Post-transcriptional coordination of immunological responses by RNA-binding proteins.

Immunological reactions are propelled by ever-changing signals that alter the translational ability of the RNA in the cells involved. Such alterations are considered to be consequential modifications in the transcriptomic decoding of the genetic blueprint. The identification of RNA-binding protein (RBP) assemblies engaged in the coordinative regulation of state-specific RNAs indicates alternative and exclusive means for determining the activation, plasticity and tolerance of cells of the immune system. Here we review current knowledge about RBP-regulated post-transcriptional events involved in the reactivity of cells of the immune system and the importance of their alteration during chronic inflammatory pathology and autoimmunity.

Polypyrimidine tract binding protein stabilizes the encephalomyocarditis virus IRES structure via binding multiple sites in a unique orientation.

Polypyrimidine tract binding (PTB) protein is a regulator of alternative pre-mRNA splicing, and also stimulates the initiation of translation dependent on many viral internal ribosome entry segments/sites (IRESs). It has four RNA-binding domains (RBDs), but although the contacts with many IRESs have been mapped, the orientation of binding (i.e., which RBD binds to which site in the IRES) is unknown. To answer this question, 16 derivatives of PTB1, each with a single cysteine flanking the RNA-binding surface in an RBD, were constructed and used in directed hydroxyl radical probing with the encephalomyocarditis virus IRES. The results, together with mass spectrometry data on the stoichiometry of PTB binding to different IRES derivatives, show that the minimal IRES binds a single PTB in a unique orientation, with RBD1 and RBD2 binding near the 3' end, and RBD3 contacting the 5' end, thereby constraining and stabilizing the three-dimensional structural fold of the IRES.

The organization of RNA contacts by PTB for regulation of FAS splicing.

Post-transcriptional steps of gene expression are regulated by RNA binding proteins. Major progress has been made in characterizing RNA-protein interactions, from high resolution structures to transcriptome-wide profiling. Due to the inherent technical challenges, less attention has been paid to the way in which proteins with multiple RNA binding domains engage with target RNAs. We have investigated how the four RNA recognition motif (RRM) domains of Polypyrimidine tract binding (PTB) protein, a major splicing regulator, interact with FAS pre-mRNA under conditions in which PTB represses FAS exon 6 splicing. A combination of tethered hydroxyl radical probing, targeted inactivation of individual RRMs and single molecule analyses revealed an unequal division of labour between the four RRMs of PTB. RNA binding by RRM4 is the most important for function despite the low intrinsic binding specificity and the complete lack of effect of disrupting individual RRM4 contact points on the RNA. The ordered RRM3-4 di-domain packing provides an extended binding surface for RNA interacting at RRM4, via basic residues in the preceding linker. Our results illustrate how multiple alternative low-specificity binding configurations of RRM4 are consistent with repressor function as long as the overall ribonucleoprotein architecture provided by appropriate di-domain packing is maintained.

The mechanism of translation initiation on Aichivirus RNA mediated by a novel type of picornavirus IRES.

Picornavirus mRNAs contain IRESs that sustain their translation during infection, when host protein synthesis is shut off. The major classes of picornavirus IRESs (Types 1 and 2) have distinct structures and sequences, but initiation on both is determined by their specific interaction with eIF4G. We report here that Aichivirus (AV), a member of the Kobuvirus genus of Picornaviridae, contains an IRES that differs structurally from Type 1 and Type 2 IRESs. Its function similarly involves interaction with eIF4G, but its eIF4G-interacting domain is structurally distinct, although it contains an apical eIF4G-interacting motif similar to that in Type 2 IRESs. Like Type 1 and Type 2 IRESs, AV IRES function is enhanced by pyrimidine tract-binding protein (PTB), but the pattern of PTB's interaction with each of these IRESs is distinct. Unlike all known IRESs, the AV IRES is absolutely dependent on DHX29, a requirement imposed by sequestration of its initiation codon in a stable hairpin.

Polypyrimidine tract-binding protein stimulates the poliovirus IRES by modulating eIF4G binding.

