SF1 Phosphorylation Enhances Specific Binding to U2AF 65 and Reduces Binding to 3′-Splice-Site RNA

Abstract

Splicing factor 1 (SF1) recognizes 3 0 splice sites of the major class of introns as a ternary complex with U2AF 65 and U2AF 35 splicing factors. A conserved SPSP motif in a coiled-coil domain of SF1 is highly phosphorylated in proliferating human cells and is required for cell proliferation. The UHM kinase 1 (UHMK1), also called KIS, double-phosphorylates both serines of this SF1 motif. Here, we use isothermal titration calorimetry to demonstrate that UHMK1 phosphorylation of the SF1 SPSP motif slightly enhances specific binding of phospho-SF1 to its cognate U2AF 65 protein partner. Conversely, quantitative fluorescence anisotropy RNA binding assays and isothermal titration calorimetry experiments establish that double-SPSP phosphorylation reduces phospho-SF1 and phospho-SF1-U2AF 65 binding affinities for either optimal or suboptimal splice-site RNAs. Domain-substitution and mutagenesis experiments further demonstrate that arginines surrounding the phosphorylated SF1 loop are required for cooperative 3 0 splice site recognition by the SF1-U2AF 65 complex (where cooperativity is defined as a nonadditive increase in RNA binding by the protein complex relative to the individual proteins). In the context of local, intracellular concentrations, the subtle effects of SF1 phosphorylation on its associations with U2AF 65 and splice-site RNAs are likely to influence pre-mRNA splicing. However, considering roles for SF1 in pre-mRNA retention and transcriptional repression, as well as in splicing, future comprehensive investigations are needed to fully explain the requirement for SF1 SPSP phosphorylation in proliferating human cells.

COX5B-Mediated Bioenergetic Alteration Regulates Tumor Growth and Migration by Modulating AMPK-UHMK1-ERK Cascade in Hepatoma

“…Despite a bundle of phospho-proteins that have been proposed as substrates [32,36,[39][40][41][42][43][44], it is still unclear regarding the key downstream signaling pathway(s) perturbed by UHMK1 during tumorigenesis and progression. However, by mining the downstream targets of UHMK1 kinase activity, ERK2 and Stathmin1 were identified harboring the most significant changes of phosphorylation status (Table 1).…”

The pre-mRNA splicing and transcription factor Tat-SF1 is a functional partner of the spliceosome SF3b1 subunit via a U2AF homology motif interface

“…To avoid overlapping molecular weights, we conversely compared GST-Tat-SF1 retention of SF3b1 ULM with SF1. The SF1 protein was phosphorylated (P) in vitro with UHMK1 as described (25,36), which subtly enhances SF1-U2AF 65 association (36,37) and is the major form of SF1 in proliferating cells (36,38).…”

UHMK1 (U2AF homology motif kinase 1)

“…Through the UHM motif, UHMK1 interacts with the splicing factors SF1 and SF3B1 (Manceau et al, 2008). Upon interaction, UHMK1 phosphorylates SF1, which enhances SF1 specific binding to U2AF 65 and reduces the SF1-U2AF 65 binding to the 3' splice site RNA (Chatrikhi et al, 2016;Manceau et al, 2006). In addition, UHMK1…”