The APT complex is involved in non-coding RNA transcription and is distinct from CPF

The 3'-ends of eukaryotic pre-mRNAs are processed in the nucleus by a large multiprotein complex, the cleavage and polyadenylation factor (CPF). CPF cleaves RNA, adds a poly(A) tail and signals transcription termination. CPF harbors four enzymatic activities essential for these processes, but how these are coordinated remains poorly understood. Several subunits of CPF, including two protein phosphatases, are also found in the related 'associated with Pta1' (APT) complex, but the relationship between CPF and APT is unclear. Here, we show that the APT complex is physically distinct from CPF. The 21 kDa Syc1 protein is associated only with APT, and not with CPF, and is therefore the defining subunit of APT. Using ChIP-seq, PAR-CLIP and RNA-seq, we show that Syc1/APT has distinct, but possibly overlapping, functions from those of CPF. Syc1/APT plays a more important role in sn/snoRNA production whereas CPF processes the 3'-ends of protein-coding pre-mRNAs. These results define distinct protein machineries for synthesis of mature eukaryotic protein-coding and non-coding RNAs

Loss of the yeast SR protein Npl3 alters gene expression due to transcription readthrough

Yeast Npl3 is a highly abundant, nuclear-cytoplasmic shuttling, RNA-binding protein, related to metazoan SR proteins. Reported functions of Npl3 include transcription elongation, splicing and RNA 3' end processing. We used UV crosslinking and analysis of cDNA (CRAC) to map precise RNA binding sites, and strand-specific tiling arrays to look at the effects of loss of Npl3 on all transcripts across the genome. We found that Npl3 binds diverse RNA species, both coding and non-coding, at sites indicative of roles in both early pre-mRNA processing and 3' end formation. Tiling arrays and RNAPII mapping data revealed 3' extended RNAPII-transcribed RNAs in the absence of Npl3, suggesting that defects in pre-mRNA packaging events result in termination readthrough. Transcription readthrough was widespread and frequently resulted in down-regulation of neighboring genes. We conclude that the absence of Npl3 results in widespread 3' extension of transcripts with pervasive effects on gene expression

Transcriptome maps of general eukaryotic RNA degradation factors.

RNA degradation pathways enable RNA processing, the regulation of RNA levels, and the surveillance of aberrant or poorly functional RNAs in cells. Here we provide transcriptome-wide RNA-binding profiles of 30 general RNA degradation factors in the yeast Saccharomyces cerevisiae. The profiles reveal the distribution of degradation factors between different RNA classes. They are consistent with the canonical degradation pathway for closed-loop forming mRNAs after deadenylation. Modeling based on mRNA half-lives suggests that most degradation factors bind intact mRNAs, whereas decapping factors are recruited only for mRNA degradation, consistent with decapping being a rate-limiting step. Decapping factors preferentially bind mRNAs with non-optimal codons, consistent with rapid degradation of inefficiently translated mRNAs. Global analysis suggests that the nuclear surveillance machinery, including the complexes Nrd1/Nab3 and TRAMP4, targets aberrant nuclear RNAs and processes snoRNAs

Transcriptome surveillance by selective termination of noncoding RNA synthesis

Pervasive transcription of eukaryotic genomes stems to a large extent from bidirectional promoters that synthesize mRNA and divergent noncoding RNA (ncRNA). Here, we show that ncRNA transcription in the yeast S. cerevisiae is globally restricted by early termination that relies on the essential RNA-binding factor Nrd1. Depletion of Nrd1 from the nucleus results in 1,526 Nrd1-unterminated transcripts (NUTs) that originate from nucleosome-depleted regions (NDRs) and can deregulate mRNA synthesis by antisense repression and transcription interference. Transcriptome-wide Nrd1-binding maps reveal divergent NUTs at most promoters and antisense NUTs in most 30 regions of genes. Nrd1 and its partner Nab3 preferentially bind RNA motifs that are depleted in mRNAs and enriched in ncRNAs and some mRNAs whose synthesis is controlled by transcription attenuation. These results define a global mechanism for transcriptome surveillance that selectively terminates ncRNA synthesis to provide promoter directionality and to suppress antisense transcription

The conserved protein Seb1 drives transcription termination by binding RNA polymerase II and nascent RNA

AbstractTermination of RNA polymerase II (Pol II) transcription is an important step in the transcription cycle, which involves the dislodgement of polymerase from DNA, leading to release of a functional transcript. Recent studies have identified the key players required for this process and showed that a common feature of these proteins is a conserved domain that interacts with the phosphorylated C-terminus of Pol II (CTD-interacting domain, CID). However, the mechanism by which transcription termination is achieved is not understood. Using genome-wide methods, here we show that the fission yeast CID-protein Seb1 is essential for termination of protein-coding and non-coding genes through interaction with S2-phosphorylated Pol II and nascent RNA. Furthermore, we present the crystal structures of the Seb1 CTD- and RNA-binding modules. Unexpectedly, the latter reveals an intertwined two-domain arrangement of a canonical RRM and second domain. These results provide important insights into the mechanism underlying eukaryotic transcription termination

The conserved protein Seb1 drives transcription termination by binding RNA polymerase II and nascent RNA.

Termination of RNA polymerase II (Pol II) transcription is an important step in the transcription cycle, which involves the dislodgement of polymerase from DNA, leading to release of a functional transcript. Recent studies have identified the key players required for this process and showed that a common feature of these proteins is a conserved domain that interacts with the phosphorylated C-terminus of Pol II (CTD-interacting domain, CID). However, the mechanism by which transcription termination is achieved is not understood. Using genome-wide methods, here we show that the fission yeast CID-protein Seb1 is essential for termination of protein-coding and non-coding genes through interaction with S2-phosphorylated Pol II and nascent RNA. Furthermore, we present the crystal structures of the Seb1 CTD- and RNA-binding modules. Unexpectedly, the latter reveals an intertwined two-domain arrangement of a canonical RRM and second domain. These results provide important insights into the mechanism underlying eukaryotic transcription termination