The human genome contains only ~20.000 genes, however, most of them encode multiple transcripts resulting from alternative promoter usage, splicing, and 3’ end selection. Gene 3’ ends can be defined by the positions of RNA 3’ cleavage, or the location where RNA polymerase II terminates transcription. Alternative 3’ ends determine the properties of the encoded protein: typically its abundance, but sometimes also domain structure – as for immunoglobulin M heavy chain which is membrane-bound or secreted depending on the 3’ cleavage site. Widespread changes in 3’ end usage are characteristic of many processes e.g. differentiation and cancer like neuroblastoma. We do not understand what drives this selectivity. In this research project I will answer the fundamental question of how the location and timing of RNA polymerase II entering into termination mode impacts on the choice of the alternative cleavage and polyadenylation site (Aim 1). I will use biochemical and genetic approaches to elucidate the sequence determinant of alternative cleavage and termination (Aim 2), and investigate sequence-independent components of alternative termination (Aim 3). I recently pioneered the measurement of 3’ cleavage positions together with locations of transcription termination by a novel transcriptomic method. I will apply this method to investigate the timing of changes in cleavage and termination relative to each other on an averaged cell population level, and use a new technique to test this for single molecules. I will also determine the baseline for cleavage site selection utilizing a newly developed in vitro system. Combining those unique integrative and separation-of-function approaches will yield a comprehensive view of alternative gene end regulation. Ultimately, understanding the complex crosstalk between RNA cleavage and transcription termination in alternative 3’ end selection will enable the manipulation of this process e.g. to alleviate diseases such as neuroblastoma.