Neuronal BC RNAs cooperate with eIF4B to mediate activity-dependent translational control

In neurons, translational regulation of gene expression has been implicated in the activity-dependent management of synapto-dendritic protein repertoires. However, the fundamentals of stimulus-modulated translational control in neurons remain poorly understood. Here we describe a mechanism in which regulatory brain cytoplasmic (BC) RNAs cooperate with eukaryotic initiation factor 4B (eIF4B) to control translation in a manner that is responsive to neuronal activity. eIF4B is required for the translation of mRNAs with structured 5′ untranslated regions (UTRs), exemplified here by neuronal protein kinase Mζ (PKMζ) mRNA. Upon neuronal stimulation, synapto-dendritic eIF4B is dephosphorylated at serine 406 in a rapid process that is mediated by protein phosphatase 2A. Such dephosphorylation causes a significant decrease in the binding affinity between eIF4B and BC RNA translational repressors, enabling the factor to engage the 40S small ribosomal subunit for translation initiation. BC RNA translational control, mediated via eIF4B phosphorylation status, couples neuronal activity to translational output, and thus provides a mechanistic basis for long-term plastic changes in nerve cells.     

Genomic SELEX for Hfq-binding RNAs identifies genomic aptamers predominantly in antisense transcripts

An unexpectedly high number of regulatory RNAs have been recently discovered that fine-tune the function of genes at all levels of expression. We employed Genomic SELEX, a method to identify protein-binding RNAs encoded in the genome, to search for further regulatory RNAs in Escherichia coli. We used the global regulator protein Hfq as bait, because it can interact with a large number of RNAs, promoting their interaction. The enriched SELEX pool was subjected to deep sequencing, and 8865 sequences were mapped to the E. coli genome. These short sequences represent genomic Hfq-aptamers and are part of potential regulatory elements within RNA molecules. The motif 5′-AAYAAYAA-3′ was enriched in the selected RNAs and confers low-nanomolar affinity to Hfq. The motif was confirmed to bind Hfq by DMS footprinting. The Hfq aptamers are 4-fold more frequent on the antisense strand of protein coding genes than on the sense strand. They were enriched opposite to translation start sites or opposite to intervening sequences between ORFs in operons. These results expand the repertoire of Hfq targets and also suggest that Hfq might regulate the expression of a large number of genes via interaction with cis-antisense RNAs.     

Characterization of a cis-acting regulatory element in the protein coding region of thymidylate synthase mRNA

Thymidylate synthase (TS) functions as an RNA-binding protein by interacting with two different sequences on its own mRNA. One site is located in the 5′-upstream region of human TS mRNA while the second site is located within the protein coding region corresponding to nt 434–634. In this paper, a 70 nt RNA sequence, corresponding to nt 480–550, was identified that binds TS protein with an affinity similar to that of full-length TS mRNA and TS434–634 RNA. In vitro translation studies confirmed that this sequence is critical for the translational autoregulatory effects of TS. To document in vivo biological significance, TS sequences contained within this region were cloned onto the 5′-end of a luciferase reporter plasmid and transient transfection experiments were performed using H630 human colon cancer cells. In cells transfected with p644/TS434–634 or p644/TS480–550, luciferase activity was decreased 2.5-fold when compared to cells transfected with p644 plasmid alone. Luciferase mRNA levels were identical for each of these conditions as determined by RNase protection and RT–PCR analysis. Immunoprecipitation of TS ribonucleoprotein complexes revealed a direct interaction between TS protein and TS480–550 RNA in transfected H630 cells. Treatment with 5-fluorouridine resulted in a nearly 2-fold increase in luciferase activity only in cells transfected with p644/TS434–634 and p644/TS480–550. This study identifies a 70 nt TS response element in the protein coding region of TS mRNA with in vitro and in vivo translational regulatory activity.     

