David Piñeiro

Alternative mechanisms to initiate translation in eukaryotic mRNAs

The composition of the cellular proteome is under the control of multiple processes, one of the most important being translation initiation. The majority of eukaryotic cellular mRNAs initiates translation by the cap-dependent or scanning mode of translation initiation, a mechanism that depends on the recognition of the m7G(5′)ppp(5′)N, known as the cap. However, mRNAs encoding proteins required for cell survival under stress bypass conditions inhibitory to cap-dependent translation; these mRNAs often harbor internal ribosome entry site (IRES) elements in their 5′UTRs that mediate internal initiation of translation. This mechanism is also exploited by mRNAs expressed from the genome of viruses infecting eukaryotic cells. In this paper we discuss recent advances in understanding alternative ways to initiate translation across eukaryotic organisms.

Selection of aptamers against KMP-11 using colloidal gold during the SELEX process

SELEX procedure is a methodology in which single stranded oligonucleotides are selected from a wide variety of sequences based on their interaction with a target molecule. We have designed a novel SELEX methodology using colloidal gold to select high affinity single stranded DNA aptamers against Leishmania infantum KMP-11. Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell membrane of kinetoplastid parasites. Although its function is not known, the fact that KMP-11 is a cytoskeleton-associated protein suggests that it may be involved in mobility or in some other aspects of the flagellar structure. We have isolated a single stranded DNA aptamer population that binds specifically to L. infantum KMP-11. This population has been characterized in a series of in vitro experiments suggesting that it may be used as a powerful tool to further investigate the role of KMP-11 during Leishmania development and/or as a diagnostic tool in Leishmania infection.

Gemin5 promotes IRES interaction and translation control through its C-terminal region

Gene expression control largely depends on ribonucleoprotein complexes regulating mRNA translation. Initiation of translation in mRNAs that overcome cap-dependent translation inhibition is often driven by internal ribosome entry site (IRES) elements, whose activity is regulated by multifunctional RNA-binding factors. Here we show that Gemin5 interacts preferentially with a specific domain of a viral IRES consisting of a hairpin flanked by A/U/C-rich sequences. RNA-binding assays using purified proteins revealed that Gemin5–IRES interaction depends on the C-terminal region of the protein. Consistent with this novel finding, the C-terminal region of Gemin5, but not the N-terminal region, impaired translation. Furthermore, RNA selective 2′hydroxyl acylation analysed by primer extension (SHAPE) reactivity demonstrated that addition of purified Gemin5 to IRES mRNA induced the specific protection of residues around the hairpin of the IRES element. We further demonstrate that Gemin5 out-competed SHAPE reactivity variations induced by the IRES-binding factor PTB, leading to a local conformational change in the IRES structure. Together, our data unveil the inhibitory mechanism of Gemin5 on IRES-mediated translation.

A DNA aptamer population specifically detects Leishmania infantum H2A antigen.

Aptamers are short single-stranded DNA or RNA oligonucleotides that are selected in vitro by their affinity and specificity for the target. Binding is a consequence of the particular tertiary structure that they are able to acquire, depending on their sequence. Parasites of the genus Leishmania belongs to the lower eukaryote order Kinetoplastida that causes leishmaniosis in man and animals. Histone genes in Leishmania are of considerable interest because these flagellates do not condense their chromatin during mitosis. Thus, the study of the structural features of histones has been considered of particular interest and, as a result, in recent years a great number of histone genes have been characterized in trypanosomatids. Histones are extremely conserved proteins, reflecting their apparent universality of function. Sequence similarity of kinetoplastid core histones those of higher eukaryotes is found predominantly in the globular region with high sequence divergences in the N- and in the C-terminal domains. These divergences indicate that they may be potential diagnostic and/or therapeutics targets. We have successfully isolated a pool of DNA sequences, named SELH2A, which specifically binds to Leishmania infantum H2A. When tested in an enzyme-linked oligonucleotide assay, slot blot and Western blot analysis, the aptamer pool exhibited specificity in its ability to bind only to H2A antigen but not to other proteins from L. infantum including other histones. Thus, it appears that this novel anti-H2A aptamer population may be of potential application as a diagnostic system for leishmaniosis.

Increased replicative fitness can lead to decreased drug sensitivity of hepatitis C virus

ABSTRACT Passage of hepatitis C virus (HCV) in human hepatoma cells resulted in populations that displayed partial resistance to alpha interferon (IFN-α), telaprevir, daclatasvir, cyclosporine, and ribavirin, despite no prior exposure to these drugs. Mutant spectrum analyses and kinetics of virus production in the absence and presence of drugs indicate that resistance is not due to the presence of drug resistance mutations in the mutant spectrum of the initial or passaged populations but to increased replicative fitness acquired during passage. Fitness increases did not alter host factors that lead to shutoff of general host cell protein synthesis and preferential translation of HCV RNA. The results imply that viral replicative fitness is a mechanism of multidrug resistance in HCV. IMPORTANCE Viral drug resistance is usually attributed to the presence of amino acid substitutions in the protein targeted by the drug. In the present study with HCV, we show that high viral replicative fitness can confer a general drug resistance phenotype to the virus. The results exclude the possibility that genomes with drug resistance mutations are responsible for the observed phenotype. The fact that replicative fitness can be a determinant of multidrug resistance may explain why the virus is less sensitive to drug treatments in prolonged chronic HCV infections that favor increases in replicative fitness.

