Biochemical characterization and three-dimensional structure analysis of the yeast cleavage and polyadenylation factor CPF

In eukaryotes, protein-encoding genes are transcribed in the nucleus by RNA polymerase II (RNAP II). Before the gene transcript (pre-mRNA) is transported to the cytoplasm and can function in translation, it has to undergo three specific maturation steps: capping, splicing and 3’ end processing. It is well established now that premRNA processing events occur cotranscritpionally, while RNAP II is still transcribing the gene. Pre-mRNA 3’ end formation is an essential step in gene expression. With the exception of mRNAs coding for replication-dependent histone proteins, all eukaryotic pre-mRNAs are processed at their 3’ end by a coupled two-step reaction that involves a specific endonucleolytic cleavage at the poly(A) site and subsequent poly(A) tail addition to the upstream cleavage fragment catalyzed by poly(A) polymerase (Pap1p). The complete maturation of the pre-mRNA 3’ end is directed by the presence of cisacting sequence elements on the pre-mRNA that recruit protein factors. Surprisingly, 3’ end processing is accomplished by a complex protein machinery, which is highly conserved from yeast to mammals. Indeed, most of the polypeptides involved in 3’ end formation in mammals have homologues in yeast. In S. cerevisiae, the cleavage and polyadenylation factor (CPF), cleavage factor IA (CF IA), cleavage factor IB (CF IB or Nab4p/Hrp1p) and the poly(A) binding protein (Pab1p) are required for specific and accurate 3’ end processing activities. The main pre-mRNA 3’ end processing factor, CPF, is a multiprotein complex that consists of 15 polypeptides and is required for both pre-mRNA 3’ end processing reactions. Most of its subunits are essential (only two are not essential) and all the components are involved in RNA recognition or protein-protein interactions within CPF or with other protein complexes, such as CF IA or RNAP II. Biochemical studies of all CPF subunits have confirmed that they belong to the complex and allowed characterization of their function in the context of pre-mRNA 3’ end formation. Affinity purification of CPF identified a novel protein that co-purified with the previously known components and was therefore proposed to be a new subunit of the complex. This putative new component is non-essential and was named Cpf11p. In this work, we showed that Cpf11p is not stably associated with CPF, as TAP tag purification of Cpf11p did not result in the purification of CPF components (Chapter 3). Furthermore, in vitro cleavage and polyadenylation assays performed with Cpf11p-depleted extracts did not show any defect, indicating that Cpf11p is not involved in pre-mRNA 3’ end formation. We also found that expression of Cpf11p, in contrast to the expression of CPF subunits, is controlled in a sugar-dependent manner. Taken together, our results strongly suggest that Cpf11p has no function in premRNA 3’ end processing. CPF plays a central role in pre-mRNA 3’ end processing. Its requirement for cleavage and polyadenylation reactions is mediated by cooperative interactions with CF IA and recognition of cis-acting polyadenylation signals on the primary transcript. Despite every polypeptide involved in pre-mRNA 3’ end processing was characterized, the mechanism by which the pre-mRNA is cleaved and polyadenylated is not known. One aim of this thesis is to provide insight into the three-dimensional structure of CPF. This would allow a better understanding in the arrangement of the subunits in the complex and provide information on the molecular mechanism of premRNA 3’ end processing. We have developed an efficient purification procedure that yields highly pure and active CPF. Here we report for the first time the 3D structure of the complex at a resolution of 25 A, determined by single-particle electron microscopy on natively purified CPF using angular reconstitution and random conical tilt (Chapter 4). The 3D model reveals a rough globular shape of the complex and a strikingly large central cavity. We discuss the possibility that the inner cavity represents a reaction chamber in which pre-mRNA 3’ end processing reactions could take place. Furthermore, we have determined the mass of CPF particles by scanning transmission electron microscopy (STEM) at approximately one megaDalton. The work reported in this thesis should contribute to a better understanding of the mechanism by which pre-mRNAs are processed at their 3’ end by presenting the first 3D model of the CPF complex, and by refining the protein composition of CPF. In addition, a part of this work is dedicated to investigation of in vivo interconnections between transcription and 3’ end processing of pre-mRNAs (Chapter 2).