The structural basis of CstF-77 modulation of cleavage and polyadenylation through stimulation of CstF-64 activity

Abstract

Cleavage and polyadenylation (C/P) of mRNA is an important cellular process that promotes increased diversity of mRNA isoforms and could change their stability in different cell types. The cleavage stimulation factor (CstF) complex, part of the C/P machinery, binds to U- and GU-rich sequences located downstream from the cleavage site through its RNA-binding subunit, CstF-64. Less is known about the function of the other two subunits of CstF, CstF-77 and CstF-50. Here, we show that the carboxy-terminus of CstF-77 plays a previously unrecognized role in enhancing C/P by altering how the RNA recognition motif (RRM) of CstF-64 binds RNA. In support of this finding, we also show that CstF-64 relies on CstF-77 to be transported to the nucleus; excess CstF-64 localizes to the cytoplasm, possibly via interaction with cytoplasmic RNAs. Reverse genetics and nuclear magnetic resonance studies of recombinant CstF-64 (RRM-Hinge) and CstF-77 (monkeytail-carboxy-terminal domain) indicate that the last 30 amino acids of CstF-77 increases the stability of the RRM, thus altering the affinity of the complex for RNA. These results provide new insights into the mechanism by which CstF regulates the location of the RNA cleavage site during C/P.

A Point Mutation in the RNA Recognition Motif of CSTF2 Associated with Intellectual Disability in Humans Causes Defects in 3′ End Processing

“…RNA-contact residues in the CSTF2 RRM play specific roles during C/P (Grozdanov et al, 2018b). Introduction of the D50A mutation into CSTF2 resulted in a small but consistent reduction in C/P efficiency (Figure 2A).…”

“…Mutations in the RNA binding site I and II of CSTF2 affect the synergetic function of CSTF2 and CSTF3 (Grozdanov et al, 2018b). Therefore, we co-expressed CSTF3 with either MCP-CSTF2 or MCP-CSTF2 D50A to determine whether the D50A mutation affects cleavage and polyadenylation in the presence of CSTF3.…”

“…CSTF3 increased SLAP for both wild type and MCP-CSTF2 D50A to a similar extent, effectively eliminating the reduction observed in the MCP-CSTF2 D50A construct expressed alone. Mutations in the CSTF2 RRM altered nuclear-tocytoplasmic localization (Grozdanov et al, 2018b). Therefore, we hypothesized that the CSTF2 D50A mutation might also affect the ratio between nuclear and cytoplasmic CSTF2.…”

“…Because the aspartic acid (D) to alanine (A) mutation changes the charge in the loop connecting the 2 and 3-strands ( Figure 4 and Figure S1, Deka et al, 2005;Grozdanov et al, 2018b), we wanted to determine whether RNA binding was altered in the CSTF2 D50A RRM mutant. To measure binding via fluorescence polarization/anisotropy, bacterially expressed CSTF2 and CSTF2 D50A RRMs (amino acids 1-107, Grozdanov et al, 2018b) were incubated with fluorescently-labeled RNA oligonucleotides from either the SV40 late transcription unit (SVL, MacDonald et al, 1994) or (GU)8 (Deka et al, 2005). The wild type RRM bound to the SVL and (GU)8 RNAs with Kd, app of 1.45 0.07 µM and 0.26 0.02 µM, respectively ( Figure 2C, D and Table S1).…”

“…CstF is the regulatory factor in C/P, consisting of three subunits, CstF-50 (gene symbol CSTF1), , and . CSTF2, the RNA-binding component of CstF, binds to U-or GU-rich sequences downstream of the cleavage site through its RNA recognition motif (RRM, Grozdanov et al, 2018b). As such, CSTF2 regulates gene expression in immune cells (Chuvpilo et al, 1999;Edwalds-Gilbert and Milcarek, 1995;Takagaki et al, 1996), spermatogenesis (Dass et al, 2007;Hockert et al, 2011;Li et al, 2012), and embryonic stem cell development .…”