Emerging roles of Gemin5: From snRNPs assembly to translation control

RNA-binding proteins (RBPs) play a pivotal role in the lifespan of RNAs. The disfunction of RBPs is frequently the cause of cell disorders which are incompatible with life. Furthermore, the ordered assembly of RBPs and RNAs in ribonucleoprotein (RNP) particles determines the function of biological complexes, as illustrated by the survival of the motor neuron (SMN) complex. Defects in the SMN complex assembly causes spinal muscular atrophy (SMA), an infant invalidating disease. This multi-subunit chaperone controls the assembly of small nuclear ribonucleoproteins (snRNPs), which are the critical components of the splicing machinery. However, the functional and structural characterization of individual members of the SMN complex, such as SMN, Gemin3, and Gemin5, have accumulated evidence for the additional roles of these proteins, unveiling their participation in other RNA-mediated events. In particular, Gemin5 is a multidomain protein that comprises tryptophan-aspartic acid (WD) repeat motifs at the N-terminal region, a dimerization domain at the middle region, and a non-canonical RNA-binding domain at the C-terminal end of the protein. Beyond small nuclear RNA (snRNA) recognition, Gemin5 interacts with a selective group of mRNA targets in the cell environment and plays a key role in reprogramming translation depending on the RNA partner and the cellular conditions. Here, we review recent studies on the SMN complex, with emphasis on the individual components regarding their involvement in cellular processes critical for cell survivalBFU2017-84492-R, Comunidad de Madrid (B2017/BMD3770

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 proteinsThis work was supported by grants PID2020-115096RB-I00 (MICIN), B2017/BMD-3770 (cofinanced by Autonomous Community of Madrid and FEDER funds), and an Institutional grant from Fundación Ramón Arece

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 protei

Gemin5, a multifunctional RNA-binding protein involved in translation control

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 05-11-2020Esta tesis tiene embargado el acceso al texto completo hasta el 05-05-2022RNA-binding proteins (RBPs) regulate all steps of RNA metabolism including protein synthesis, which is an essential stage of gene expression. Gemin5 is an RBP involved in the assembly of splicing machinery, gene expression reprogramming, and translation control. This protein is organized in domains that interact with distinct cellular targets. The N-terminal region comprises 14WD repeat motifs (residues 1-739) that recognize small nuclear RNAs, and bind to the ribosome, while the C-terminal end bears a bipartite RNA-binding site, designated as RBS1 and RBS2 (residues 1287-1508). RBS1 is a non-canonical RNA-binding domain (RBD) that recognizes a sequence located within the coding region of Gemin5 mRNA, termed H12. Remarkably, Gemin5 stimulates its own translation through direct interaction between RBS1 and H12, providing a regulatory feedback loop that allows to fine-tune its cellular levels. In this thesis, we have conducted an RNA-protein coevolution study that predicted the coevolving pairs between H12 and RBS1. The H12 RNA shows a stable secondary structure consisting of two stem-loops, SL1 and SL2. SL1 contains all coevolving nucleotides. Analysis of the RNAprotein interactions in vitro confirmed that SL1 is the main motif recognized by RBS1 in H12. Biochemical and functional assays revealed that the PXSS motif in RBS1 is involved in the recognition of H12 through SL1. This study paves the way for the recognition of non-canonical RBDs carrying similar motifs. In addition, we have solved the structure of a tetratricopeptide (TPR)-like domain in the middle region of Gemin5 (residues 845-1097) that self-assembles into a compact canoe-shaped dimer and acts as a protein-protein interaction platform. Despite the tight association of this dimer, a single point mutation (A951E) at the closest intersubunit distance was sufficient to destabilize it. We have proven that this domain drives the dimerization of Gemin5 in living cells. These data indicate that the recruitment of the endogenous Gemin5 by p85, a viral cleavage fragment that includes the TPR and the RBS domains, might prevent the role of Gemin5 in translation control. The presence of distinct RBDs in Gemin5 facilitates the interaction with a wide variety of cellular mRNAs. In order to identify the mRNAs selectively translated by Gemin5, we performed a genome-wide analysis of polysome-associated mRNAs in Gemin5-depleted cells relative to control cells. Among the transcripts displaying enhanced association to polysomes, there are mRNAs encoding for ribosomal proteins, histones, mitochondrial ribosomal proteins, proteins of cytochrome P450, and Sm and like-Sm proteins. Remarkably, we have found that Gemin5 stimulates translation of the TOP mRNA family, which includes all ribosomal protein transcripts. Finally, the relevance of RNA structure for RNA-protein interactions prompted us to explore a new method based on tRNA scaffold to identify RBPs associated to RNA structural elements. This novel approach will allow the identification of RBPs critical in different steps of RNA metabolis

