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In vitro reconstitution of the human pre-mRNA 3′ end processing machinery and mechanistic insights into endonuclease activation



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Boreikaite, Vytaute  ORCID logo


Maturation of protein-coding transcripts in eukaryotes involves several processing steps, including 5′ capping, splicing, and 3′ end processing. The latter entails endonucleolytic cleavage of the nascent pre-mRNA and addition of a poly(A) tail to the resultant free 3′ end. The poly(A) tail then facilitates nuclear export of mRNAs and controls their stability and translational efficiency in the cytoplasm. Pre-mRNA 3′ end processing is also tightly coupled to transcription termination. Defects in 3′ end processing cause a variety of human diseases, highlighting its critical role in gene expression.

In humans, 3′ end processing of most pre-mRNAs is carried out by a seven-subunit protein complex known as cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). The polyadenylation activity of CPSF has been studied in detail biochemically, but our understanding of how CPSF cleaves the pre-mRNA remains limited. CPSF is an inherently inactive endonuclease on its own and was believed to require additional protein factors for its activation, enabling tight regulation of 3′ cleavage. To gain insight into the activation mechanism of the 3′ endonuclease, a minimal in vitro system with a well-defined protein composition is required, but this has eluded researchers for decades.

In this dissertation, I have reconstituted specific and efficient pre-mRNA cleavage activity by the human CPSF complex with purified recombinant proteins. I have determined that activation of the CPSF endonuclease requires three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm), and, importantly, a multidomain protein RBBP6. The role of RBBP6 in 3′ end processing in humans has been largely overlooked, and therefore, I studied this protein in more detail. The yeast orthologue of RBBP6, Mpe1, senses pre-mRNA binding and is a constitutive subunit of CPF. In contrast, by purifying endogenous CPSF from human cells, I show that RBBP6 is not a stable component of the human complex. Instead, my biochemical studies reveal that RBBP6 is likely recruited to CPSF in an RNA-dependent manner and that it also interacts with the CFIIm cleavage factor complex. My sequence and mutational analyses suggest that the role of RBBP6 in activating the CPSF endonuclease is conserved from yeast to human. I have also performed cryo-electron microscopy studies of some protein complexes involved in pre-mRNA 3′ end processing in humans, aiming towards an atomic-level understanding of CPSF cleavage activity, which remains the major outstanding knowledge gap in the field of 3′ end processing.

Overall, the reconstitution of human pre-mRNA 3′ end processing with purified proteins described in this dissertation has enabled detailed mechanistic studies of CPSF structure and function, and may also facilitate the development of new therapeutics.





Passmore, Lori


mRNA, Endonuclease, Cleavage, Polyadenylation, CPSF, RNA processing, Biochemistry


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge