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1.
Comput Struct Biotechnol J ; 20: 6182-6191, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36420152

RESUMEN

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.

2.
Nat Commun ; 13(1): 5166, 2022 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-36056043

RESUMEN

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.


Asunto(s)
ARN Nuclear Pequeño , Ribonucleoproteínas Nucleares Pequeñas , Ligandos , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas del Complejo SMN/metabolismo
3.
Cell Mol Life Sci ; 79(9): 490, 2022 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-35987821

RESUMEN

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.


Asunto(s)
Histonas , ARN , Histonas/genética , Histonas/metabolismo , Polirribosomas/metabolismo , Biosíntesis de Proteínas , ARN/metabolismo , ARN Mensajero/metabolismo
4.
Proc Natl Acad Sci U S A ; 119(35): e2204752119, 2022 08 30.
Artículo en Inglés | MEDLINE | ID: mdl-35994673

RESUMEN

p38γ and p38δ (p38γ/p38δ) regulate inflammation, in part by controlling tumor progression locus 2 (TPL2) expression in myeloid cells. Here, we demonstrate that TPL2 protein levels are dramatically reduced in p38γ/p38δ-deficient (p38γ/δ-/-) cells and tissues without affecting TPL2 messenger ribonucleic acid (mRNA) expression. We show that p38γ/p38δ posttranscriptionally regulates the TPL2 amount at two different levels. p38γ/p38δ interacts with the TPL2/A20 Binding Inhibitor of NF-κB2 (ABIN2)/Nuclear Factor κB1p105 (NF-κB1p105) complex, increasing TPL2 protein stability. Additionally, p38γ/p38δ regulates TPL2 mRNA translation by modulating the repressor function of TPL2 3' Untranslated region (UTR) mediated by its association with aconitase-1 (ACO1). ACO1 overexpression in wild-type cells increases the translational repression induced by TPL2 3'UTR and severely decreases TPL2 protein levels. p38δ binds to ACO1, and p38δ expression in p38γ/δ-/- cells fully restores TPL2 protein to wild-type levels by reducing the translational repression of TPL2 mRNA. This study reveals a unique mechanism of posttranscriptional regulation of TPL2 expression, which given its central role in innate immune response, likely has great relevance in physiopathology.


Asunto(s)
Aconitato Hidratasa , Quinasas Quinasa Quinasa PAM , Proteína Quinasa 12 Activada por Mitógenos , Proteína Quinasa 13 Activada por Mitógenos , Aconitato Hidratasa/genética , Aconitato Hidratasa/metabolismo , Regulación de la Expresión Génica , Inmunidad Innata , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Proteína Quinasa 12 Activada por Mitógenos/genética , Proteína Quinasa 12 Activada por Mitógenos/metabolismo , Proteína Quinasa 13 Activada por Mitógenos/genética , Proteína Quinasa 13 Activada por Mitógenos/metabolismo , ARN Mensajero/genética
5.
Life Sci Alliance ; 5(7)2022 07.
Artículo en Inglés | MEDLINE | ID: mdl-35393353

RESUMEN

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.


Asunto(s)
Enfermedades del Sistema Nervioso , Proteínas de Unión al ARN , Ribosomas , Humanos , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/metabolismo , ARN/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Proteínas del Complejo SMN/genética , Proteínas del Complejo SMN/metabolismo
6.
Front Cell Dev Biol ; 10: 783762, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35295849

RESUMEN

The hereditary ataxias are a heterogenous group of disorders with an increasing number of causative genes being described. Due to the clinical and genetic heterogeneity seen in these conditions, the majority of such individuals endure a diagnostic odyssey or remain undiagnosed. Defining the molecular etiology can bring insights into the responsible molecular pathways and eventually the identification of therapeutic targets. Here, we describe the identification of biallelic variants in the GEMIN5 gene among seven unrelated families with nine affected individuals presenting with spastic ataxia and cerebellar atrophy. GEMIN5, an RNA-binding protein, has been shown to regulate transcription and translation machinery. GEMIN5 is a component of small nuclear ribonucleoprotein (snRNP) complexes and helps in the assembly of the spliceosome complexes. We found that biallelic GEMIN5 variants cause structural abnormalities in the encoded protein and reduce expression of snRNP complex proteins in patient cells compared with unaffected controls. Finally, knocking out endogenous Gemin5 in mice caused early embryonic lethality, suggesting that Gemin5 expression is crucial for normal development. Our work further expands on the phenotypic spectrum associated with GEMIN5-related disease and implicates the role of GEMIN5 among patients with spastic ataxia, cerebellar atrophy, and motor predominant developmental delay.

