Understanding RNA Binding by the Nonclassical Zinc Finger Protein CPSF30, a Key Factor in Polyadenylation during Pre-mRNA Processing

Cleavage
and polyadenylation specificity factor 30 (CPSF30) is
a zinc finger protein that regulates pre-mRNA processing. CPSF30 contains
five CCCH domains and one CCHC domain and recognizes two conserved
3′ pre-mRNA sequences: an AU hexamer and a U-rich motif. AU
hexamer motifs are common in pre-mRNAs and are typically defined as
AAUAAA. Variations within the AAUAAA hexamer occur in certain pre-mRNAs
and can affect polyadenylation efficiency or be linked to diseases.
The effects of disease-related variations on CPSF30/pre-mRNA binding
were determined using a construct of CPSF30 that contains just the
five CCCH domains (CPSF30-5F). Bioinformatics was utilized to identify
the variability within the AU hexamer sequence in pre-mRNAs. The effects
of this sequence variability on CPSF30-5F/RNA binding affinities were
measured. Bases at positions 1, 2, 4, and 5 within the AU hexamer
were found to be important for RNA binding. Bioinformatics revealed
that the three bases flanking the AU hexamer at the 5′ and
3′ ends are twice as likely to be adenine or uracil as guanine
and cytosine. The presence of A and U residues in these flanking regions
was determined to promote higher-affinity CPSF30-5F/RNA binding than
G and C residues. The addition of the zinc knuckle domain to CPSF30-5F
(CPSF30-FL) restored binding to AU hexamer variants. This restoration
of binding is connected to the presence of a U-rich sequence within
the pre-mRNA to which the zinc knuckle binds. A mechanism of differential
RNA binding by CPSF30, modulated by accessibility of the two RNA binding
sites, is proposed.