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Rosario Francisco‐Velilla

Centro de Biología Molecular Severo Ochoa

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Encarnación Martı́nez-Salas

Universidad Autónoma de Madrid

Azman Embarc‐Buh

MRC University of Glasgow Centre for Virus Research

Javier Fernández-Chamorro

Universidad Autónoma de Madrid

Salvador Abellan

Centro de Biología Molecular Severo Ochoa

Gloria Lozano

Centro de Biología Molecular Severo Ochoa

Santiago Ramón‐Maiques

Instituto de Biomedicina de Valencia

Jorge Ramajo

Centro de Biología Molecular Severo Ochoa

Miguel Remacha

Universidad Autónoma de Madrid

Rosa Díaz-Toledano

Centro de Biología Molecular Severo Ochoa

Francisco del Caño‐Ochoa

Instituto de Biomedicina de Valencia

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Consejo Superior de Investigaciones Científicas

Universidad Autónoma de Madrid

Centro de Biología Molecular Severo Ochoa

Instituto de Salud Carlos III

Centre for Biomedical Network Research on Rare Diseases

University of Science and Technology of China

Centro de Investigación Biomédica en Red

Instituto de Investigación de Enfermedades Raras

Hefei National Center for Physical Sciences at Nanoscale

Centro Nacional de Biotecnología

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Alternative splicing events driven by altered levels of GEMIN5 undergo translation

RNA Biology (2024)

Rosario Francisco‐Velilla Salvador Abellan Juan Antonio García-Martín Juan Carlos Oliveros Encarnación Martínez‐Salas

GEMIN5 is a multifunctional protein involved in various aspects of RNA biology, including biogenesis of snRNPs and translation control. Reduced levels of GEMIN5 confer a differential translation to selective groups of mRNAs, and biallelic variants reducing protein stability or inducing structural conformational changes are associated with neurological disorders. Here, we show that upregulation of GEMIN5 can be detrimental as it modifies the steady state of mRNAs and enhances alternative splicing (AS) events of genes involved in a broad range of cellular processes. RNA-Seq identification of the mRNAs associated with polysomes in cells with high levels of GEMIN5 revealed that a significant fraction of the differential AS events undergo translation. The association of mRNAs with polysomes was dependent on the type of AS event, being more frequent in the case of exon skipping. However, there were no major differences in the percentage of genes showing open-reading frame disruption. Importantly, differential AS events in mRNAs engaged in polysomes, eventually rendering non-functional proteins, encode factors controlling cell growth. The broad range of mRNAs comprising AS events engaged in polysomes upon GEMIN5 upregulation supports the notion that this multifunctional protein has evolved as a gene expression balancer, consistent with its dual role as a member of the SMN complex and as a modulator of protein synthesis, ultimately impinging on cell homoeostasis.

Polysome

10.1080/15476286.2024.2394755

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Citations (1)

Rab1b and ARF5 are novel RNA-binding proteins involved in FMDV IRES–driven RNA localization

Life Science Alliance (2019)

Javier Fernández-Chamorro Rosario Francisco‐Velilla Jorge Ramajo Encarnación Martínez‐Salas

Internal ribosome entry site (IRES) elements are organized in domains that guide internal initiation of translation. Here, we have combined proteomic and imaging analysis to study novel foot-and-mouth disease virus IRES interactors recognizing specific RNA structural subdomains. Besides known picornavirus IRES–binding proteins, we identified novel factors belonging to networks involved in RNA and protein transport. Among those, Rab1b and ARF5, two components of the ER-Golgi, revealed direct binding to IRES transcripts. However, whereas Rab1b stimulated IRES function, ARF5 diminished IRES activity. RNA-FISH studies revealed novel features of the IRES element. First, IRES-RNA formed clusters within the cell cytoplasm, whereas cap-RNA displayed disperse punctate distribution. Second, the IRES-driven RNA localized in close proximity with ARF5 and Rab1b, but not with the dominant-negative of Rab1b that disorganizes the Golgi. Thus, our data suggest a role for domain 3 of the IRES in RNA localization around ER-Golgi, a ribosome-rich cellular compartment.

10.26508/lsa.201800131

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Citations (16)

RNA-Binding Proteins Impacting on Internal Initiation of Translation

International Journal of Molecular Sciences (2013)

Encarnación Martínez‐Salas Gloria Lozano Javier Fernández-Chamorro Rosario Francisco‐Velilla Alfonso Galan

RNA-binding proteins (RBPs) are pivotal regulators of all the steps of gene expression. RBPs govern gene regulation at the post-transcriptional level by virtue of their capacity to assemble ribonucleoprotein complexes on certain RNA structural elements, both in normal cells and in response to various environmental stresses. A rapid cellular response to stress conditions is triggered at the step of translation initiation. Two basic mechanisms govern translation initiation in eukaryotic mRNAs, the cap-dependent initiation mechanism that operates in most mRNAs, and the internal ribosome entry site (IRES)-dependent mechanism activated under conditions that compromise the general translation pathway. IRES elements are cis-acting RNA sequences that recruit the translation machinery using a cap-independent mechanism often assisted by a subset of translation initiation factors and various RBPs. IRES-dependent initiation appears to use different strategies to recruit the translation machinery depending on the RNA organization of the region and the network of RBPs interacting with the element. In this review we discuss recent advances in understanding the implications of RBPs on IRES-dependent translation initiation.

