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

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.

Structural basis for Gemin5 decamer-mediated mRNA binding.

Gemin5 in the Survival Motor Neuron (SMN) complex serves as the RNA-binding protein to deliver small nuclear RNAs (snRNAs) to the small nuclear ribonucleoprotein Sm complex via its N-terminal WD40 domain. Additionally, the C-terminal region plays an important role in regulating RNA translation by directly binding to viral RNAs and cellular mRNAs. Here, we present the three-dimensional structure of the Gemin5 C-terminal region, which adopts a homodecamer architecture comprised of a dimer of pentamers. By structural analysis, mutagenesis, and RNA-binding assays, we find that the intact pentamer/decamer is critical for the Gemin5 C-terminal region to bind cognate RNA ligands and to regulate mRNA translation. The Gemin5 high-order architecture is assembled via pentamerization, allowing binding to RNA ligands in a coordinated manner. We propose a model depicting the regulatory role of Gemin5 in selective RNA binding and translation. Therefore, our work provides insights into the SMN complex-independent function of Gemin5.

Gemin5-dependent RNA association with polysomes enables selective translation of ribosomal and histone mRNAs.

Selective translation allows to orchestrate the expression of specific proteins in response to different signals through the concerted action of cis-acting elements and RNA-binding proteins (RBPs). Gemin5 is a ubiquitous RBP involved in snRNP assembly. In addition, Gemin5 regulates translation of different mRNAs through apparently opposite mechanisms of action. Here, we investigated the differential function of Gemin5 in translation by identifying at a genome-wide scale the mRNAs associated with polysomes. Among the mRNAs showing Gemin5-dependent enrichment in polysomal fractions, we identified a selective enhancement of specific transcripts. Comparison of the targets previously identified by CLIP methodologies with the polysome-associated transcripts revealed that only a fraction of the targets was enriched in polysomes. Two different subsets of these mRNAs carry unique cis-acting regulatory elements, the 5' terminal oligopyrimidine tracts (5'TOP) and the histone stem-loop (hSL) structure at the 3' end, respectively, encoding ribosomal proteins and histones. RNA-immunoprecipitation (RIP) showed that ribosomal and histone mRNAs coprecipitate with Gemin5. Furthermore, disruption of the TOP motif impaired Gemin5-RNA interaction, and functional analysis showed that Gemin5 stimulates translation of mRNA reporters bearing an intact TOP motif. Likewise, Gemin5 enhanced hSL-dependent mRNA translation. Thus, Gemin5  promotes polysome association of only a subset of its targets, and as a consequence, it favors translation of the ribosomal and the histone mRNAs. Together, the results presented here unveil Gemin5 as a novel translation regulator of mRNA subsets encoding proteins involved in fundamental cellular processes.

Functional and structural deficiencies of Gemin5 variants associated with neurological disorders.

Dysfunction of RNA-binding proteins is often linked to a wide range of human disease, particularly with neurological conditions. Gemin5 is a member of the survival of the motor neurons (SMN) complex, a ribosome-binding protein and a translation reprogramming factor. Recently, pathogenic mutations in Gemin5 have been reported, but the functional consequences of these variants remain elusive. Here, we report functional and structural deficiencies associated with compound heterozygosity variants within the Gemin5 gene found in patients with neurodevelopmental disorders. These clinical variants are located in key domains of Gemin5, the tetratricopeptide repeat (TPR)-like dimerization module and the noncanonical RNA-binding site 1 (RBS1). We show that the TPR-like variants disrupt protein dimerization, whereas the RBS1 variant confers protein instability. All mutants are defective in the interaction with protein networks involved in translation and RNA-driven pathways. Importantly, the TPR-like variants fail to associate with native ribosomes, hampering its involvement in translation control and establishing a functional difference with the wild-type protein. Our study provides insights into the molecular basis of disease associated with malfunction of the Gemin5 protein.

Picornavirus translation strategies.

The genome of viruses classified as picornaviruses consists of a single monocistronic positive strand RNA. The coding capacity of these RNA viruses is rather limited, and thus, they rely on the cellular machinery for their viral replication cycle. Upon the entry of the virus into susceptible cells, the viral RNA initially competes with cellular mRNAs for access to the protein synthesis machinery. Not surprisingly, picornaviruses have evolved specialized strategies that successfully allow the expression of viral gene products, which we outline in this review. The main feature of all picornavirus genomes is the presence of a heavily structured RNA element on the 5´UTR, referred to as an internal ribosome entry site (IRES) element, which directs viral protein synthesis as well and, consequently, triggers the subsequent steps required for viral replication. Here, we will summarize recent studies showing that picornavirus IRES elements consist of a modular structure, providing sites of interaction for ribosome subunits, eIFs, and a selective group of RNA-binding proteins.