Role and dynamics of the ribosomal protein p0 and its related trans-acting factor Mrt4 during ribosome assembly in Saccharomyces cerevisiae

Mrt4 is a nucleolar component of the ribosome assembly machinery that shares notable similarity and competes for binding to the 25S rRNA GAR domain with the ribosomal protein P0. Here, we show that loss of function of either P0 or Mrt4 results in a deficit in 60S subunits, which is apparently due to impaired rRNA processing of 27S precursors. Mrt4, which shuttles between the nucleus and the cytoplasm, defines medium pre-60S particles. In contrast, P0 is absent from medium but present in late/cytoplasmic pre-60S complexes. The absence of Mrt4 notably increased the amount of P0 in nuclear Nop7–TAP complexes and causes P0 assembly to medium pre-60S particles. Upon P0 depletion, Mrt4 is relocated to the cytoplasm within aberrant 60S subunits. We conclude that Mrt4 controls the position and timing of P0 assembly. In turn, P0 is required for the release of Mrt4 and exchanges with this factor at the cytoplasm. Our results also suggest other P0 assembly alternatives

Deconstructing internal ribosome entry site elements: An update of structural motifs and functional divergences

Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/ 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 wideThis work was supported by MINECO (BFU2017-84492-R) and an Institutional grant from Fundación Ramón Arece

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

RNA-protein interaction methods to study viral IRES elements

Translation control often takes place through the mRNA untranslated regions, involving direct interactions with RNA-binding proteins (RBPs). Internal ribosome entry site elements (IRESs) are cis-acting RNA regions that promote translation initiation using a cap-independent mechanism. A subset of positive-strand RNA viruses harbor IRESs as a strategy to ensure efficient viral protein synthesis. IRESs are organized in modular structural domains with a division of functions. However, viral IRESs vary in nucleotide sequence, secondary RNA structure, and transacting factor requirements. Therefore, in-depth studies are needed to understand how distinct types of viral IRESs perform their function. In this review we describe methods to isolate and identify RNA-binding proteins important for IRES activity, and to study the impact of RNA structure and RNA-protein interactions on IRES activity.BFU2011-25437 and CSD2009-00080 from MINECO (Ministerio de Economia y Competitividad), and by an Institutional grant from Fundación Ramón Areces.Peer Reviewe

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

Estudio del ensamblaje pre-ribosómico de las proteínas P de Saccharomyces cerevisiae

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 19-06-2009The stalk is an evolutionary conserved lateral flexible protuberance of the large ribosomal subunit that is essential for the recruitment of translation factors and for the enhancement of their GTPase activity. In Saccharomyces cerevisiae the stalk is composed of four acidic phosphoproteins (P1α, P1β, P2α and P2β) with an average molecular weight of 11 KDa, bound to a larger protein (P0, 32 KDa), that is essential for cellular viability. Using the two‐hybrid approach, we have studied the detailed interactions between any possible combinations of the P proteins. As a result, relevant aspects of the puzzling interactions between these proteins have been clarified. Regarding the interactions with P0, neither P1 nor P2 interact directly as they need the endogenous heterodimer partner to be expressed in the cell; in addition, a P2 free amino end is a requisite for these heterodimers binding to P0. With regards the twohybrid interactions between P1 and P2, the known canonical P1α/P2β and P1β/P2α interactions do not depend on either a free amino end or the presence of native P0, P1 or P2 proteins. Furthermore, the non‐canonical P1β/P2β behaves similarly, although this interaction is weaker and a free amino end in P0 is needed. Interestingly, P1α/P2α, P1α/P1β and P2α/P2β interactions were also detected, being the partner endogenous P proteins involved (including P0). Thus, these positive two hybrid interactions are the consequence of the interaction between two canonical heterodimers and P0, thus forming a pentameric complex. This complex has been purified from the cytosol when a P0 mutant protein unable to bind to the ribosome was expressed in the cell, suggesting the possibility that this is the normal way in which the P proteins assemble to the ribosome. Moreover, different nuclear or cytoplasmic pre‐ribosomal particles have been purified using TAP‐tag system, these particles containing both P0 and P1/P2 proteins. These results open the possibility that the assembly of the pentameric complex may take place also in the nucleus. However, the P proteins appear accumulated in the cytosol when visualized by fluorescence microscopy in conditions where the subunit transport is inhibited. All these results indicate that, in yeast, all five P proteins may simultaneously assemble to the nascent 60S subunit during the biogenesis of ribosomes, and this binding can be occur either in the nucleus than or the cytosol

