Deciphering the Akt1-HuD interaction in HuD-mediated neuronal differentiation.

The RNA-binding protein HuD/ELAVL4 is essential for neuronal development and synaptic plasticity by governing various post-transcriptional processes of target mRNAs, including stability, translation, and localization. We previously showed that the linker region and poly(A)-binding domain of HuD play a pivotal role in promoting translation and inducing neurite outgrowth. In addition, we found that HuD interacts exclusively with the active form of Akt1, through the linker region. Although this interaction is essential for neurite outgrowth, HuD is not a substrate for Akt1, raising questions about the dynamics between HuD-mediated translational stimulation and its association with active Akt1. Here, we demonstrate that active Akt1 interacts with the cap-binding complex via HuD. We identify key amino acids in linker region of HuD responsible for Akt1 interaction, leading to the generation of two point-mutated HuD variants: one that is incapable of binding to Akt1 and another that can interact with Akt1 regardless of its phosphorylation status. In vitro translation assays using these mutants reveal that HuD-mediated translation stimulation is independent of its binding to Akt1. In addition, it is evident that the interaction between HuD and active Akt1 is essential for HuD-induced neurite outgrowth, whereas a HuD mutant capable of binding to any form of Akt1 leads to aberrant neurite development. Collectively, our results revisit the understanding of the HuD-Akt1 interaction in translation and suggest that this interaction contributes to HuD-mediated neurite outgrowth via a unique molecular mechanism distinct from translation regulation.

Disruption of the Mammalian Ccr4-Not Complex Contributes to Transcription-Mediated Genome Instability.

The mammalian Ccr4-Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. It is involved in the control of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, and nuclear RNA surveillance; the Ccr4-Not complex also plays a central role in the regulation of mRNA decay. Growing evidence suggests that gene transcription has a vital role in shaping the landscape of genome replication and is also a potent source of replication stress and genome instability. Here, we have examined the effects of the inactivation of the Ccr4-Not complex, via the depletion of the scaffold subunit CNOT1, on DNA replication and genome integrity in mammalian cells. In CNOT1-depleted cells, the elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which, together with R-loop accumulation, results in replication fork slowing, DNA damage, and senescence. Furthermore, we have shown that the stability of TBP mRNA increases in the absence of CNOT1, which may explain its elevated protein expression in CNOT1-depleted cells. Finally, we have shown the activation of mitogen-activated protein kinase signalling as evidenced by ERK1/2 phosphorylation in the absence of CNOT1, which may be responsible for the observed cell cycle arrest at the border of G1/S.

Neuronal RNA-Binding Protein HuD Interacts with Translation Initiation Factor eIF3.

Translation initiation is the rate-limiting step of protein synthesis and is the main target of translation regulation. RNA-binding proteins (RBPs) are key mediators of the spatiotemporal control of translation and are critical for cell proliferation, development, and differentiation. We have previously shown that HuD, one of the neuronal RBPs, enhances cap-dependent translation through the direct interaction with eukaryotic initiation factor 4A (eIF4A) and poly(A) tail using a HeLa-derived in vitro translation system. We have also found that translation stimulation of HuD is essential for HuD-induced neurite outgrowth in PC12 cells. However, it remains unclear how HuD is involved in the regulation of translation initiation. Here, we report that HuD binds to eukaryotic initiation factor 3 (eIF3) via the eIF3b subunit, which belongs to the functional core of mammalian eIF3. eIF3 plays an essential role in recruiting the 40S ribosomal subunit onto mRNA in translation initiation. We hypothesize that the interaction between HuD and eIF3 stabilizes the translation initiation complex and increases translation efficiency. We also showed that the linker region of HuD is required for the interaction with eIF3b. Moreover, we found that eIF3b-binding region of HuD is conserved in all Hu proteins (HuB, HuC, HuD, and HuR). These data might also help to explain how Hu proteins stimulate translation in a cap- and poly(A)-dependent way.

Regulation of CCR4-NOT complex deadenylase activity and cellular responses by MK2-dependent phosphorylation of CNOT2.