7.
FEBS Open Bio ; 12(6): 1125-1141, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35313388

RESUMEN

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.


Asunto(s)
Picornaviridae , Sitios Internos de Entrada al Ribosoma/genética , Picornaviridae/genética , Picornaviridae/metabolismo , ARN Mensajero/genética , ARN Viral/genética , Replicación Viral
8.
RNA Biol ; 18(sup1): 496-506, 2021 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-34424823

RESUMEN

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.


Asunto(s)
Arginina/metabolismo , Motivos de Unión al ARN , ARN/química , ARN/metabolismo , Proteínas del Complejo SMN/química , Proteínas del Complejo SMN/metabolismo , Triptófano/metabolismo , Secuencia de Aminoácidos , Arginina/química , Arginina/genética , Sitios de Unión , Humanos , Modelos Moleculares , Unión Proteica , ARN/genética , Proteínas del Complejo SMN/genética , Homología de Secuencia , Triptófano/química , Triptófano/genética
9.
Viruses ; 13(6)2021 05 21.
Artículo en Inglés | MEDLINE | ID: mdl-34064059

RESUMEN

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.


Asunto(s)
Interacciones Huésped-Patógeno , Virus ARN/fisiología , ARN Viral/genética , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Elementos de Respuesta , Transporte Biológico , Gránulos Citoplasmáticos/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Biosíntesis de Proteínas , Infecciones por Virus ARN/metabolismo , Infecciones por Virus ARN/virología , ARN Viral/química , Estrés Fisiológico , Vesículas Transportadoras/metabolismo , Replicación Viral
10.
Methods Mol Biol ; 2323: 109-119, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34086277

RESUMEN

RNA motifs guide the interaction with specific proteins leading to the assembly of ribonucleoprotein complexes that perform key functions in cellular processes. Internal ribosome entry site (IRES) elements are organized in structural domains that determine internal initiation of translation. In this chapter we describe a pull-down assay using streptavidin-aptamer tagged RNAs that combines RNA structure-dependent protein isolation with proteomic analysis to identify novel interactors recognizing RNA structural domains. This approach takes advantage of tRNA-scaffold guided expression, allowing the identification of factors belonging to networks involved in RNA and protein metabolism.


Asunto(s)
Motivos de Nucleótidos , Proteínas de Unión al ARN/aislamiento & purificación , Aptámeros de Nucleótidos , Electroforesis en Gel de Poliacrilamida , Humanos , Sitios Internos de Entrada al Ribosoma , Espectrometría de Masas , Motivos de Nucleótidos/genética , Biosíntesis de Proteínas , Proteómica/métodos , ARN/aislamiento & purificación , ARN/metabolismo , ARN de Transferencia/biosíntesis , ARN de Transferencia/química , Proteínas de Unión al ARN/metabolismo , Estreptavidina , Especificidad por Sustrato
11.
Int J Mol Sci ; 21(11)2020 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-32485878

RESUMEN

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.


Asunto(s)
Neuronas Motoras/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas del Complejo SMN/metabolismo , Animales , Humanos , Neuronas Motoras/patología , Biosíntesis de Proteínas , Multimerización de Proteína , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/genética , Ribosomas/metabolismo , Proteínas del Complejo SMN/química , Proteínas del Complejo SMN/genética
12.
RNA Biol ; 17(9): 1331-1341, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32476560

RESUMEN

Regulation of protein synthesis is an essential step of gene expression. This process is under the control of cis-acting RNA elements and trans-acting factors. Gemin5 is a multifunctional RNA-binding protein organized in distinct domains. The protein bears a non-canonical RNA-binding site, designated RBS1, at the C-terminal end. Among other cellular RNAs, the RBS1 region recognizes a sequence located within the coding region of Gemin5 mRNA, termed H12. Expression of RBS1 stimulates translation of RNA reporters carrying the H12 sequence, counteracting the negative effect of Gemin5 on global protein synthesis. A computational analysis of RBS1 protein and H12 RNA variability across the evolutionary scale predicts coevolving pairs of amino acids and nucleotides. RBS1 footprint and gel-shift assays indicated a positive correlation between the identified coevolving pairs and RNA-protein interaction. The coevolving residues of RBS1 contribute to the recognition of stem-loop SL1, an RNA structural element of H12 that contains the coevolving nucleotides. Indeed, RBS1 proteins carrying substitutions on the coevolving residues P1297 or S1299S1300, drastically reduced SL1-binding. Unlike the wild type RBS1 protein, expression of these mutant proteins in cells failed to enhance translation stimulation of mRNA reporters carrying the H12 sequence. Therefore, the PXSS motif within the RBS1 domain of Gemin5 and the RNA structural motif SL1 of its mRNA appears to play a key role in fine-tuning the expression level of this essential protein.