Tethered hydroxyl-radical probing has been used to determine the orientation of binding of polypyrimidine tract-binding protein (PTB) to the poliovirus type 1 (Mahoney) (PV-1(M)) internal ribosome entry site/segment (IRES)-the question of which RNA-binding domain (RBD) binds to which sites on the IRES. The results show that under conditions in which PTB strongly stimulates IRES activity, a single PTB is binding to the IRES, a finding which was confirmed by mass spectrometry of PTB/IRES complexes. RBDs1 and 2 interact with the basal part of the Domain V irregular stem loop, very close to the binding site of eIF4G, and RBDs3 and 4 interact with the single-stranded regions flanking Domain V. The binding of PTB is subtly altered in the presence of the central domain (p50) of eIF4G, and p50 binding is likewise modified if PTB is present. This suggests that PTB stimulates PV-1(M) IRES activity by inducing eIF4G to bind in the optimal position and orientation to promote internal ribosome entry, which, in PV-1(M), is at an AUG triplet 30 nt downstream of the base of Domain V.

Activation of picornaviral IRESs by PTB shows differential dependence on each PTB RNA-binding domain.

Polypyrimidine tract binding protein (PTB) is an RNA-binding protein with four RNA-binding domains (RBDs). It is a major regulator of alternative splicing and also stimulates translation initiation at picornavirus IRESs (internal ribosome entry sites). The sites of interaction of each RBD with two picornaviral IRESs have previously been mapped. To establish which RBD-IRES interactions are essential for IRES activation, point mutations were introduced into the RNA-binding surface of each RBD. Three such mutations were sufficient to inactivate RNA-binding by any one RBD, but the sites of the other three RBD-IRES interactions remained unperturbed. Poliovirus IRES activation was abrogated by inactivation of RBD1, 2, or 4, but the RBD3-IRES interaction was superfluous. Stimulation of the encephalomyocarditis virus IRES was reduced by inactivation of RBD1, 3, or 4, and abrogated by mutation of RBD2, or both RBDs 3 and 4. Surprisingly, therefore, the binding of PTB in its normal orientation does not guarantee IRES activation; three native RBDs are sufficient for correct binding but not for activation if the missing RBD-IRES interaction is critical.

IQGAP1 mediates the communication between the nucleus and the mitochondria via NDUFS4 alternative splicing.

Constant communication between mitochondria and nucleus ensures cellular homeostasis and adaptation to mitochondrial stress. Anterograde regulatory pathways involving a large number of nuclear-encoded proteins control mitochondrial biogenesis and functions. Such functions are deregulated in cancer cells, resulting in proliferative advantages, aggressive disease and therapeutic resistance. Transcriptional networks controlling the nuclear-encoded mitochondrial genes are known, however alternative splicing (AS) regulation has not been implicated in this communication. Here, we show that IQGAP1, a scaffold protein regulating AS of distinct gene subsets in gastric cancer cells, participates in AS regulation that strongly affects mitochondrial respiration. Combined proteomic and RNA-seq analyses of IQGAP1KO and parental cells show that IQGAP1KO alters an AS event of the mitochondrial respiratory chain complex I (CI) subunit NDUFS4 and downregulates a subset of CI subunits. In IQGAP1KO cells, CI intermediates accumulate, resembling assembly deficiencies observed in patients with Leigh syndrome bearing NDUFS4 mutations. Mitochondrial CI activity is significantly lower in KO compared to parental cells, while exogenous expression of IQGAP1 reverses mitochondrial defects of IQGAP1KO cells. Our work sheds light to a novel facet of IQGAP1 in mitochondrial quality control that involves fine-tuning of CI activity through AS regulation in gastric cancer cells relying highly on mitochondrial respiration.

Isoform changes of action potential regulators in the ventricles of arrhythmogenic phospholamban-R14del humanized mouse hearts.