CCR4-NOT complex-mediated mRNA deadenylation serves critical functions in multiple biological processes, yet how this activity is regulated is not fully understood. Here, we show that osmotic stress induces MAPKAPK-2 (MK2)-mediated phosphorylation of CNOT2. Programmed cell death is greatly enhanced by osmotic stress in CNOT2-depleted cells, indicating that CNOT2 is responsible for stress resistance of cells. Although wild-type (WT) and non-phosphorylatable CNOT2 mutants reverse this sensitivity, a phosphomimetic form of CNOT2, in which serine at the phosphorylation site is replaced with glutamate, does not have this function. We also show that mRNAs have elongated poly(A) tails in CNOT2-depleted cells and that introduction of CNOT2 WT or a non-phosphorylatable mutant, but not phosphomimetic CNOT2, renders their poly(A) tail lengths comparable to those in control HeLa cells. Consistent with this, the CCR4-NOT complex containing phosphomimetic CNOT2 exhibits less deadenylase activity than that containing CNOT2 WT. These data suggest that CCR4-NOT complex deadenylase activity is regulated by post-translational modification, yielding dynamic control of mRNA deadenylation.

Translation Initiation Regulated by RNA-Binding Protein in Mammals: The Modulation of Translation Initiation Complex by Trans-Acting Factors.

Protein synthesis is tightly regulated at each step of translation. In particular, the formation of the basic cap-binding complex, eukaryotic initiation factor 4F (eIF4F) complex, on the 5' cap structure of mRNA is positioned as the rate-limiting step, and various cis-elements on mRNA contribute to fine-tune spatiotemporal protein expression. The cis-element on mRNAs is recognized and bound to the trans-acting factors, which enable the regulation of the translation rate or mRNA stability. In this review, we focus on the molecular mechanism of how the assembly of the eIF4F complex is regulated on the cap structure of mRNAs. We also summarize the fine-tuned regulation of translation initiation by various trans-acting factors through cis-elements on mRNAs.

Corrigendum: Emerging Evidence of Translational Control by AU-Rich Element-Binding Proteins.

[This corrects the article DOI: 10.3389/fgene.2019.00332.].

Sequestration of the PKC ortholog Pck2 in stress granules as a feedback mechanism of MAPK signaling in fission yeast.

Protein kinase C (PKC) signaling is a highly conserved signaling module that plays a central role in a myriad of physiological processes, ranging from cell proliferation to cell death, via various signaling pathways, including MAPK signaling. Stress granules (SGs) are non-membranous cytoplasmic foci that aggregate in cells exposed to environmental stresses. Here, we explored the role of SGs in PKC/MAPK signaling activation in fission yeast. High-heat stress (HHS) induced Pmk1 MAPK activation and Pck2 translocation from the cell tips into poly(A)-binding protein (Pabp)-positive SGs. Pck2 dispersal from the cell tips required Pck2 kinase activity, and constitutively active Pck2 exhibited increased translocation to SGs. Importantly, Pmk1 deletion impaired Pck2 recruitment to SGs, indicating that MAPK activation stimulates Pck2 SG translocation. Consistently, HHS-induced SGs delayed Pck2 relocalization at the cell tips, thereby blocking subsequent Pmk1 reactivation after recovery from HHS. HHS partitioned Pck2 into the Pabp-positive SG-containing fraction, which resulted in reduced Pck2 abundance and kinase activity in the soluble fraction. Taken together, these results indicate that MAPK-dependent Pck2 SG recruitment serves as a feedback mechanism to intercept PKC/MAPK activation induced by HHS, which might underlie PKC-related diseases.

ARE-binding protein ZFP36L1 interacts with CNOT1 to directly repress translation via a deadenylation-independent mechanism.