Asunto(s)
Sitios de Unión , Motivos de Unión al ARN , Proteínas de Unión al ARN/química , ARN/química , Proteínas del Complejo SMN/química , Secuencia de Aminoácidos , Evolución Biológica , Secuencia Conservada , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , ARN/genética , ARN/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas del Complejo SMN/metabolismo
13.
Nucleic Acids Res ; 48(2): 788-801, 2020 01 24.
Artículo en Inglés | MEDLINE | ID: mdl-31799608

RESUMEN

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.


Asunto(s)
Biosíntesis de Proteínas , Proteínas de Unión al ARN/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Proteínas del Complejo SMN/genética , Humanos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Multimerización de Proteína/genética , Ribonucleoproteínas Nucleares Pequeñas/química , Proteínas del Complejo SMN/química
14.
Bioessays ; 41(4): e1800241, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30919488

RESUMEN

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.


Asunto(s)
Biosíntesis de Proteínas , ARN/metabolismo , Proteínas del Complejo SMN/química , Proteínas del Complejo SMN/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , Modelos Biológicos , Dominios Proteicos , ARN/química
15.
Life Sci Alliance ; 2(1)2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30655362

RESUMEN

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.


Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Virus de la Fiebre Aftosa/metabolismo , Sitios Internos de Entrada al Ribosoma , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Factores de Ribosilacion-ADP/genética , Animales , Retículo Endoplásmico/metabolismo , Fiebre Aftosa/virología , Silenciador del Gen , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Unión Proteica , Dominios Proteicos , Proteómica/métodos , Caperuzas de ARN , Transfección , Proteínas de Unión al GTP rab1/genética
16.
Open Biol ; 8(11)2018 11 28.
Artículo en Inglés | MEDLINE | ID: mdl-30487301

RESUMEN

Beyond the general cap-dependent translation initiation, eukaryotic organisms use alternative mechanisms to initiate protein synthesis. Internal ribosome entry site (IRES) elements are cis-acting RNA regions that promote internal initiation of translation using a cap-independent mechanism. However, their lack of primary sequence and secondary RNA structure conservation, as well as the diversity of host factor requirement to recruit the ribosomal subunits, suggest distinct types of IRES elements. In spite of this heterogeneity, conserved motifs preserve sequences impacting on RNA structure and RNA-protein interactions important for IRES-driven translation. This conservation brings the question of whether IRES elements could consist of basic building blocks, which upon evolutionary selection result in functional elements with different properties. Although RNA-binding proteins (RBPs) perform a crucial role in the assembly of ribonucleoprotein complexes, the versatility and plasticity of RNA molecules, together with their high flexibility and dynamism, determines formation of macromolecular complexes in response to different signals. These properties rely on the presence of short RNA motifs, which operate as modular entities, and suggest that decomposition of IRES elements in short modules could help to understand the different mechanisms driven by these regulatory elements. Here we will review evidence suggesting that model IRES elements consist of the combination of short modules, providing sites of interaction for ribosome subunits, eIFs and RBPs, with implications for definition of criteria to identify novel IRES-like elements genome wide.


Asunto(s)
Sitios Internos de Entrada al Ribosoma/fisiología , Motivos de Nucleótidos/fisiología , Iniciación de la Cadena Peptídica Traduccional/fisiología , Animales , Humanos , Proteínas de Unión al ARN/metabolismo
17.
Hum Mutat ; 39(10): 1338-1343, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30011114

RESUMEN

McArdle disease is a disorder of muscle glycogen metabolism caused by mutations in the PYGM gene, encoding for the muscle-specific isoform of glycogen phosphorylase (M-GP). The activity of this enzyme is completely lost in patients' muscle biopsies, when measured with a standard biochemical test which, does not allow to determine M-GP protein levels. We aimed to determine M-GP protein levels in the muscle of McArdle patients, by studying biopsies of 40 patients harboring a broad spectrum of PYGM mutations and 22 controls. Lack of M-GP protein was found in muscle in the vast majority (95%) of patients, irrespective of the PYGM genotype, including those carrying missense mutations, with few exceptions. M-GP protein biosynthesis is not being produced by PYGM mutations inducing premature termination codons (PTC), neither by most PYGM missense mutations. These findings explain the lack of PYGM genotype-phenotype correlation and have important implications for the design of molecular-based therapeutic approaches.


