ANALYSIS AND MAPPING OF RNA POLYMERASE II TERMINATION FACTORS
Schaughency, Paul Michael
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Termination of transcription is an important, but poorly understood, cellular process. RNA polymerase II (Pol II) terminates coding transcripts and non-coding transcripts (ncRNAs) through two distinct mechanisms in yeast. In the non-poly(A) dependent pathway, Nrd1 and Nab3 bind to their recognition sites on the nascent RNA of ncRNAs to cause termination. This is done through an unknown mechanism, but is known to involve the RNA-DNA helicase Sen1. In our first study, we have mapped Pol II distribution before and after depletion of different termination factors. This was done, by depleting each factor from the nucleus and then using PAR-CLIP to assess the position of Pol II. Nrd1 depletion leads to widespread runaway transcription at non-poly(A) terminators. In contrast, depletion of Sen1 or Ysh1, an endonuclease involved in the poly(A) dependent pathway, does not lead to elevated readthrough transcription. These differences allowed us to map all the non-poly(A) termination regions throughout the yeast genome. Our second study follows the human homolog of Nrd1, RBM16. RBM16 shares a common protein domain architecture and a conserved CTD interacting domain with Nrd1. Although it is unclear whether there is a similar non-poly(A) termination pathway in human cells, we show that there are at least similarities between the classes of RNAs that are bound to both Nrd1 and RBM16. The final study focuses on the unique methodologies used to analyze PAR-CLIP datasets. It describes computer programs and bioinformatic pipelines written to provide answers to basic questions based on PAR-CLIP data. The functions created to answer these questions could be applicable to a wide variety of data. The studies presented here provide a foundation so that fundamental mechanisms of termination can be elucidated.