Structural basis for regulation of RNA-binding proteins by phosphorylation

Abstract

Ribonucleoprotein complexes involved in pre-mRNA splicing and mRNA decay are often regulated by phosphorylation of RNA-binding proteins. Cells use phosphorylation-dependent signaling pathways to turn on and off gene expression. Not much is known about how phosphorylation-dependent signals transmitted by exogenous factors or cell cycle checkpoints regulate RNA-mediated gene expression at the atomic level. Several human diseases are linked to an altered phosphorylation state of an RNA binding protein. Understanding the structural response to the phosphorylation "signal" and its effect on ribonucleoprotein assembly provides mechanistic understanding, as well as new information for the design of novel drugs. In this review, I highlight recent structural studies that reveal the mechanisms by which phosphorylation can regulate protein-protein and protein-RNA interactions in ribonucleoprotein complexes.

Figure 1

Crystal structures of phosphorylated SFRS1 bound to Transporting 3 and SRPK1. (A) Domain structure of SFRS1 is depicted. (B) The sequence of the RS domain consisting of the N-terminal RS1, which is phosphorylated by SRPK1, is shown. The C-terminal RS2 region is phosphorylated by Clk/Sty kinases. (C) The 2.9 Å crystal structure of the SRPK1- SFRS1 complex (PDB code 3BEG) is shown. The kinase is in blue ribbon and SFRS1 in red. (D) Specific interactions between protein side chains of SRPK1 and the RS1 domain of SFRS1 in the docking site are depicted. (E) Cartoon representation of the 2.6 Å crystal structure of Transportin 3 (in blue) bound to the SFRS1 RRM2-RS1 region (in red) (PDB code 4C0O). Heat repeats 12–16 (shown in green) primarily interact with RS1 (in gold). (F) The specific interactions between protein side chains of Transportin 3 and the RS1 domain of SFRS1 is depicted. An extensive arginine-zipper-like interface is observed. The Transportin 3 helices are in gray and the RS1 domain in magenta. Arginines (R206, R208, and R210) from SFRS1 are shown in magenta/blue stick, the phosphoserines (S209 and S207) are in orange. Residues from Transportin 3 that interact with the RS1 peptide are in green stick. Black dashes denote salt bridge interactions between arginines from Transportin 3 and red dashes denote salt bridge interactions between arginines from SFRS1.

Figure 2

X-ray crystallographic and NMR structures of phosphorylated SF1. (A) Schematic showing the domain organization and interactions between SF1, U2AF, and the RNA. (B) Solution NMR structural ensemble (PDB code 2M09) of the free helix hairpin (HH) motif consisting of two α-helices in an antiparallel arrangement connected by a flexible linker is shown. The serines that are phosphorylated are in magenta in a dynamic SPSP loop. (C) The 2.29 Å crystal structure of phosphorylated SF1 HH bound to the U2AF UHM (PDB code 4FXW) is shown. SF1 is in red, and U2AF in blue. The phosphorylated serines are depicted in magenta. (D) Interactions of the phosphates with neighboring arginines to form an “arginine claw” are shown.

Figure 3

X-ray crystal structure of phosphorylated human SLBP. (A) Schematic showing the domain organization of human SLBP (hSLBP) and Drosophila SLBP (dSLBP). The RNA binding domain is designated the “L-motif” and is followed by an acidic region, rich in Asp and Glu residues, that is also phosphorylated. The N-terminal domain is involved in translation activation (TAD). Phosphorylation sites that have been mapped in vivo(76,80,124) are indicated. (B) The T171 phosphorylated SLBP L-motif is shown with a characteristic L-shape as seen in the crystal structure of the hSLBP/histone mRNA/3′hExo ternary complex (PDB code 4QOZ). The fold consists of three α-helices connected by a 20-residue flexible loop that has the site of phosphorylation (shown in stick). Hydrophobic residues at the junction of the helices are shown in yellow (inset). (C) Hydrogen bonding interactions mediated by the phosphothreonine with R163, R169, K146, Y151, and W190 (via a water molecule) are shown. The structured loop that is disordered in the unphosphorylated SLBP structure is fully ordered in phosphorylated SLBP. The unphosphorylated structure is shown in blue and the phosphorylated structure in red ribbon. (D) Residues in helix-2 and the structured loop that undergo a conformational change upon SLBP phosphorylation and have been implicated in RNA processing are highlighted.

Figure 4

Structures of phosphorylated mRNA decay factors that bind 14–3–3 or 14–3–3-like domains. (A) Schematic showing domain organization of KSRP. The four KH domains are shown in red and the nuclear localization signals are in blue. (B) Solution NMR structure of the first KH domain (PDB code 2OPU) of KSRP when unphosphorylated. The site of phosphorylation, Ser193, is shown in stick. (C) Comparison of the folding topologies of 14–3–3ζ with 14–3–3-like domains from SMG6, SMG7, and the SMG5-SMG7 complex is shown in purple. The phosphoserine binding site is indicated. (D) Interaction of a phosphoserine peptide with 14–3–3ζ as seen in the crystal structure of the cocomplex (PDB code 1QJB) is depicted. The phosphoserine binds in an extended conformation to 14–3–3 proteins.