Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5'-terminal regions of cap0-, cap1- and 5'ppp- mRNAs.

Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA's 5'-terminal 'cap'. The minimal 'cap0' consists of N7-methylguanosine linked to the first nucleotide via a 5'-5' triphosphate (ppp) bridge. Cap0 is further modified by 2'-O-methylation of the next two riboses, yielding 'cap1' (m7GpppNmN) and 'cap2' (m7GpppNmNm). However, some viral RNAs lack 2'-O-methylation, whereas others contain only ppp- at their 5'-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5'ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2'-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5'-terminal regions of 5'ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5'-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5'ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations

YB-1 promotes microtubule assembly in vitro through interaction with tubulin and microtubules

<p>Abstract</p> <p>Background</p> <p>YB-1 is a major regulator of gene expression in eukaryotic cells. In addition to its role in transcription, YB-1 plays a key role in translation and stabilization of mRNAs.</p> <p>Results</p> <p>We show here that YB-1 interacts with tubulin and microtubules and stimulates microtubule assembly <it>in vitro</it>. High resolution imaging via electron and atomic force microscopy revealed that microtubules assembled in the presence of YB-1 exhibited a normal single wall ultrastructure and indicated that YB-1 most probably coats the outer microtubule wall. Furthermore, we found that YB-1 also promotes the assembly of MAPs-tubulin and subtilisin-treated tubulin. Finally, we demonstrated that tubulin interferes with RNA:YB-1 complexes.</p> <p>Conclusion</p> <p>These results suggest that YB-1 may regulate microtubule assembly <it>in vivo </it>and that its interaction with tubulin may contribute to the control of mRNA translation.</p

Suppression of Her2/neu expression through ILK inhibition is regulated by a pathway involving TWIST and YB-1

In a previous study it was found that the therapeutic effects of QLT0267, a small molecule inhibitor of integrin-linked kinase (ILK), were influenced by Her2/neu expression. To understand how inhibition or silencing of ILK influences Her2/neu expression, Her2/neu signaling was evaluated in six Her2/neu-positive breast cancer cell lines (LCC6Her2, MCF7Her2, SKBR3, BT474, JIMT-1 and KPL-4). Treatment with QLT0267 engendered suppression (32–87%) of total Her2/neu protein in these cells. Suppression of Her2/neu was also observed following small interfering RNA-mediated silencing of ILK expression. Time course studies suggest that ILK inhibition or silencing caused transient decreases in P-AKTser473, which were not temporally related to Her2/neu downregulation. Attenuation of ILK activity or expression was, however, associated with decreases in YB-1 (Y-box binding protein-1) protein and transcript levels. YB-1 is a known transcriptional regulator of Her2/neu expression, and in this study it is demonstrated that inhibition of ILK activity using QLT0267 decreased YB-1 promoter activity by 50.6%. ILK inhibition was associated with changes in YB-1 localization, as reflected by localization of cytoplasmic YB-1 into stress granules. ILK inhibition also suppressed TWIST (a regulator of YB-1 expression) protein expression. To confirm the role of ILK on YB-1 and TWIST, cells were engineered to overexpress ILK. This was associated with a fourfold increase in the level of YB-1 in the nucleus, and a 2- and 1.5-fold increase in TWIST and Her2/neu protein levels, respectively. Taken together, these data indicate that ILK regulates the expression of Her2/neu through TWIST and YB-1, lending support to the use of ILK inhibitors in the treatment of aggressive Her2/neu-positive tumors

Formation of Amyloid-Like Fibrils by Y-Box Binding Protein 1 (YB-1) Is Mediated by Its Cold Shock Domain and Modulated by Disordered Terminal Domains

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions

A Conserved Stem Loop Motif in the 5′Untranslated Region Regulates Transforming Growth Factor-β1 Translation

