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

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 the CPF including two protein phosphatases are also found in a related complex, the '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 has functions separable from those of CPF. Syc1 plays a 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. Overall design: In this study, we used photoactivatable ribonucleoside-enhanced crosslinking and immuno-precipitation (PAR-CLIP) and ChIP-seq to map direct protein-RNA interactions and protein-DNA interactions, respectively, in vivo. We applied these methods to subunits of the CPF and APT complexes of the yeast Saccharomyces cerevisiae. Both ChIP and PAR-CLIP occupancy profiles suggested that the APT defining subunit Syc1 is preferentially associated with sn/snoRNA genes and transcripts. Thus, we also investigated RNA synthesis in vivo using metabolic labeling with 4-thiouracil, followed by sequencing of the newly synthesized, labeled RNA (4tU-seq) and compared syc1 knock-out cells to wild-type.

4

Lidschreiber M, Easter AD, Battaglia S, Rodríguez-Molina JB, Casañal A, Carminati M, Baejen C, Grzechnik P, Maier KC, Cramer P, Passmore LA