A missense mutation in the CSTF2 gene that impairs the function of the RNA recognition motif and causes defects in 3′ end processing is associated with intellectual disability in humans

Abstract

CSTF2 encodes an RNA-binding protein that is essential for mRNA cleavage and polyadenylation (C/P). No disease-associated mutations have been described for this gene. Here, we report a mutation in the RNA recognition motif (RRM) of CSTF2 that changes an aspartic acid at position 50 to alanine (p.D50A), resulting in intellectual disability in male patients. In mice, this mutation was sufficient to alter polyadenylation sites in over 1300 genes critical for brain development. Using a reporter gene assay, we demonstrated that C/P efficiency of CSTF2D50A was lower than wild type. To account for this, we determined that p.D50A changed locations of amino acid side chains altering RNA binding sites in the RRM. The changes modified the electrostatic potential of the RRM leading to a greater affinity for RNA. These results highlight the significance of 3′ end mRNA processing in expression of genes important for brain plasticity and neuronal development.

Electrostatic Interactions between CSTF2 and pre-mRNA Drive Cleavage and Polyadenylation

“…binding site residues and the disease-associated mutation D50A had an effect on the SLAP signal (9,13). Here, we also observed differences in SLAP upon mutation of surface electrostatic residues (Fig.…”

“…Given the importance of electrostatics and enthalpy-entropy compensation in RRM binding to SVL RNA, we investigated the changes in fast timescale motions resulting from RNA binding to various RRM mutants. As previously demonstrated for the RRM D50A mutant, the enthalpy-entropy compensation led to an increase in the amplitude of fast timescale RRM backbone dynamics (13). Accordingly, 15 N R 1 , R 2 , and { 1 H}- 15 N heteronuclear NOE relaxation data were recorded for several mutants at 14.1 T (600 MHz 1 H frequency) to study the nanosecond timescale dynamics that report on the backbone flexibility of RRM constructs.…”

“…For NMR experiments, transformed Rosetta (DE3) pLysS cells were grown in 2x minimal M9 media (22) using 15 NH 4 Cl (1 g/L) and unlabeled D-glucose (3 g/L) as sole nitrogen and carbon sources, respectively. Protein expression was induced with 0.5 mM isopropyl-b-d-thiogalactopyranoside for 18 h at 25 C. Protein purification proceeded as previously described (9,13).…”

“…NMR titration data reveal that changes in RRM surface charge alter both the on-and off-rates of RNA binding Using 2D line shape analysis of NMR titration experiments, we previously showed that the D50A mutation in the RRM increased the apparent on-rate for SVL binding without significantly affecting the apparent off-rate, resulting in a lower K d (13). For both the wild-type and D50A mutant, the apparent on-rates were faster than the rate of diffusion (4.35 x 10 8 and 5.83 x 10 8 M À1 sec À1 , respectively, under these sample conditions), which we attributed to electrostatic attraction between the positively charged RRM and negatively charged RNA.…”

Cleavage Stimulation Factor Subunit 2: Function Across Cancers and Potential Target for Chemotherapeutic Drugs

“…CSTF2 encodes a 557-amino acid protein that contains a conserved RNA recognition motif (RRM) RNA binding domain at its N-terminus, a long proline region rich in glycine, and a pentapeptide repeat region that forms an extended alpha-helix (Steber et al, 2019). In the RRM of CSTF2, there is a U dinucleotide specific binding site and a highly mobilized protein: through its RRM, CSTF2 binds to the U or GU rich sequence downstream of the cleavage site to form a stable complex, affecting the processing of pre-mRNA (Grozdanov et al, 2020). Therefore, CSTF2 is essential for the cleavage of mRNA and polyadenosine (C/P).…”