Title

In Vivo Characterization of the Drosophila mRNA 3’-end Processing Core Cleavage Complex: Poly(A) mRNA & Histone mRNA 3’-end Processing

Document Type

Dissertation

Degree

Doctor of Philosophy

Major

Biology

Date of Defense

3-12-2017

Graduate Advisor

Wendy Olivas, PhD

Committee

Wendy Olivas

Dr. Teresa Thiel

Dr. Michael Hughes

Dr. Lon Chubiz

Abstract

A core cleavage complex (CCC) consisting of CPSF73, CPSF100 and Symplekin is required for co-transcriptional 3’ end processing of all metazoan pre-mRNAs, yet little is known about the in vivo molecular interactions within this complex. The CCC is a component of two distinct complexes, the cleavage/polyadenylation complex and the complex that processes non-polyadenylated histone pre-mRNAs. RNAi-depletion of CCC factors in Drosophila culture cells causes reduction of CCC processing activity on histone mRNAs, resulting in read through transcription. In contrast, RNAi-depletion of factors only required for histone mRNA processing allows use of downstream cryptic polyadenylation signals to produce polyadenylated histone mRNAs. We used Dmel-2 tissue culture cells stably expressing tagged CCC components to determine that amino acids 272-1080 of Symplekin and the C-terminal ~200 amino acids of both CPSF73 and CPSF100 are required for efficient CCC formation in vivo. Additional experiments reveal that the C-terminal 241 amino acids of CPSF100 are sufficient for histone mRNA processing indicating that the first 524 amino acids of CPSF100 are dispensable for both CCC formation and histone mRNA 3’-end processing. CCCs containing deletions of Symplekin lacking the first 271 amino acids resulted in a significantly increased use of downstream polyadenylation sites for histone mRNA 3’-end processing similar to RNAi-depletion of histone specific 3’-end processing factors FLASH, SLBP and U7 snRNA. We propose a model in which CCC formation is mediated by CPSF73, CPSF100 and Symplekin C-termini, and the N-terminal region of Symplekin facilitates co-transcriptional 3’-end processing of histone mRNAs.

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