Noemí Fernández

A novel role for Gemin5 in mRNA translation

In eukaryotic cells translation initiation occurs through two alternative mechanisms, a cap-dependent operating in the majority of mRNAs, and a 5′-end-independent driven by internal ribosome entry site (IRES) elements, specific for a subset of mRNAs. IRES elements recruit the translation machinery to an internal position in the mRNA through a mechanism involving the IRES structure and several trans-acting factors. Here, we identified Gemin5 protein bound to the foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) IRES using two independent approaches, riboproteomic analysis and immunoprecipitation of photocroslinked factors. Functional analysis performed in Gemin5 shRNA-depleted cells, or in in vitro translation reactions, revealed an unanticipated role of Gemin5 in translation control as a down-regulator of cap-dependent and IRES-driven translation initiation. Consistent with this, pull-down assays showed that Gemin5 forms part of two distinct complexes, a specific IRES-ribonucleoprotein complex and an IRES-independent protein complex containing eIF4E. Thus, beyond its role in snRNPs biogenesis, Gemin5 also functions as a modulator of translation activity.

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.

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.

Magnesium‐dependent folding of a picornavirus IRES element modulates RNA conformation and eIF4G interaction

Internal ribosome entry site (IRES) elements are high‐order RNA structures that promote internal initiation of translation to allow protein synthesis under situations that compromise the general cap‐dependent translation mechanism. Picornavirus IRES elements are highly efficient elements with a modular RNA structure organization. Here we investigated the effect of Mg2+ concentration on the local flexibility and solvent accessibility of the foot‐and‐mouth disease virus (FMDV) IRES element measured on the basis of selective 2′‐hydroxyl acylation analyzed by primer extension (SHAPE) reactivity and hydroxyl radical cleavage. We have found that Mg2+ concentration affects the organization of discrete IRES regions, mainly the apical region of domain 3, the 10 nt loop of domain 4, and the pyrimidine tract of domain 5. In support of the effect of RNA structure on IRES activity, substitution or deletion mutants of the 10 nt loop of domain 4 impair internal initiation. In addition, divalent cations affect the binding of eIF4G, a eukaryotic initiation factor that is essential for IRES‐dependent translation that interacts with domain 4. Binding of eIF4G is favored by the local RNA flexibility adopted at low Mg2+ concentration, while eIF4B interacts with the IRES independently of the compactness of the RNA structure. Our study shows that the IRES element adopts a near‐native structure in the absence of proteins, shedding light on the influence of Mg2+ ions on the local flexibility and binding of eIF4G in a model IRES element.

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.

Functional and structural analysis of maize hsp101 IRES.

Maize heat shock protein of 101 KDa (HSP101) is essential for thermotolerance induction in this plant. The mRNA encoding this protein harbors an IRES element in the 5′UTR that mediates cap-independent translation initiation. In the current work it is demonstrated that hsp101 IRES comprises the entire 5′UTR sequence (150 nts), since deletion of 17 nucleotides from the 5′ end decreased translation efficiency by 87% compared to the control sequence. RNA structure analysis of maize hsp101 IRES revealed the presence of three stem-loops toward its 5′ end, whereas the remainder sequence contains a great proportion of unpaired nucleotides. Furthermore, HSP90 protein was identified by mass spectrometry as the protein preferentially associated with the maize hsp101 IRES. In addition, it has been found that eIFiso4G rather than eIF4G initiation factor mediates translation of the maize hsp101 mRNA.

Molecular Determinants of Negative Regulation of the Bradyrhizobium diazoefficiens Transcription Factor FixK2

FixK2 is a key regulator involved in the control of different lifestyles of the facultative soybean endosymbiont Bradyrhizobium diazoefficiens. Expression of the fixK 2 gene is activated by the superimposed two-component regulatory system FixLJ in response to low oxygen (at or below 5 % oxygen in the gas phase) and repressed by its own product (directly or indirectly) by a still unknown mechanism. FixK2 is an unusual member of the CRP/FNR family of bacterial transcription factors , because it is active in vitro without an additional effector molecule and is negatively regulated posttranslationally by the oxidation of its singular cysteine residue (Cys-183). FixK2 is also regulated by proteolysis by the ClpAP1 chaperone-protease system. Remarkably, the recently solved FixK2 structure in complex with DNA revealed the molecular basis for the posttranslational control of FixK2. Specifically, both Cys-183 and the last 12 amino acids of FixK2 are surface-exposed, rendering the protein accessible for both oxidation and proteolysis, respectively. Here, we compile the updated knowledge about the critical elements involved in the negative control of fixK 2 gene expression and FixK2 protein activity.

Riboproteomic Approaches to Understanding IRES Elements

Initiation of translation is a key step of protein synthesis. Two basic mechanisms govern translation initiation in eukaryotic mRNAs, the cap-dependent initiation mechanism that operates in the vast majority of mRNAs and the internal ribosome entry site (IRES)-dependent mechanism. IRES elements, first discovered in picornaviruses, are cis-acting RNA sequences that form secondary and tertiary structures and recruit the translation machinery using a 5′ end-independent mechanism, usually assisted by a subset of translation initiation factors and various RNA-binding proteins termed IRES transacting factors (ITAFs). RNA-binding proteins are pivotal regulators of gene expression, including internal initiation of translation, in response to numerous stresses. This chapter discusses recent advances on riboproteomic approaches aimed to identify ITAFs and the relationship between RNA–protein interaction and IRES activity.