Asunto(s)
Estudios de Asociación Genética , Enfermedad del Almacenamiento de Glucógeno Tipo V/genética , Mutación Missense , Adolescente , Adulto , Anciano , Alelos , Biopsia , Femenino , Genotipo , Glucógeno Fosforilasa de Forma Muscular/genética , Enfermedad del Almacenamiento de Glucógeno Tipo V/diagnóstico , Humanos , Masculino , Persona de Mediana Edad , Isoformas de Proteínas , Adulto Joven
18.
Nucleic Acids Res ; 46(14): 7339-7353, 2018 08 21.
Artículo en Inglés | MEDLINE | ID: mdl-29771365

RESUMEN

Gemin5 is a predominantly cytoplasmic protein that downregulates translation, beyond controlling snRNPs assembly. The C-terminal region harbors a non-canonical RNA-binding site consisting of two domains, RBS1 and RBS2, which differ in RNA-binding capacity and the ability to modulate translation. Here, we show that these domains recognize distinct RNA targets in living cells. Interestingly, the most abundant and exclusive RNA target of the RBS1 domain was Gemin5 mRNA. Biochemical and functional characterization of this target demonstrated that RBS1 polypeptide physically interacts with a predicted thermodynamically stable stem-loop upregulating mRNA translation, thereby counteracting the negative effect of Gemin5 protein on global protein synthesis. In support of this result, destabilization of the stem-loop impairs the stimulatory effect on translation. Moreover, RBS1 stimulates translation of the endogenous Gemin5 mRNA. Hence, although the RBS1 domain downregulates global translation, it positively enhances translation of RNA targets carrying thermodynamically stable secondary structure motifs. This mechanism allows fine-tuning the availability of Gemin5 to play its multiple roles in gene expression control.


Asunto(s)
Retroalimentación Fisiológica , Biosíntesis de Proteínas , ARN/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Sitios de Unión/genética , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Células K562 , Unión Proteica , Dominios Proteicos , ARN/química , ARN/metabolismo , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Proteínas del Complejo SMN
19.
Sci Rep ; 8(1): 5545, 2018 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-29615727

RESUMEN

Internal ribosome entry site (IRES) elements are RNA regions that recruit the translation machinery internally. Here we investigated the conformational changes and RNA dynamics of a picornavirus IRES upon incubation with distinct ribosomal fractions. Differential SHAPE analysis of the free RNA showed that nucleotides reaching the final conformation on long timescales were placed at domains 4 and 5, while candidates for long-range interactions were located in domain 3. Salt-washed ribosomes induced a fast RNA local flexibility modification of domains 2 and 3, while ribosome-associated factors changed domains 4 and 5. Consistent with this, modeling of the three-dimensional RNA structure indicated that incubation of the IRES with native ribosomes induced a local rearrangement of the apical region of domain 3, and a reorientation of domains 4 and 5. Furthermore, specific motifs within domains 2 and 3 showed a decreased flexibility upon incubation with ribosomal subunits in vitro, and presence of the IRES enhanced mRNA association to the ribosomal subunits in whole cell lysates. The finding that RNA modules can provide direct IRES-ribosome interaction suggests that linking these motifs to additional sequences able to recruit trans-acting factors could be useful to design synthetic IRESs with novel activities.


Asunto(s)
Virus de la Fiebre Aftosa/genética , Sitios Internos de Entrada al Ribosoma/genética , Biosíntesis de Proteínas , ARN Viral/química , ARN Viral/genética , Ribosomas/metabolismo , Secuencia de Bases , Virus de la Fiebre Aftosa/crecimiento & desarrollo , Virus de la Fiebre Aftosa/metabolismo , Conformación de Ácido Nucleico , ARN Mensajero/genética , ARN Mensajero/metabolismo , Subunidades Ribosómicas Grandes de Eucariotas/genética , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/genética
20.
Front Microbiol ; 8: 2629, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29354113

RESUMEN

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.

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