Carboxy terminal modifications of the P0 protein reveal alternative mechanisms of nuclear ribosomal stalk assembly

The P0 scaffold protein of the ribosomal stalk is mainly incorporated into pre-ribosomes in the cytoplasm where it replaces the assembly factor Mrt4. In analyzing the role of the P0 carboxyl terminal domain (CTD) during ribosomal stalk assembly, we found that its complete removal yields a protein that is functionally similar to Mrt4, whereas a chimeric Mrt4 containing the P0 CTD behaves more like P0. Deleting the P0 binding sites for the P1 and P2 proteins provoked the nuclear accumulation of P0ΔAB induced by either leptomycin B-mediated blockage of nuclear export or Mrt4 deletion. This effect was reversed by removing P1/P2 from the cell, whereas nuclear accumulation was restored on reintroduction of these proteins. Together, these results indicate that the CTD determines the function of the P0 in stalk assembly. Moreover, they indicate that in cells lacking Mrt4, P0 and its stalk base partner, the L12 protein, bind to pre-ribosomes in the nucleus, a complex that is then exported to the cytoplasm by a mechanism assisted by the interaction with P1/P2 proteins. Furthermore, in wild-type cells, the presence of nuclear pre-ribosome complexes containing P0 but not L12 is compatible with the existence of an alternative stalk assembly process. © 2013 The Author(s). Published by Oxford University Press.Spanish Ministry of Science and Innovation (BFU2009-09738); Fundacion Ramon ArecesPeer Reviewe

The RNA-binding protein Gemin5 binds directly to the ribosome and regulates global translation

RNA-binding proteins (RBPs) play crucial roles in all organisms. The protein Gemin5 harbors two functional domains. The N-terminal domain binds to snRNAs targeting them for snRNPs assembly, while the C-terminal domain binds to IRES elements through a non-canonical RNA-binding site. Here we report a comprehensive view of the Gemin5 interactome; most partners copurified with the N-terminal domain via RNA bridges. Notably, Gemin5 sediments with the subcellular ribosome fraction, and His-Gemin5 binds to ribosome particles via its N-terminal domain. The interaction with the ribosome was lost in F381A and Y474A Gemin5 mutants, but not in W14A and Y15A. Moreover, the ribosomal proteins L3 and L4 bind directly with Gemin5, and conversely, Gemin5 mutants impairing the binding to the ribosome are defective in the interaction with L3 and L4. The overall polysome profile was affected by Gemin5 depletion or overexpression, concomitant to an increase or a decrease, respectively, of global protein synthesis. Gemin5, and G5-Nter as well, were detected on the polysome fractions. These results reveal the ribosome-binding capacity of the N-ter moiety, enabling Gemin5 to control global protein synthesis. Our study uncovers a crosstalk between this protein and the ribosome, and provides support for the view that Gemin5 may control translation elongation.MINECO [BFU2011-25437, BFU2014-54564]; Institutional grant from Fundación Ramón Areces. Funding for open access charge: MINECO [BFU2014-54564].Peer Reviewe

Gemin 5: a multitasking RNA-binding protein involved in translational control

17 páginas; contiene gráficos y tablasGemin5 is a RNA-binding protein (RBP) that was first identified as a peripheral component of the survival of motor neurons (SMN) complex. This predominantly cytoplasmic protein recognises the small nuclear RNAs (snRNAs) through its WD repeat domains, allowing assembly of the SMN complex into small nuclear ribonucleoproteins (snRNPs). Additionally, the amino-terminal end of the protein has been reported to possess cap-binding capacity and to interact with the eukaryotic initiation factor 4E (eIF4E). Gemin5 was also shown to downregulate translation, to be a substrate of the picornavirus L protease and to interact with viral internal ribosome entry site (IRES) elements via a bipartite non-canonical RNA-binding site located at its carboxy-terminal end. These features link Gemin5 with translation control events. Thus, beyond its role in snRNPs biogenesis, Gemin5 appears to be a multitasking protein cooperating in various RNA-guided processes. In this review, we will summarise current knowledge of Gemin5 functions. We will discuss the involvement of the protein on translation control and propose a model to explain how the proteolysis fragments of this RBP in picornavirus-infected cells could modulate protein synthesis.David Piñeiro is supported by Biotechnology and Biological Sciences Research Council (BBSRC) Grant BB/M006700/1. Work at Encarna Martinez-Salas’s laboratory was supported by Grants CSD2009-00080, BFU2011-25437 from Ministerio de Economía y Competitividad (MINECO) and by an institutional grant from Fundación Ramón ArecesWe acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe