UCLA

Termination of pre-mRNA transcription by RNA polymerase II occurs in two steps: a decrease of elongation rate (pause), followed by the dissociation of the polymerase from the DNA template (release). While the pause can be triggered solely by the AAUAAA hexamer in the nascent transcript, the release can only occur in presence of a complete poly(A) signal, which also requires a GU-rich sequence downstream the hexamer. The hexamer and the GU-rich element are specifically recognized by CPSF and CstF respectively. Contradicting views exist about whether CPSF and CstF define the complete poly(A) signal in a sequential recruitment manner or as a preassemble complex. In Chapter 2, taking advantage of an in vitro system in HeLa nuclear extract, in which pre-mRNA 3'-end processing is coupled to transcription, we found that the functional poly(A) signal is defined by a preassembled CPSF-CstF complex, which potentially captures the upcoming GU-rich transcript more efficiently.

The complete poly(A) signal, together with its associated protein apparatus, is capable of releasing the polymerase from the DNA. But it has been controversial for decades what is the prerequisite of the release step: the poly(A) site cleavage and the subsequent degradation of the downstream RNA, or a particular conformational rearrangement of the transcription complex. In Chapter 3, based on the same in vitro system, we developed a transcription termination assay, which can measure the termination and concurrent poly(A) site cleavage simultaneously. Through this assay, we established that the release of the polymerase does not require poly(A) site cleavage. Rather, the transcription complex experiences a conformational rearrangement immediately after crossing the poly(A) signal, followed by a slow dissociation. The dissociation of the transcription complex can be fully inhibited by α-amanitin, presumably by disruption of the poly(A)-induced conformational rearrangement.