RBBP6 activates the pre-mRNA 3' end processing machinery in humans.

Thomas S Elliott,Lori A Passmore,Vytaute Boreikaite,Jason W Chin

doi:10.1101/gad.349223.121

Thomas S Elliott, Lori A Passmore + Show 2 more

Open Access

https://doi.org/10.1101/gad.349223.121

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Abstract

3' end processing of most human mRNAs is carried out by the cleavage and polyadenylation specificity factor (CPSF; CPF in yeast). Endonucleolytic cleavage of the nascent pre-mRNA defines the 3' end of the mature transcript, which is important for mRNA localization, translation, and stability. Cleavage must therefore be tightly regulated. Here, we reconstituted specific and efficient 3' endonuclease activity of human CPSF with purified proteins. This required the seven-subunit CPSF as well as three additional protein factors: cleavage stimulatory factor (CStF), cleavage factor IIm (CFIIm), and, importantly, the multidomain protein RBBP6. Unlike its yeast homolog Mpe1, which is a stable subunit of CPF, RBBP6 does not copurify with CPSF and is recruited in an RNA-dependent manner. Sequence and mutational analyses suggest that RBBP6 interacts with the WDR33 and CPSF73 subunits of CPSF. Thus, it is likely that the role of RBBP6 is conserved from yeast to humans. Overall, our data are consistent with CPSF endonuclease activation and site-specific pre-mRNA cleavage being highly controlled to maintain fidelity in mRNA processing.

Highlights

Eukaryotic protein-coding pre-mRNAs undergo multiple processing steps during transcription by RNA polymerase II
Cleavage stimulatory factor (CStF) and cleavage factor IIm (CFIIm) are both multisubunit protein complexes implicated in GENES & DEVELOPMENT 36:1–15 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/22; www.genesdev.org cleavage (Takagaki et al 1990; De Vries et al 2000)
cleavage stimulatory factor (CStF), CFIIm, and RBBP6 are required for activation of cleavage and polyadenylation specificity factor (CPSF) endonuclease

Summary

Introduction

Eukaryotic protein-coding pre-mRNAs undergo multiple processing steps during transcription by RNA polymerase II. Structures of mPSF/polymerase module in apo and RNA-bound states have recently been elucidated (Casañal et al 2017; Clerici et al 2017, 2018; Sun et al 2018) These showed how the CPSF30 and WDR33 subunits recognize the hexameric polyadenylation signal (PAS) sequence, most commonly AAUAAA, thereby recruiting CPSF to cleavage sites on pre-mRNAs. The poly(A) polymerase enzyme (PAP) is not a stable subunit of human CPSF but is instead recruited to cleaved transcripts by hFip (Kaufmann et al 2004; Chan et al 2014). Whether it plays a direct role in the cleavage reaction remains unclear

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