Identification of novel non-canonical RNA-binding sites in Gemin5 involved in internal initiation of translation

Ribonucleic acid (RNA)-binding proteins are key players of gene expression control. We have shown that Gemin5 interacts with internal ribosome entry site (IRES) elements and modulates initiation of translation. However, little is known about the RNA-binding sites of this protein. Here we show that the C-terminal region of Gemin5 bears two non-canonical bipartite RNA-binding sites, encompassing amino acids 1297–1412 (RBS1) and 1383–1508 (RBS2). While RBS1 exhibits greater affinity for RNA than RBS2, it does not affect IRES-dependent translation in G5-depleted cells. In solution, the RBS1 three-dimensional structure behaves as an ensemble of flexible conformations rather than having a defined tertiary structure. However, expression of the polypeptide G51383–1508, bearing the low RNA-binding affinity RBS2, repressed IRES-dependent translation. A comparison of the RNA-binding capacity and translation control properties of constructs expressed in mammalian cells to that of the Gemin5 proteolysis products observed in infected cells reveals that non-repressive products accumulated during infection while the repressor polypeptide is not stable. Taken together, our results define the low affinity RNA-binding site as the minimal element of the protein being able to repress internal initiation of translation.

Gemin5 proteolysis reveals a novel motif to identify L protease targets

Translation of picornavirus RNA is governed by the internal ribosome entry site (IRES) element, directing the synthesis of a single polyprotein. Processing of the polyprotein is performed by viral proteases that also recognize as substrates host factors. Among these substrates are translation initiation factors and RNA-binding proteins whose cleavage is responsible for inactivation of cellular gene expression. Foot-and-mouth disease virus (FMDV) encodes two proteases, Lpro and 3Cpro. Widespread definition of Lpro targets suffers from the lack of a sufficient number of characterized substrates. Here, we report the proteolysis of the IRES-binding protein Gemin5 in FMDV-infected cells, but not in cells infected by other picornaviruses. Proteolysis was specifically associated with expression of Lpro, yielding two stable products, p85 and p57. In silico search of putative L targets within Gemin5 identified two sequences whose potential recognition was in agreement with proteolysis products observed in infected cells. Mutational analysis revealed a novel Lpro target sequence that included the RKAR motif. Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and Lpro-expressing cells. This protein carries a RRLR motif whose substitution to EELR abrogated Lpro recognition. Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of Lpro in host factors.

Evolutionary conserved motifs constrain the RNA structure organization of picornavirus IRES

Picornavirus RNAs initiate translation using a 5′ end‐independent mechanism based on internal ribosome entry site (IRES) elements. Despite performing similar functions, IRES elements present in genetically distant RNAs differ in primary sequence, RNA secondary structure and trans‐acting factors requirement. The lack of conserved features amongst IRESs represents obstacles for the understanding of the internal initiation process. RNA structure is tightly linked to picornavirus IRES activity, consistent with the conservation of RNA motifs. This study extends the functional relevance of evolutionary conserved motifs of foot‐and‐mouth disease virus (FMDV) IRES. SHAPE structural analysis of mutant IRESs revealed local changes in RNA flexibility indicating the existence of an interactive structure constrained by lateral bulges that maintain the RNA conformation necessary for IRES‐mediated translation.

Gemin5: A Multitasking RNA-Binding Protein Involved in Translation Control

Gemin5 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.

Analysis of the protein expression changes during taxol-induced apoptosis under translation inhibition conditions

Taxol is currently used in chemotherapeutic treatments of different types of cancers. In this article, we demonstrate that taxol induces apoptosis and translation down-regulation in human embryonic kidney (HEK293T) cells. Antibody arrays are a promising new tool for the analysis of protein levels changes in cells responding to different stimuli. Using this approach, we have identified changes in the expression of 38 proteins (20 down-regulated and 18 up-regulated), implicated in several cellular processes mainly in apoptosis, cell cycle and signal transduction pathways, and also cytoskeleton proteins. Among them, we have confirmed a considerable decrease in the expression of p14ARF and a significant increase in the levels of dystrophin and c-Myc. It is known that c-Myc mRNA has an internal ribosome entry segment (IRES) element in its 5′UTR that could regulate its expression under global protein synthesis inhibition conditions. We demonstrate that after taxol treatment, the c-Myc IRES activity is maintained meanwhile cap-dependent activity is inhibited. In addition, an increase in c-Myc mRNA was also observed after taxol treatment. We conclude that taxol-induced c-Myc expression is regulated at both transcriptional and translational levels, the last of them by a mechanism mediated by IRES.