Emerging Roles of Gemin5: From snRNPs Assembly to Translation Control

RNA-binding proteins (RBPs) play a pivotal role in the lifespan of RNAs. The disfunction of RBPs is frequently the cause of cell disorders which are incompatible with life. Furthermore, the ordered assembly of RBPs and RNAs in ribonucleoprotein (RNP) particles determines the function of biological complexes, as illustrated by the survival of the motor neuron (SMN) complex. Defects in the SMN complex assembly causes spinal muscular atrophy (SMA), an infant invalidating disease. This multi-subunit chaperone controls the assembly of small nuclear ribonucleoproteins (snRNPs), which are the critical components of the splicing machinery. However, the functional and structural characterization of individual members of the SMN complex, such as SMN, Gemin3, and Gemin5, have accumulated evidence for the additional roles of these proteins, unveiling their participation in other RNA-mediated events. In particular, Gemin5 is a multidomain protein that comprises tryptophan-aspartic acid (WD) repeat motifs at the N-terminal region, a dimerization domain at the middle region, and a non-canonical RNA-binding domain at the C-terminal end of the protein. Beyond small nuclear RNA (snRNA) recognition, Gemin5 interacts with a selective group of mRNA targets in the cell environment and plays a key role in reprogramming translation depending on the RNA partner and the cellular conditions. Here, we review recent studies on the SMN complex, with emphasis on the individual components regarding their involvement in cellular processes critical for cell survival.MINECO (grant BFU2017-84492-R), Comunidad de Madrid (B2017/BMD-3770) and an Institutional grant from Fundación Ramón Areces

Rna-binding proteins at the host-pathogen interface targeting viral regulatory elements

Viral RNAs contain the information needed to synthesize their own proteins, to replicate, and to spread to susceptible cells. However, due to their reduced coding capacity RNA viruses rely on host cells to complete their multiplication cycle. This is largely achieved by the concerted action of regulatory structural elements on viral RNAs and a subset of host proteins, whose dedicated function across all stages of the infection steps is critical to complete the viral cycle. Importantly, not only the RNA sequence but also the RNA architecture imposed by the presence of specific structural domains mediates the interaction with host RNA-binding proteins (RBPs), ultimately affecting virus multiplication and spreading. In marked difference with other biological systems, the genome of positive strand RNA viruses is also the mRNA. Here we focus on distinct types of positive strand RNA viruses that differ in the regulatory elements used to promote translation of the viral RNA, as well as in the mechanisms used to evade the series of events connected to antiviral response, including translation shutoff induced in infected cells, assembly of stress granules, and trafficking stress.BFU2017-84492-R (MINECO), B2017/BMD-3770 (cofinanced by Autonomous Community of Madrid and FEDER funds), and an Institutional grant from Fundación Ramón Arece

The RBS1 domain of Gemin5 is intrinsically unstructured and interacts with RNA through conserved Arg and aromatic residues

11 pags., 6 figs., 1 tab.Gemin5 is a multifaceted RNA-binding protein that comprises distinct structural domains, including a WD40 and TPR-like for which the X-ray structure is known. In addition, the protein contains a non-canonical RNA-binding domain (RBS1) towards the C-terminus. To understand the RNA binding features of the RBS1 domain, we have characterized its structural characteristics by solution NMR linked to RNA-binding activity. Here we show that a short version of the RBS1 domain that retains the ability to interact with RNA is predominantly unfolded even in the presence of RNA. Furthermore, an exhaustive mutational analysis indicates the presence of an evolutionarily conserved motif enriched in R, S, W, and H residues, necessary to promote RNA-binding via ¿-¿ interactions. The combined results of NMR and RNA-binding on wild-type and mutant proteins highlight the importance of aromatic and arginine residues for RNA recognition by RBS1, revealing that the net charge and the ¿-amino acid density of this region of Gemin5 are key factors for RNA recognitionThis work was supported by grants from MINECO (CTQ2018-84371) to JMP-C, BFU2017-84492-R (to EMS), and B2017/BMD-3770 cofinanced by Autonomous Community of Madrid and FEDER funds to EMS and JMP-C

Structural basis for the dimerization of Gemin5 and its role in protein recruitment and translation control

In all organisms, a selected type of proteins accomplishes critical roles in cellular processes that govern gene expression. The multifunctional protein Gemin5 cooperates in translation control and ribosome binding, besides acting as the RNA-binding protein of the survival of motor neuron (SMN) complex. While these functions reside on distinct domains located at each end of the protein, the structure and function of the middle region remained unknown. Here, we solved the crystal structure of an extended tetratricopeptide (TPR)-like domain in human Gemin5 that self-Assembles into a previously unknown canoe-shaped dimer. We further show that the dimerization module is functional in living cells driving the interaction between the viral-induced cleavage fragment p85 and the full-length Gemin5, which anchors splicing and translation members. Disruption of the dimerization surface by a point mutation in the TPR-like domain prevents this interaction and also abrogates translation enhancement induced by p85. The characterization of this unanticipated dimerization domain provides the structural basis for a role of the middle region of Gemin5 as a central hub for protein-protein interactions.MINECO [BFU2016-80570-R, BFU2017-84492-R, RTI2018-098084-B-I00, AEI/FEDER, UE]; Comunidad de Madrid [B2017/BMD-3770]; Institutional grant from Fundación Ramón Arece

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

10 páginas, 7 figurasGemin5 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.This work was supported by the Ministerio de Ciencia e Innovación (MICIN) and Fondo Europeo de Desarrollo Regional (AEI/FEDER UE) (PID2020-115096RB-I00), Comunidad de Madrid (B2017/BMD-3770) and an Institutional grant from Fundación Ramón Areces. FdC is a postdoctoral fellow of the Generalitat Valenciana (APOSTD 2021).Peer reviewe