Eukaryotic gene expression can be spatiotemporally tuned at the post-transcriptional level by cis-regulatory elements in mRNA sequences. An important example is the AU-rich element (ARE), which induces mRNA destabilization in a variety of biological contexts in mammals and can also mediate translational control. Regulation is mediated by trans-acting factors that recognize the ARE, such as Tristetraprolin (TTP) and BRF1/ZFP36L1. Although both proteins can destabilize their target mRNAs through the recruitment of the CCR4-NOT deadenylation complex, TTP also directly regulates translation. Whether ZFP36L1 can directly repress translation remains unknown. Here, we used an in vitro translation system derived from mammalian cell lines to address this key mechanistic issue in ARE regulation by ZFP36L1. Functional assays with mutant proteins reveal that ZFP36L1 can repress translation via AU-Rich elements independent of deadenylation. ZFP36L1-mediated translation repression requires interaction between ZFP36L1 and CNOT1, suggesting that it might use a repression mechanism similar to either TPP or miRISC. However, several lines of evidence suggest that the similarity ends there. Unlike, TTP, it does not efficiently interact with either 4E-HP or GIGYF2, suggesting it does not repress translation by recruiting these proteins to the mRNA cap. Moreover, ZFP36L1 could not repress ECMV-IRES driven translation and was resistant to pharmacological eIF4A inhibitor silvestrol, suggesting fundamental differences with miRISC repression via eIF4A. Collectively, our results reveal that ZFP36L1 represses translation directly and suggest that it does so via a novel mechanism distinct from other translational regulators that interact with the CCR4-NOT deadenylase complex.

Codon bias confers stability to human mRNAs.

Codon bias has been implicated as one of the major factors contributing to mRNA stability in several model organisms. However, the molecular mechanisms of codon bias on mRNA stability remain unclear in humans. Here, we show that human cells possess a mechanism to modulate RNA stability through a unique codon bias. Bioinformatics analysis showed that codons could be clustered into two distinct groups-codons with G or C at the third base position (GC3) and codons with either A or T at the third base position (AT3): the former stabilizing while the latter destabilizing mRNA. Quantification of codon bias showed that increased GC3-content entails proportionately higher GC-content. Through bioinformatics, ribosome profiling, and in vitro analysis, we show that decoupling the effects of codon bias reveals two modes of mRNA regulation, one GC3- and one GC-content dependent. Employing an immunoprecipitation-based strategy, we identify ILF2 and ILF3 as RNA-binding proteins that differentially regulate global mRNA abundances based on codon bias. Our results demonstrate that codon bias is a two-pronged system that governs mRNA abundance.

ARF6 and AMAP1 are major targets of KRAS and TP53 mutations to promote invasion, PD-L1 dynamics, and immune evasion of pancreatic cancer.

Although KRAS and TP53 mutations are major drivers of pancreatic ductal adenocarcinoma (PDAC), the incurable nature of this cancer still remains largely elusive. ARF6 and its effector AMAP1 are often overexpressed in different cancers and regulate the intracellular dynamics of integrins and E-cadherin, thus promoting tumor invasion and metastasis when ARF6 is activated. Here we show that the ARF6-AMAP1 pathway is a major target by which KRAS and TP53 cooperatively promote malignancy. KRAS was identified to promote eIF4A-dependent ARF6 mRNA translation, which contains a quadruplex structure at its 5'-untranslated region, by inducing TEAD3 and ETV4 to suppress PDCD4; and also eIF4E-dependent AMAP1 mRNA translation, which contains a 5'-terminal oligopyrimidine-like sequence, via up-regulating mTORC1. TP53 facilitated ARF6 activation by platelet-derived growth factor (PDGF), via its known function to promote the expression of PDGF receptor ß (PDGFRß) and enzymes of the mevalonate pathway (MVP). The ARF6-AMAP1 pathway was moreover essential for PDGF-driven recycling of PD-L1, in which KRAS, TP53, eIF4A/4E-dependent translation, mTOR, and MVP were all integral. We moreover demonstrated that the mouse PDAC model KPC cells, bearing KRAS/TP53 mutations, express ARF6 and AMAP1 at high levels and that the ARF6-based pathway is closely associated with immune evasion of KPC cells. Expression of ARF6 pathway components statistically correlated with poor patient outcomes. Thus, the cooperation among eIF4A/4E-dependent mRNA translation and MVP has emerged as a link by which pancreatic driver mutations may promote tumor cell motility, PD-L1 dynamics, and immune evasion, via empowering the ARF6-based pathway and its activation by external ligands.

Emerging Evidence of Translational Control by AU-Rich Element-Binding Proteins.

