Phosphorylation of T897 in the dimerization domain of Gemin5 modulates protein interactions and translation regulation

Abstract

Gemin5 is a multifunctional RNA binding protein (RBP) organized in domains with a distinctive structural organization. The protein is a hub for several protein networks performing diverse RNA-dependent functions including regulation of translation, and recognition of small nuclear RNAs (snRNAs). Here we sought to identify the presence of phosphoresidues on the C-terminal half of Gemin5, a region of the protein that harbors a tetratricopeptide repeat (TPR)-like dimerization domain and a non-canonical RNA binding site (RBS1). We identified two phosphoresidues in the purified protein: P-T897 in the dimerization domain and P-T1355 in RBS1. Replacing T897 and T1355 with alanine led to decreased translation, and mass spectrometry analysis revealed that mutation T897A strongly abrogates the association with cellular proteins related to the regulation of translation. In contrast, the phosphomimetic substitutions to glutamate partially rescued the translation regulatory activity. The structural analysis of the TPR dimerization domain indicates that local rearrangements caused by phosphorylation of T897 affect the conformation of the flexible loop 2-3, and propagate across the dimerization interface, impacting the position of the C-terminal helices and the loop 12-13 shown to be mutated in patients with neurological disorders. Computational analysis of the potential relationship between post-translation modifications and currently known pathogenic variants indicates a lack of overlapping of the affected residues within the functional domains of the protein and provides molecular insights for the implication of the phosphorylated residues in translation regulation.

Keywords: BiNGO, Biological Networks Gene Ontology application; CHX, cycloheximide; Gemin5 interactome; Human variants; IRES, internal ribosome entry site; LC-MS/MS, liquid chromatography-mass spectrometry; MD, molecular dynamics; NEDCAM, neurological disorders with cerebellar atrophy and motor dysfunction; Neurological disease; Phosphoresidues; Protein structure modeling; Protein synthesis; RBP, RNA-binding protein; RBS1, RNA-binding site1; RNA-binding proteins; SGs, stress granules; SMN complex; SMN, survival of motor neurons; TAP, tandem affinity purification; TPR-like, tetratricopeptide repeat-like domain; WD-40, tryptophan-aspartic repeat motif; eIF4E, eukaryotic initiation factor 4E; snRNAs, small nuclear RNAs.

Gemin5 functional domains

Fig. 1

Gemin5 organization and conservation. Schematic representing the structural domains of Gemin5 protein. Gray bars denote the regions encompassing the WD40 domain, the TPR-like dimerization module, and the non-canonical RNA binding sites RBS1 and RBS2. Numbers denote amino acid positions. Two phosphoresidues, T897 (in TPR) and T1355 (in RBS1), were identified in the Gemin5_845-1508 protein produced in HEK293 cells and are depicted in red. Alignment of Gemin5 sequences spanning parts of the TPR-like and RBS1 domains encompassing the identified phosphopeptides. Residues are colored according to their degree of conservation, from blue to white. The detected phosphopeptides (P-threonine depicted in red) are indicated over the sequences. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Translation activity of phosphorylation defective mutants. A) Schematic representation of the bicistronic RNA expressed from the construct CMVpBIC. HEK293 cells expressing Xpress-G5_845-1508-WT or the mutants T897A, T897E (B) or T1355A, and T1355E (C) were cotransfected with the CMVpBIC bicistronic plasmid to monitor IRES- and cap-dependent translation. Protein expression was monitored by WB using anti-Gemin5, and tubulin was used as the loading control. In all cases, values are normalized to cells expressing Xpress-G5_845-1508-WT conducted side by side. Values represent the mean ± SEM obtained in three independent assays. Asterisks denote P-values (**P < 0.01, ***P < 0.001), “ns” denotes not statistically significant.

Fig. 3

Structural modeling of T897 modifications. A-D) Cartoon representations of the TPR-like dimer (PDB ID 6RNQ) centered on the position of residue 897 with subunits colored in blue and orange. The thicker blue dashed lines represent a flexible loop between helices 2 and 3 that was not observed in the crystal structure. A) Structure of the WT protein with T897 (in yellow) and nearby residues shown in sticks. The side chain of T897 makes a H-bond (shown as a thin dashed line) with the carbonyl O of L893, being both residues in helix 4. B, C) Modeling of the mutations T897A (B) and T897E (C) on the WT crystal structure. Both mutations cause the loss of the H-bond, and the glutamate causes a steric clash of the negatively charged side chain with surrounding hydrophobic residues. In the rotamer drawn for the glutamate in panel C, the negatively charged side chain of T897E collides with the side chain of V887, and other possible rotamers would also crash against nearby elements. D) Modeling of the phosphorylation of T897 on the crystal structure of the WT protein. As for mutation T897E, the phosphate falls in a hydrophobic environment, and thus, this posttranslational modification is concomitant to local structural rearrangements. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Mutations T897A and T897E do not affect protein stability. HEK293 cells expressing the wild-type version of G5_845-1508, side by side with mutants T897A and T897E for 12 h were treated (+) or not (−) with cycloheximide (CHX) for additional 16 h. Samples were taken at 0, 12, and 16 h post–CHX treatment. The intensity of each protein at the indicated time was determined by WB. Bars represent the protein intensity (mean ± SEM) of three independent experiments relative to time 0 in each case. The differences between values were not significant in all cases.

Fig. 5

The protein interactome is differentially affected by T897A and T897E. Venn diagrams showing the overlap (%) of proteins identified for the WT (blue), T897A (pink), and T897E (violet). STRING protein–protein networks associated with G5_845-1508 WT, T897A, and T897E depicting proteins identified in all the cases (grey border) or exclusive factors identified in G5_845-1508 WT (blue border), T897A (pink border), and T897E (violet border). Interactors with WD-score > 1 are denoted by colored circles as above. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Protein networks associated with Gemin5 T897 mutants. A) Gene ontology analysis of the proteins associated with the WT and mutant proteins, according to P-value, colored as in Fig. 5. B) Heatmap representing differential interactions of representative RBPs with mutants T897A and T897E exclusively included in Regulation of translation and RNA localization GO groups according to their Z-score.

Fig. 7

Diagram of Gemin5 phosphorylation sites and naturally variant residues. Red triangles depict the position of the phosphoresidues deposited in phosphosite (www.phosphosite.org) on the functional domains of the protein. Blue arrowheads denote the position of mutations found in patients with neurological disease. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)