Alternative gene ends: the crosstalk of RNA cleavage and transcription termination

Introduction

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.

Importance of Alternative 3' Ends

Alternative 3’ ends determine the properties of the encoded protein: typically its abundance, but sometimes also domain structure. For example, immunoglobulin M heavy chain can be membrane-bound or secreted depending on the 3’ cleavage site. Widespread changes in 3’ end usage are characteristic of many processes, such as differentiation and cancer, including neuroblastoma. We do not understand what drives this selectivity.

Research Aims

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 the choice of the alternative cleavage and polyadenylation site (Aim 1).

Biochemical and Genetic Approaches

I will use biochemical and genetic approaches to elucidate the sequence determinants of alternative cleavage and termination (Aim 2), and investigate sequence-independent components of alternative termination (Aim 3).

Methodology

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. Additionally, I will use a new technique to test this for single molecules.

In Vitro System

I will also determine the baseline for cleavage site selection utilizing a newly developed in vitro system. Combining these unique integrative and separation-of-function approaches will yield a comprehensive view of alternative gene end regulation.

Conclusion

Ultimately, understanding the complex crosstalk between RNA cleavage and transcription termination in alternative 3’ end selection will enable the manipulation of this process, for example, to alleviate diseases such as neuroblastoma.