RNA-binding proteins (RBPs) are key regulators of posttranscriptional gene expression and control many important biological processes including cell proliferation, development, and differentiation. RBPs bind specific motifs in their target mRNAs and regulate mRNA fate at many steps. The AU-rich element (ARE) is one of the major cis-regulatory elements in the 3' untranslated region (UTR) of labile mRNAs. Many of these encode factors requiring very tight regulation, such as inflammatory cytokines and growth factors. Disruption in the control of these factors' expression can cause autoimmune diseases, developmental disorders, or cancers. Therefore, these mRNAs are strictly regulated by various RBPs, particularly ARE-binding proteins (ARE-BPs). To regulate mRNA metabolism, ARE-BPs bind target mRNAs and affect some factors on mRNAs directly, or recruit effectors, such as mRNA decay machinery and protein kinases to target mRNAs. Importantly, some ARE-BPs have stabilizing roles, whereas others are destabilizing, and ARE-BPs appear to compete with each other when binding to target mRNAs. The function of specific ARE-BPs is modulated by posttranslational modifications (PTMs) including methylation and phosphorylation, thereby providing a means for cellular signaling pathways to regulate stability of specific target mRNAs. In this review, we summarize recent studies which have revealed detailed molecular mechanisms of ARE-BP-mediated regulation of gene expression and also report on the importance of ARE-BP function in specific physiological contexts and how this relates to disease. We also propose an mRNP regulatory network based on competition between stabilizing ARE-BPs and destabilizing ARE-BPs.

Translation of Hepatitis A Virus IRES Is Upregulated by a Hepatic Cell-Specific Factor.

Many viruses strongly prefer to infect certain cell types, a phenomenon known as "tropism." Understanding tropism's molecular basis is important for the design of vaccines and antiviral therapy. A common mechanism involves viral protein interactions with cell-specific surface receptors, but intracellular mechanisms involving translation have also been described. In this report, we focus on Hepatitis A Virus (HAV) tissue tropism from the standpoint of the translational machinery. HAV genomic RNA, like other positive stranded RNA viruses, is devoid of a cap structure and its translation is driven by highly structured RNA sequences termed internal ribosome entry site (IRES) in the 5' untranslated region (UTR). Unlike most viral IRESs, HAV IRES-mediated translation requires eIF4E and the 3' end of HAV RNA is polyadenylated. However, the molecular mechanism of HAV IRES-mediated translation initiation remains poorly understood. We analyzed HAV-IRES-mediated translation in a cell-free system derived from either non-hepatic cells (HeLa) or hepatoma cells (Huh-7) that enables investigation of the contribution of the cap and the poly(A) tail. This revealed that HAV IRES-mediated translation activity in hepatoma cell extracts is higher as compared to extracts derived from a non-hepatic line. Our data suggest that HAV IRES-mediated translation is upregulated by a hepatic cell-specific activator in a poly(A) tail-independent manner.

Identification of matrix metalloproteinase 9-interacting sequences in staphylococcal superantigen-like protein 5.

Staphylococcal superantigen like 5 (SSL5) is an exotoxin produced by S. aureus and has a strong inhibitory effect on MMP-9 enzymatic activity. However, the mechanism of inhibition remains unclear. We sought to identify the responsible regions of SSL5 for the interaction with MMP-9 by comparing a series of domain swap and deletion mutants of SSL5. Binding analyses revealed that SSL5 had two regions for binding to MMP-9 catalytic domain, ß1-3 region (25SKELKNVTGY RYSKGGKHYL IFDKNRKFTR VQIFGK60) in N-terminal half and α4ß9 region (138KELDFKLRQY LIQNFDLYKK FPKDSKIKVI MKD170) in C-terminal half. The collagen binding domain and zinc-chelating histidine residues of MMP-9 were not essential for the specific binding to SSL5. The domain swap mutants of SSL5 that conserved ß1-3 but not α4ß9 region inhibited the gelatinolysis by MMP-9, and the mutant of SSL7 that substituted ß1-3 region to that of SSL5 acquired the binding and inhibitory activity. Furthermore, the polypeptide that harbored ß1-3 region of SSL5 inhibited gelatinolysis by MMP-9. Taken together, SSL5 inhibits the MMP9 activity through binding to the catalytic domain, and the ß1-3 region is responsible for the inhibition of proteolytic activity of MMP-9.