Preferential translation of Hsp83 in Leishmania requires a thermosensitive polypyrimidine-rich element in the 3′ UTR and involves scanning of the 5′ UTR

Heat shock proteins (HSPs) provide a useful system for studying developmental patterns in the digenetic Leishmania parasites, since their expression is induced in the mammalian life form. Translation regulation plays a key role in control of protein coding genes in trypanosomatids, and is directed exclusively by elements in the 3′ untranslated region (UTR). Using sequential deletions of the Leishmania Hsp83 3′ UTR (888 nucleotides [nt]), we mapped a region of 150 nt that was required, but not sufficient for preferential translation of a reporter gene at mammalian-like temperatures, suggesting that changes in RNA structure could be involved. An advanced bioinformatics package for prediction of RNA folding (UNAfold) marked the regulatory region on a highly probable structural arm that includes a polypyrimidine tract (PPT). Mutagenesis of this PPT abrogated completely preferential translation of the fused reporter gene. Furthermore, temperature elevation caused the regulatory region to melt more extensively than the same region that lacked the PPT. We propose that at elevated temperatures the regulatory element in the 3′ UTR is more accessible to mediators that promote its interaction with the basal translation components at the 5′ end during mRNA circularization. Translation initiation of Hsp83 at all temperatures appears to proceed via scanning of the 5′ UTR, since a hairpin structure abolishes expression of a fused reporter gene.     

Posttranscriptional regulation of angiotensin II type 1 receptor expression by glyceraldehyde 3-phosphate dehydrogenase

Regulation of angiotensin II type 1 receptor (AT1R) has a pathophysiological role in hypertension, atherosclerosis and heart failure. We started from an observation that the 3′-untranslated region (3′-UTR) of AT1R mRNA suppressed AT1R translation. Using affinity purification for the separation of 3′-UTR-binding proteins and mass spectrometry for their identification, we describe glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an AT1R 3′-UTR-binding protein. RNA electrophoretic mobility shift analysis with purified GAPDH further demonstrated a direct interaction with the 3′-UTR while GAPDH immunoprecipitation confirmed this interaction with endogenous AT1R mRNA. GAPDH-binding site was mapped to 1–100 of 3′-UTR. GAPDH-bound target mRNAs were identified by expression array hybridization. Analysis of secondary structures shared among GAPDH targets led to the identification of a RNA motif rich in adenines and uracils. Silencing of GAPDH increased the expression of both endogenous and transfected AT1R. Similarly, a decrease in GAPDH expression by H₂O₂ led to an increased level of AT1R expression. Consistent with GAPDH having a central role in H₂O₂-mediated AT1R regulation, both the deletion of GAPDH-binding site and GAPDH overexpression attenuated the effect of H₂O₂ on AT1R mRNA. Taken together, GAPDH is a translational suppressor of AT1R and mediates the effect of H₂O₂ on AT1R mRNA.     

The 5'-leader of tobacco mosaic virus promotes translation through enhanced recruitment of eIF4F

The 5′-leader sequence (called Ω) of tobacco mosaic virus (TMV) functions as a translational enhancer in plants. A poly(CAA) region within Ω is responsible for the translation enhancement and serves as a binding site for the heat shock protein, HSP101, which is required for the translational enhancement. Genetic analysis of the HSP101-mediated enhancement of translation from Ω-containing mRNA suggested that two eukaryotic initiation factors (eIFs), i.e. eIF4G and eIF3, were necessary. In this study, the functional interaction between Ω and other RNA elements known to participate in the recruitment of eIF4G, i.e. the 5′-cap and the poly(A) tail, was examined. Ω exhibited functional overlap with the 5′-cap and the poly(A) tail but not with the native TMV 3′-UTR which contains an independent translational enhancer. Consistent with the role of HSP101 in mediating the translational function of Ω, the enhancement afforded by Ω increased following a heat shock, which elevates expression of HSP101. The use of a fractionated translation lysate revealed that of the two eIF4F proteins present in plants, eIF4F was specifically required for the activity of Ω. The data suggest that Ω is functionally similar to a 5′-cap and a poly(A) tail in that it serves to recruit eIF4F in order to enhance translation from an mRNA.     

Identification of DEAD-box RNA Helicase 6 (DDX6) as a Cellular Modulator of Vascular Endothelial Growth Factor Expression under Hypoxia