Eukaryotic translation

Translational regulation

Eukaryotic initiation factor

10.3390/ijms141121705

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Citations (62)

Emerging Roles of Gemin5: From snRNPs Assembly to Translation Control

International Journal of Molecular Sciences (2020)

Encarnación Martínez‐Salas Azman Embarc‐Buh Rosario Francisco‐Velilla

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.

snRNP

Small nuclear RNA

Small nuclear ribonucleoprotein

Chaperone (clinical)

10.3390/ijms21113868

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Citations (35)

RNA–protein interaction methods to study viral IRES elements

Rosario Francisco‐Velilla Javier Fernández-Chamorro Gloria Lozano Rosa Díaz-Toledano Encarnación Martínez‐Salas

RNA Silencing

Nucleic acid structure

10.1016/j.ymeth.2015.06.023

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

Computational and Structural Biotechnology Journal (2022)

Rosario Francisco‐Velilla Azman Embarc‐Buh Salvador Abellan Francisco del Caño‐Ochoa Santiago Ramón‐Maiques

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.

Posttranslational modification

10.1016/j.csbj.2022.11.018

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Functional and structural deficiencies of Gemin5 variants associated with neurological disorders

Life Science Alliance (2022)

Rosario Francisco‐Velilla Azman Embarc‐Buh Francisco del Caño‐Ochoa Salvador Abellan Marçal Vilar

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.

10.26508/lsa.202201403

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Citations (11)

Insights into Structural and Mechanistic Features of Viral IRES Elements

Frontiers in Microbiology (2018)

Encarnación Martínez‐Salas Rosario Francisco‐Velilla Javier Fernández-Chamorro Azman M. Embarek

Internal ribosome entry site (IRES) elements are cis-acting RNA regions that promote internal initiation of protein synthesis using cap-independent mechanisms. However, distinct types of IRES elements present in the genome of various RNA viruses perform the same function despite lacking conservation of sequence and secondary RNA structure. Likewise, IRES elements differ in host factor requirement to recruit the ribosomal subunits. In spite of this diversity, evolutionarily conserved motifs in each family of RNA viruses preserve sequences impacting on RNA structure and RNA-protein interactions important for IRES activity. Indeed, IRES elements adopting remarkable different structural organizations contain RNA structural motifs that play an essential role in recruiting ribosomes, initiation factors and/or RNA-binding proteins using different mechanisms. Therefore, given that a universal IRES motif remains elusive, it is critical to understand how diverse structural motifs deliver functions relevant for IRES activity. This will be useful for understanding the molecular mechanisms beyond cap-independent translation, as well as the evolutionary history of these regulatory elements. Moreover, it could improve the accuracy to predict IRES-like motifs hidden in genome sequences. This review summarizes recent advances on the diversity and biological relevance of RNA structural motifs for viral IRES elements.

Nucleic acid structure

Structural motif

Eukaryotic translation

10.3389/fmicb.2017.02629

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Citations (130)

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

Figshare (2021)

Azman Embarc‐Buh Rosario Francisco‐Velilla Sergio Camero José Manuel Pérez‐Cañadillas Encarnación Martínez‐Salas

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 π-π 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 π-amino acid density of this region of Gemin5 are key factors for RNA recognition.

10.6084/m9.figshare.16415770.v2

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Impact of RNA–Protein Interaction Modes on Translation Control: The Versatile Multidomain Protein Gemin5

BioEssays (2019)

Rosario Francisco‐Velilla Azman Embarc‐Buh Encarnación Martínez‐Salas

The fate of cellular RNAs is largely dependent on their structural conformation, which determines the assembly of ribonucleoprotein (RNP) complexes. Consequently, RNA‐binding proteins (RBPs) play a pivotal role in the lifespan of RNAs. The advent of highly sensitive in cellulo approaches for studying RNPs reveals the presence of unprecedented RNA‐binding domains (RBDs). Likewise, the diversity of the RNA targets associated with a given RBP increases the code of RNA–protein interactions. Increasing evidence highlights the biological relevance of RNA conformation for recognition by specific RBPs and how this mutual interaction affects translation control. In particular, noncanonical RBDs present in proteins such as Gemin5, Roquin‐1, Staufen, and eIF3 eventually determine translation of selective targets. Collectively, recent studies on RBPs interacting with RNA in a structure‐dependent manner unveil new pathways for gene expression regulation, reinforcing the pivotal role of RNP complexes in genome decoding.

10.1002/bies.201800241

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Citations (21)