Lay summary
The recent observations that alternative pre-mRNA 3' end processing results in a global decreased susceptibility of mRNAs to micro RNA-dependent inhibition in dividing cells including cancer cells caused renewed interest in 3' end processing. The mechanism by which a change in the use of proximal versus distal polyadenylation sites in resting compared to dividing cells occurs is not known. Our previous work contributed to the elucidation of the dynamics of 3' end processing factors within the 3' end processing complex itself and within the transcriptomes of selected mammalian cell systems and is of relevance to the unsolved puzzles concerning the systematic changes in 3' end processing in relation to the cellular state. In collaboration with the group of Professor Mihaela Zavolan at the Biozentrum, we recently initiated a study of binding sites of various 3' end processing factors in a number of cell types and under various conditions. We could show that the binding sites that we identified reflect the known sequence specificities of the 3' end processing factors and their relative location within individual transcripts is consistent with earlier reports.The second project concerns the structure of the cleavage and polyadenylation factor CPF from yeast cells. In collaboration with the group of Professor Andreas Engel at the Biozentrum, we have determined the molecular mass of this complex and the stoichiometry of its subunits by scanning transmission electron microscopy. Professor Holger Stark (Max-Planck-Institute for Biophysical Chemistry, Göttingen) has provided us with his newly developed protein crosslinking-gradient centrifugation procedure "GraFix" to purify CPF preparations suitable for electron microscopy and he has established the three-dimensional structure of the complex at a resolution of 25Å in negatively stained preparations and by single-particle cryo-electron microscopy. CPF has a complex asymmetric architecture in which an outer protein wall surrounds a large inner cavity. Moreover, three GFP-tagged subunits could be localized within the structure and the X-ray structure of poly(A) polymerase could be fitted to the complex. Professor Stark is currently analyzing more images by cryo-electron microscopy to improve the resolution of the structure. We hope that our experiments will contribute to the understanding of the mechanism of 3' end processing and of how this fundamental step of mRNA metabolism interfaces with other cellular processes such as transcription, alternative splicing and polyadenylation as well as with the susceptibility to cotranscriptional and post-transcriptional regulation.