Transforming growth factor-β1 (TGF-β1) regulates cellular proliferation, differentiation, migration, and survival. The human TGF-β1 transcript is inherently poorly translated, and translational activation has been documented in relation to several stimuli. In this paper, we have sought to identify in cis regulatory elements within the TGF-β1 5′Untranslated Region (5′UTR). In silico analysis predicted formation of stable secondary structure in a G/C-rich element between nucleotides +77 to +106, and demonstrated that this element is highly conserved across species. Circular dichroism spectroscopy confirmed the presence of secondary structure in this region. The proximal 5′UTR was inhibitory to translation in reporter gene experiments, and mutation of the secondary structure motif increased translational efficiency. Translational regulation of TGF-β1 mRNA is linked to altered binding of YB-1 protein to its 5′UTR. Immunoprecipitation-RT-qPCR demonstrated a high basal association of YB-1 with TGF-β1 mRNA. However, mutation of the secondary structure motif did not prevent interaction of YB-1 with the 5′UTR, suggesting that YB-1 binds to this region due to its G/C-rich composition, rather than a specific, sequence-dependent, binding site. These data identify a highly conserved element within the TGF-β1 5′UTR that forms stable secondary structure, and is responsible for the inherent low translation efficiency of this cytokine

Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling

Eukaryotic translation initiation begins with ribosomal recruitment of aminoacylated initiator tRNA (Met-tRNAMeti) by eukaryotic initiation factor eIF2. In cooperation with eIF3, eIF1, and eIF1A, Met-tRNAMeti/eIF2/GTP binds to 40S subunits yielding 43S preinitiation complexes that attach to the 5′-terminal region of mRNAs and then scan to the initiation codon to form 48S initiation complexes with established codon–anticodon base-pairing. Stress-activated phosphorylation of eIF2α reduces the level of active eIF2, globally inhibiting translation. However, translation of several viral mRNAs, including Sindbis virus (SV) 26S mRNA and mRNAs containing hepatitis C virus (HCV)-like IRESs, is wholly or partially resistant to inhibition by eIF2 phosphorylation, despite requiring Met-tRNAMeti. Here we report the identification of related proteins that individually (Ligatin) or together (the oncogene MCT-1 and DENR, which are homologous to N-terminal and C-terminal regions of Ligatin, respectively) promote efficient eIF2-independent recruitment of Met-tRNAMeti to 40S/mRNA complexes, if attachment of 40S subunits to the mRNA places the initiation codon directly in the P site, as on HCV-like IRESs and, as we show here, SV 26S mRNA. In addition to their role in initiation, Ligatin and MCT-1/DENR can promote release of deacylated tRNA and mRNA from recycled 40S subunits after ABCE1-mediated dissociation of post-termination ribosomes

Interplay between Y-box-binding protein 1 (YB-1) and poly(A) binding protein (PABP) in specific regulation of YB-1 mRNA translation

YB-1 is a DNA- and RNA-binding protein that regulates expression of many important genes. Its deficiency or excess may pose threats, including malignant cellular transformation and metastasis, which explains the necessity of strict control over its amount at every level. As we showed previously, the 3′ untranslated region (UTR) of YB-1 mRNA contains a regulatory element specifically binding to YB-1 and PABP (PABPC1). Also, we showed that YB-1 selectively inhibits YB-1 mRNA translation, while PABP stimulates it in a poly(A) tail-independent manner. It was suggested that regulation of YB-1 mRNA translation involves competition between PABP and YB-1 for binding to the regulatory element. Here we offer cogent evidence for this model and add novel details to the mechanism of regulation of YB-1 synthesis. In experiments on regulatory element deletion we showed that it is this element that is responsible for a specific effect of YB-1 and PABP on YB-1 mRNA translation. Mutations eliminating only specific YB-1 affinity for this element suppressed the inhibitory effect of YB-1 and concurrently dramatically decreased the PABP stimulating effect. Mutations reducing only specific PABP affinity for this element, as well as spatial separation of the YB-1- and PABP binding sites, did not affect the YB-1 inhibitory action but completely abolished the positive PABP effect. Together, these results unambiguously prove direct inhibitory action of YB-1 on its mRNA translation, while the positive effect of PABP is realized through displacing YB-1 from the regulatory element