The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function.

Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis.

Spatial regulation of the KH domain RNA-binding protein Rnc1 mediated by a Crm1-independent nuclear export system in Schizosaccharomyces pombe.

RNA-binding proteins (RBPs) play important roles in the posttranscriptional regulation of gene expression, including mRNA stability, transport and translation. Fission yeast rnc1+ encodes a K Homology (KH)-type RBP, which binds and stabilizes the Pmp1 MAPK phosphatase mRNA thereby suppressing the Cl- hypersensitivity of calcineurin deletion and MAPK signaling mutants. Here, we analyzed the spatial regulation of Rnc1 and discovered a putative nuclear export signal (NES)Rnc1 , which dictates the cytoplasmic localization of Rnc1 in a Crm1-independent manner. Notably, mutations in the NESRnc1 altered nucleocytoplasmic distribution of Rnc1 and abolished its function to suppress calcineurin deletion, although the Rnc1 NES mutant maintains the ability to bind Pmp1 mRNA. Intriguingly, the Rnc1 NES mutant destabilized Pmp1 mRNA, suggesting the functional importance of the Rnc1 cytoplasmic localization. Mutation in Rae1, but not Mex67 deletion or overproduction, induced Rnc1 accumulation in the nucleus, suggesting that Rnc1 is exported from the nucleus to the cytoplasm via the mRNA export pathway involving Rae1. Importantly, mutations in the Rnc1 KH-domains abolished the mRNA-binding ability and induced nuclear localization, suggesting that Rnc1 may be exported from the nucleus together with its target mRNAs. Collectively, the functional Rae1-dependent mRNA export system may influence the cytoplasmic localization and function of Rnc1.

Kribellosides, novel RNA 5'-triphosphatase inhibitors from the rare actinomycete Kribbella sp. MI481-42F6.

Yeast capping enzymes differ greatly from those of mammalian, both structurally and mechanistically. Yeast-type capping enzyme repressors are therefore candidate antifungal drugs. The 5'-guanine-N7 cap structure of mRNAs are an essential feature of all eukaryotic organisms examined to date and is the first co-transcriptional modification of cellular pre-messenger RNA. Inhibitors of the RNA 5'-triphosphatase in yeast are likely to show fungicidal effects against pathogenic yeast such as Candida. We discovered a new RNA 5'-triphosphatase inhibitor, designated as the kribellosides, by screening metabolites from actinomycetes. Kribellosides belong to the alkyl glyceryl ethers. These novel compounds inhibit the activity of Cet1p (RNA 5'-triphosphatase) from Saccharomyces cerevisiae in vitro with IC50s of 5-8 µM and show antifungal activity with MICs ranging from 3.12 to 100 µg ml-1 against S. cerevisiae.

The coupled and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation.

In mammals, spatiotemporal control of protein synthesis plays a key role in the post-transcriptional regulation of gene expression during cell proliferation, development and differentiation and RNA-binding proteins (RBPs) and microRNAs (miRNAs) are required for this phenomenon. RBPs and miRNAs control the levels of mRNA protein products by regulating mRNA stability and translation. Recent studies have shown that RBPs and miRNAs simultaneously regulate mRNA stability and translation, and that the differential functions of RBPs and miRNAs are dependent on their interaction partners. Here, we summarize the coupled- and uncoupled mechanisms by which trans-acting factors regulate mRNA stability and translation.

PI4K-beta and MKNK1 are regulators of hepatitis C virus IRES-dependent translation.

Cellular translation is down-regulated by host antiviral responses. Picornaviridae and Flaviviridae including hepatitis C virus (HCV) evade this process using internal ribosomal entry sequences (IRESs). Although HCV IRES translation is a prerequisite for HCV replication, only few host factors critical for IRES activity are known and the global regulator network remains largely unknown. Since signal transduction is an import regulator of viral infections and the host antiviral response we combined a functional RNAi screen targeting the human signaling network with a HCV IRES-specific reporter mRNA assay. We demonstrate that the HCV host cell cofactors PI4K and MKNK1 are positive regulators of HCV IRES translation representing a novel pathway with a functional relevance for the HCV life cycle and IRES-mediated translation of viral RNA.