the upstream cleavage product. Polyadenylation activity is restored by adding polyadenylation factor I (PF I). Antibodies directed against FIP1 specifically recognize a polypeptide in these fractions. Coimmunoprecipitation experiments reveal that RNA14, a subunit of cleavage factor I (CF I), directly interacts with FIP1, but not with PAP1. We propose a model in which PF I tethers PAP1 to CF I, thereby conferring specificity to poly(A) polymerase for pre-mRNA substrates.

Polyadenylation is the second step in 3′ end formation of most eukaryotic mRNAs. In Saccharomyces cerevisiae, this step requires three trans‐acting factors: poly(A) polymerase (Pap1p), cleavage factor I (CF I) and polyadenylation factor I (PF I). Here, we describe the purification and subunit composition of a multiprotein complex containing Pap1p and PF I activities. PF I–Pap1p was purified to homogeneity by complementation of extracts mutant in the Fip1p subunit of PF I. In addition to Fip1p and Pap1p, the factor comprises homologues of all four subunits of mammalian cleavage and polyadenylation specificity factor (CPSF), as well as Pta1p, which previously has been implicated in pre‐tRNA processing, and several as yet uncharacterized proteins. As expected for a PF I subunit, pta1‐1 mutant extracts are deficient for polyadenylation in vitro. PF I also appears to be functionally related to CPSF, as it polyadenylates a substrate RNA more efficiently than Pap1p alone. Possibly, the observed interaction of the complex with RNA tethers Pap1p to its substrate.

Efforts to catalog eukaryotic transcripts have uncovered many small RNAs (sRNAs) derived from gene termini and splice sites. Their biogenesis pathways are largely unknown, but a mechanism based on backtracking of RNA polymerase II (RNAPII) has been suggested. By sequencing transcripts 12-100 nucleotides in length from cells depleted of major RNA degradation enzymes and RNAs associated with Argonaute (AGO1/2) effector proteins, we provide mechanistic models for sRNA production. We suggest that neither splice site-associated (SSa) nor transcription start site-associated (TSSa) RNAs arise from RNAPII backtracking. Instead, SSa RNAs are largely degradation products of splicing intermediates, whereas TSSa RNAs probably derive from nascent RNAs protected by stalled RNAPII against nucleolysis. We also reveal new AGO1/2-associated RNAs derived from 3' ends of introns and from mRNA 3' UTRs that appear to draw from noncanonical microRNA biogenesis pathways.

PROMoter uPstream Transcripts (PROMPTs) were identified as a new class of human RNAs, which are heterologous in length and produced only upstream of the promoters of active protein-coding genes. Here, we show that PROMPTs carry 3′-adenosine tails and 5′-cap structures. However, unlike mRNAs, PROMPTs are largely nuclear and rapidly turned over by the RNA exosome. PROMPT-transcribing DNA is occupied by RNA polymerase II (RNAPII) complexes with serine 2 phosphorylated C-terminal domains (CTDs), mimicking that of the associated genic region. Thus, the inefficient elongation capacity of PROMPT transcription cannot solely be assigned to poor CTD phosphorylation. Conditions that reduce gene transcription increase RNAPII occupancy of the upstream PROMPT region, suggesting that they reside in a common transcription compartment. Surprisingly, gene promoters that are actively transcribed by RNAPI or RNAPIII also produce PROMPTs that are targeted by the exosome. RNAPIII PROMPTs bear hallmarks of RNAPII promoter-associated RNAs, explaining the physical presence of RNAPII upstream of many RNAPIII-transcribed genes. We propose that RNAPII activity upstream gene promoters are wide-spread and integral to the act of gene transcription.