Arrhythmogenic cardiomyopathy (ACM) is characterized by life-threatening ventricular arrhythmias and sudden cardiac death and affects hundreds of thousands of patients worldwide. The deletion of Arginine 14 (p.R14del) in the phospholamban (PLN) gene has been implicated in the pathogenesis of ACM. PLN is a key regulator of sarcoplasmic reticulum (SR) Ca2+ cycling and cardiac contractility. Despite global gene and protein expression studies, the molecular mechanisms of PLN-R14del ACM pathogenesis remain unclear. Using a humanized PLN-R14del mouse model and human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs), we investigated the transcriptome-wide mRNA splicing changes associated with the R14del mutation. We identified >200 significant alternative splicing (AS) events and distinct AS profiles were observed in the right (RV) and left (LV) ventricles in PLN-R14del compared to WT mouse hearts. Enrichment analysis of the AS events showed that the most affected biological process was associated with "cardiac cell action potential", specifically in the RV. We found that splicing of 2 key genes, Trpm4 and Camk2d, which encode proteins regulating calcium homeostasis in the heart, were altered in PLN-R14del mouse hearts and human iPSC-CMs. Bioinformatical analysis pointed to the tissue-specific splicing factors Srrm4 and Nova1 as likely upstream regulators of the observed splicing changes in the PLN-R14del cardiomyocytes. Our findings suggest that aberrant splicing may affect Ca2+-homeostasis in the heart, contributing to the increased risk of arrythmogenesis in PLN-R14del ACM.

Defining the roles and interactions of PTB.

PTB (polypyrimidine tract-binding protein) is an abundant and widely expressed RNA-binding protein with four RRM (RNA recognition motif) domains. PTB is involved in numerous post-transcriptional steps in gene expression in both the nucleus and cytoplasm, but has been best characterized as a regulatory repressor of some ASEs (alternative splicing events), and as an activator of translation driven by IRESs (internal ribosome entry segments). We have used a variety of approaches to characterize the activities of PTB and its molecular interactions with RNA substrates and protein partners. Using splice-sensitive microarrays we found that PTB acts not only as a splicing repressor but also as an activator, and that these two activities are determined by the location at which PTB binds relative to target exons. We have identified minimal splicing repressor and activator domains, and have determined high resolution structures of the second RRM domain of PTB binding to peptide motifs from the co-repressor protein Raver1. Using single-molecule techniques we have determined the stoichiometry of PTB binding to a regulated splicing substrate in whole nuclear extracts. Finally, we have used tethered hydroxyl radical probing to determine the locations on viral IRESs at which each of the four RRM domains bind. We are now combining tethered probing with single molecule analyses to gain a detailed understanding of how PTB interacts with pre-mRNA substrates to effect either repression or activation of splicing.

The scaffold protein IQGAP1 links heat-induced stress signals to alternative splicing regulation in gastric cancer cells.

In response to oncogenic signals, Alternative Splicing (AS) regulators such as SR and hnRNP proteins show altered expression levels, subnuclear distribution and/or post-translational modification status, but the link between signals and these changes remains unknown. Here, we report that a cytosolic scaffold protein, IQGAP1, performs this task in response to heat-induced signals. We show that in gastric cancer cells, a nuclear pool of IQGAP1 acts as a tethering module for a group of spliceosome components, including hnRNPM, a splicing factor critical for the response of the spliceosome to heat-shock. IQGAP1 controls hnRNPM's sumoylation, subnuclear localisation and the relevant response of the AS machinery to heat-induced stress. Genome-wide analyses reveal that IQGAP1 and hnRNPM co-regulate the AS of a cell cycle-related RNA regulon in gastric cancer cells, thus favouring the accelerated proliferation phenotype of gastric cancer cells. Overall, we reveal a missing link between stress signals and AS regulation.

AgtA, the dicarboxylic amino acid transporter of Aspergillus nidulans, is concertedly down-regulated by exquisite sensitivity to nitrogen metabolite repression and ammonium-elicited endocytosis.

We identified agtA, a gene that encodes the specific dicarboxylic amino acid transporter of Aspergillus nidulans. The deletion of the gene resulted in loss of utilization of aspartate as a nitrogen source and of aspartate uptake, while not completely abolishing glutamate utilization. Kinetic constants showed that AgtA is a high-affinity dicarboxylic amino acid transporter and are in agreement with those determined for a cognate transporter activity identified previously. The gene is extremely sensitive to nitrogen metabolite repression, depends on AreA for its expression, and is seemingly independent from specific induction. We showed that the localization of AgtA in the plasma membrane necessitates the ShrA protein and that an active process elicited by ammonium results in internalization and targeting of AgtA to the vacuole, followed by degradation. Thus, nitrogen metabolite repression and ammonium-promoted vacuolar degradation act in concert to downregulate dicarboxylic amino acid transport activity.