Vascular endothelial growth factor A (VEGF) is a crucial proangiogenic factor, which regulates blood vessel supply under physiologic and pathologic conditions. The VEGF mRNA 5′-untranslated region (5′-UTR) bears internal ribosome entry sites (IRES), which confer sustained VEGF mRNA translation under hypoxia when 5′-cap-dependent mRNA translation is inhibited. VEGF IRES-mediated initiation of translation requires the modulated interaction of trans-acting factors. To identify trans-acting factors that control VEGF mRNA translation under hypoxic conditions we established an in vitro translation system based on human adenocarcinoma cells (MCF-7). Cytoplasmic extracts of MCF-7 cells grown under hypoxia (1% oxygen) recapitulate VEGF IRES-mediated reporter mRNA translation. Employing the VEGF mRNA 5′-UTR and 3′-UTR in an RNA affinity approach we isolated interacting proteins from translational active MCF-7 extract prepared from cells grown under normoxia or hypoxia. Interestingly, mass spectrometry analysis identified the DEAD-box RNA helicase 6 (DDX6) that interacts with the VEGF mRNA 5′-UTR. Recombinant DDX6 inhibits VEGF IRES-mediated translation in normoxic MCF-7 extract. Under hypoxia the level of DDX6 declines, and its interaction with VEGF mRNA is diminished in vivo. Depletion of DDX6 by RNAi further promotes VEGF expression in MCF-7 cells. Increased secretion of VEGF from DDX6 knockdown cells positively affects vascular tube formation of human umbilical vein endothelial cells (HUVEC) in vitro. Our results indicate that the decrease of DDX6 under hypoxia contributes to the activation of VEGF expression and promotes its proangiogenic function.     

Translation of stable hepadnaviral mRNA cleavage fragments induced by the action of phosphorothioate-modified antisense oligodeoxynucleotides

Phosphorothioate-modified antisense oligodeoxynucleotides (ASOs) are used to suppress gene expression by inducing RNase H-mediated cleavage with subsequent degradation of the target mRNA. However, previous observations suggest that ASO/RNase H can also result in the generation of stable mRNA cleavage fragments and expression of truncated proteins. Here, we addressed the underlying translational mechanisms in more detail using hepadnavirus-transfected hepatoma cells as a model system of antisense therapy. Generation of stable mRNA cleavage fragments was restricted to the ASO/RNase H pathway and not observed upon cotransfection of isosequential small interfering RNA or RNase H-incompetent oligonucleotides. Furthermore, direct evidence for translation of mRNA fragments was established by polysome analysis. Polysome-associated RNA contained cleavage fragments devoid of a 5′ cap structure indicating that translation was, at least in part, cap-independent. Further analysis of the uncapped cleavage fragments revealed that their 5′ terminus and initiation codon were only separated by a few nucleotides suggesting a 5′ end-dependent mode of translation, whereas internal initiation could be ruled out. However, the efficiency of translation was moderate compared to uncleaved mRNA and amounted to 13–24% depending on the ASO used. These findings provide a rationale for understanding the translation of mRNA fragments generated by ASO/RNase H mechanistically.     

Distinct 5′ UTRs regulate XIAP expression under normal growth conditions and during cellular stress

X-chromosome linked inhibitor of apoptosis, XIAP, is cellular caspase inhibitor and a key regulator of apoptosis. We and others have previously shown that XIAP expression is regulated primarily at the level of protein synthesis; the 5′ untranslated region (UTR) of XIAP mRNA contains an Internal Ribosome Entry Site (IRES) that supports cap-independent expression of XIAP protein during conditions of pathophysiological stress, such as serum deprivation or gamma irradiation. Here, we show that XIAP is encoded by two distinct mRNAs that differ in their 5′ UTRs. We further show that the dominant, shorter, 5′ UTR promotes a basal level of XIAP expression under normal growth conditions. In contrast, the less abundant longer 5′ UTR contains an IRES and supports cap-independent translation during stress. Our data suggest that the combination of alternate regulatory regions and distinct translational initiation modes is critical in maintaining XIAP levels in response to cellular stress and may represent a general mechanism of cellular adaptation.     

Multiple mechanisms repress N-Bak mRNA translation in the healthy and apoptotic neurons

N-Bak is a neuron-specific BH3-only splice variant of pro-apoptotic Bcl-2 family member Bak. We have shown that its mRNA is stable in the neurons, whereas the protein cannot be detected by antibodies, suggesting a strong translational arrest of the mRNA. Here we identify two regulatory elements in the N-Bak mRNA that significantly repress translation in the luciferase reporter assay: an upstream open reading frame in the 5′-untranslated region (UTR) and naturally spliced exon–exon junction downstream of the premature translation termination codon in the 3′UTR. We also show that N-Bak mRNA is stored in granular structures in the sympathetic neurons and stays in these granules during intrinsic apoptosis. Finally, we confirm the absence of N-Bak protein by quantitative mass spectrometry analysis in the healthy, apoptotic or stressed sympathetic and cortical neurons. We conclude that N-Bak mRNA is translationally repressed by multiple mechanisms, and the protein does not participate in the classical apoptosis or cellular stress response.