Molecular mechanism for the interaction between human CPSF30 and hFip1

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Eukaryotic pre-mRNA requires 3 -end cleavage and polyadenylation for nuclear export.
• The cleavage and polyadenylation specificity factor (CPSF) complex, including CPSF30, is essential for this process.
• CPSF30's ZF4-ZF5 domains are implicated in recruiting the hFip1 subunit, but the interaction mechanism was unclear.

Purpose of the Study:

• To elucidate the molecular basis of the interaction between CPSF30's ZF4-ZF5 domains and hFip1.
• To characterize the stoichiometry and binding affinity of the CPSF30-hFip1 complex.
• To investigate the functional implications of this interaction in mRNA 3 -end polyadenylation.

Main Methods:

• X-ray crystallography to determine the structure of human CPSF30 ZF4-ZF5 in complex with hFip1.
• Site-directed mutagenesis to assess the importance of binding sites.
• Fluorescence polarization binding assays to measure binding affinity.
• In vitro assays to study the recruitment of poly(A) polymerase (PAP) and support of polyadenylation.

Main Results:

• The crystal structure revealed a conserved 1:2 stoichiometry, with one hFip1 molecule binding to each of CPSF30's ZF4 and ZF5 domains.
• Mutagenesis studies confirmed the 1:2 stoichiometry and showed that mutating both sites abrogated hFip1 binding.
• Fluorescence polarization assays indicated a higher affinity of ZF4 for hFip1 (Kd = 1.8 nM).
• The CPSF30-hFip1 complex recruited two copies of PAP in vitro, and both hFip1 binding sites supported polyadenylation.

Conclusions:

• The study reveals the structural basis for the 1:2 interaction between CPSF30 ZF4-ZF5 and hFip1.
• This interaction is critical for the recruitment of PAP and efficient mRNA 3 -end polyadenylation.
• The findings provide insights into the regulation of mRNA processing by the CPSF complex.