Interaction of yeast eIF4G with spliceosome components: implications in pre-mRNA processing events.

As evidenced from mammalian cells the eukaryotic translation initiation factor eIF4G has a putative role in nuclear RNA metabolism. Here we investigate whether this role is conserved in the yeast Saccharomyces cerevisiae. Using a combination of in vitro and in vivo methods, we show that, similar to mammalian eIF4G, yeast eIF4G homologues, Tif4631p and Tif4632p, are present both in the nucleus and the cytoplasm. We show that both eIF4G proteins interact efficiently in vitro with UsnRNP components of the splicing machinery. More specifically, Tif4631p and Tif4632p interact efficiently with U1 snRNA in vitro. In addition, Tif4631p and Tif4632p associate with protein components of the splicing machinery, namely Snu71p and Prp11p. To further delineate these interactions, we map the regions of Tif4631p and Tif4632p that are important for the interaction with Prp11p and Snu71p and we show that addition of these regions to splicing reactions in vitro has a dominant inhibitory effect. The observed interactions implicate eIF4G in aspects of pre-mRNA processing. In support of this hypothesis, deletion of one of the eIF4G isoforms results in accumulation of un-spliced precursors for a number of endogenous genes, in vivo. In conclusion these observations are suggestive of the involvement of yeast eIF4G in pre-mRNA metabolism.

Decoding the functions of post-transcriptional regulators in the determination of inflammatory states: focus on macrophage activation.

Inflammation involves a continuum of intercellular interactions and cellular responses targeting infectious or tissue damage while maintaining homeostasis. At its core, this continuum encompasses the alternating phenotypes of innate immune cells; each phenotype is typified by the expression of molecules which either support host defence or aid tissue restoration and the resolution of inflammation. The aberrant persistence of any such phenotype can drive chronic inflammatory pathology. For macrophages, these phenotypes arise as changes in cellular plasticity because of adaptation. As such their underlying gene expression programs may not be determined by robust transcriptomic and epigenetic programs but by more flexible means like post-transcriptional mechanisms affecting mRNA use. These mechanisms require the assemblies of RNA-binding proteins (RBPs) and non-coding RNAs onto specific elements on their RNA targets in Ribonucleoprotein particles (RNPs) which control mRNA maturation, turnover and translation. The collection of RNPs within a cell defines the ribonome, that is, a high order system of coordinative post-transcriptional determination. mRNAs involved in the definition of different macrophage activation phenotypes share elements of RBP recognition rendering them amenable to ribonomic regulation. The molecular features of their cognitive RBPs and the pathologies developing in the corresponding mouse mutants support their involvement in inflammatory reactions. We view this information in the context of macrophage activation states to propose that these states can be determined via differential--synergistic or antagonistic--RNP associations. In doing so, we substantiate the need for the use of systems platforms to model RNP hierarchies controlling the continuum of inflammation.

The proline permease of Aspergillus nidulans: functional replacement of the native cysteine residues and properties of a cysteine-less transporter.

The major proline transporter (PrnB) of Aspergillus nidulans belongs to the Amino acid Polyamine Organocation (APC) transporter superfamily. Members of this family have not been subjected to systematic structure-function relationship studies. In this report, we examine the functional replacement of the three native Cys residues (Cys54, Cys352 and Cys530) of PrnB and the properties of an engineered Cys-less PrnB protein, as background for employing a Cys-scanning mutagenesis approach. We show that simultaneous replacement of Cys54 with Ala, Cys352 with Ala and Cys530 with Ser results in a functional Cys-less PrnB transporter. We also introduce the use of a biotin-acceptor domain tag to quantitate protein levels of the engineered PrnB mutants by Western blot analysis. Finally, by using the background of the Cys-less PrnB transporter, we evaluate the functional importance of amino acids Q219, K245 and F248 of PrnB, which our previous data had suggested to be involved in the mechanism of PrnB-mediated proline uptake. In the current study, we show that K245 and F248 but not Q219 are critical for PrnB-mediated proline uptake.