Lev P. Ovchinnikov

Translational Activation of Snail1 and Other Developmentally Regulated Transcription Factors by YB-1 Promotes an Epithelial-Mesenchymal Transition

Increased expression of the transcription/translation regulatory protein Y-box binding protein-1 (YB-1) is associated with cancer aggressiveness, particularly in breast carcinoma. Here we establish that YB-1 levels are elevated in invasive breast cancer cells and correlate with reduced expression of E-cadherin and poor patient survival. Enforced expression of YB-1 in noninvasive breast epithelial cells induced an epithelial-mesenchymal transition (EMT) accompanied by enhanced metastatic potential and reduced proliferation rates. YB-1 directly activates cap-independent translation of messenger RNAs encoding Snail1 and other transcription factors implicated in downregulation of epithelial and growth-related genes and activation of mesenchymal genes. Hence, translational regulation by YB-1 is a restriction point enabling coordinated expression of a network of EMT-inducing transcription factors, likely acting together to promote metastatic spread.

The major mRNA‐associated protein YB‐1 is a potent 5′ cap‐dependent mRNA stabilizer

mRNA silencing and storage play an important role in gene expression under diverse circumstances, such as throughout early metazoan development and in response to many types of environmental stress. Here we demonstrate that the major mRNA‐associated protein YB‐1, also termed p50, is a potent cap‐dependent mRNA stabilizer. YB‐1 addition or overexpression dramatically increases mRNA stability in vitro and in vivo, whereas YB‐1 depletion results in accelerated mRNA decay. The cold shock domain of YB‐1 is responsible for the mRNA stabilizing activity, and a blocked mRNA 5′ end is required for YB‐1‐mediated stabilization. Significantly, exogenously added YB‐1 destabilizes the interaction of the cap binding protein, eIF4E, with the mRNA cap structure. Conversely, sequestration of eIF4E from the cap increases the association of endogenous YB‐1 with mRNA at or near the cap, and significantly enhances mRNA stability. These data support a model whereby down‐regulation of eIF4E activity or increasing the YB‐1 mRNA binding activity or concentration in cells activates a general default pathway for mRNA stabilization.

Akt-Mediated YB-1 Phosphorylation Activates Translation of Silent mRNA Species

ABSTRACT YB-1 is a broad-specificity RNA-binding protein that is involved in regulation of mRNA transcription, splicing, translation, and stability. In both germinal and somatic cells, YB-1 and related proteins are major components of translationally inactive messenger ribonucleoprotein particles (mRNPs) and are mainly responsible for storage of mRNAs in a silent state. However, mechanisms regulating the repressor activity of YB-1 are not well understood. Here we demonstrate that association of YB-1 with the capped 5′ terminus of the mRNA is regulated via phosphorylation by the serine/threonine protein kinase Akt. In contrast to its nonphosphorylated form, phosphorylated YB-1 fails to inhibit cap-dependent but not internal ribosome entry site-dependent translation of a reporter mRNA in vitro. We also show that similar to YB-1, Akt is associated with inactive mRNPs and that activated Akt may relieve translational repression of the YB-1-bound mRNAs. Using Affymetrix microarrays, we found that many of the YB-1-associated messages encode stress- and growth-related proteins, raising the intriguing possibility that Akt-mediated YB-1 phosphorylation could, in part, increase production of proteins regulating cell proliferation, oncogenic transformation, and stress response.

Nucleocytoplasmic transport of proteins.

In eukaryotic cells, the movement of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)—a large protein complex spanning the nuclear envelope. The nuclear transport of proteins is usually mediated by a family of transport receptors known as karyopherins. Karyopherins bind to their cargoes via recognition of nuclear localization signal (NLS) for nuclear import or nuclear export signal (NES) for export to form a transport complex. Its transport through NPC is facilitated by transient interactions between the karyopherins and NPC components. The interactions of karyopherins with their cargoes are regulated by GTPase Ran. In the current review, we describe the NPC structure, NLS, and NES, as well as the model of classic Ran-dependent transport, with special emphasis on existing alternative mechanisms; we also propose a classification of the basic mechanisms of protein transport regulation.

Y-box-binding protein 1 (YB-1) and its functions

This review describes the structure and functions of Y-box binding protein 1 (YB-1) and its homologs. Interactions of YB-1 with DNA, mRNAs, and proteins are considered. Data on the participation of YB-1 in DNA reparation and transcription, mRNA splicing and translation are systematized. Results on interactions of YB-1 with cytoskeleton components and its possible role in mRNA localization are discussed. Data on intracellular distribution of YB-1, its redistribution between the nucleus and the cytoplasm, and its secretion and extracellular functions are summarized. The effect of YB-1 on cell differentiation, its involvement in extra- and intracellular signaling pathways, and its role in early embryogenesis are described. The mechanisms of regulation of YB-1 expression in the cell are presented. Special attention is paid to the involvement of YB-1 in oncogenic cell transformation, multiple drug resistance, and dissemination of tumors. Both the oncogenic and antioncogenic activities of YB-1 are reviewed. The potential use of YB-1 in diagnostics and therapy as an early cancer marker and a molecular target is discussed.

General RNA binding proteins render translation cap dependent.

Translation in rabbit reticulocyte lysate is relatively independent of the presence of the mRNA m7G cap structure and the cap binding protein, eIF‐4E. In addition, initiation occurs frequently at spurious internal sites. Here we show that a critical parameter which contributes to cap‐dependent translation is the amount of general RNA binding proteins in the extract. Addition of several general RNA binding proteins, such as hnRNP A1, La autoantigen, pyrimidine tract binding protein (hnRNP I/PTB) and the major core protein of cytoplasmic mRNP (p50), rendered translation in a rabbit reticulocyte lysate cap dependent. These proteins drastically inhibited the translation of an uncapped mRNA, but had no effect on translation of a capped mRNA. Based on these and other results, we suggest that one function of general mRNA binding proteins in the cytoplasm is to promote ribosome binding by a 5′ end, cap‐mediated mechanism, and prevent spurious initiations at aberrant translation start sites.

Proteasome System of Protein Degradation and Processing

In eukaryotic cells, degradation of most intracellular proteins is realized by proteasomes. The substrates for proteolysis are selected by the fact that the gate to the proteolytic chamber of the proteasome is usually closed, and only proteins carrying a special “label” can get into it. A polyubiquitin chain plays the role of the “label”: degradation affects proteins conjugated with a ubiquitin (Ub) chain that consists at minimum of four molecules. Upon entering the proteasome channel, the polypeptide chain of the protein unfolds and stretches along it, being hydrolyzed to short peptides. Ubiquitin per se does not get into the proteasome, but, after destruction of the “labeled” molecule, it is released and labels another molecule. This process has been named “Ub-dependent protein degradation”. In this review we systematize current data on the Ub-proteasome system, describe in detail proteasome structure, the ubiquitination system, and the classical ATP/Ub-dependent mechanism of protein degradation, as well as try to focus readers’ attention on the existence of alternative mechanisms of proteasomal degradation and processing of proteins. Data on damages of the proteasome system that lead to the development of different diseases are given separately.

Proteasome-mediated cleavage of the Y-box-binding protein 1 is linked to DNA-damage stress response

YB‐1 is a DNA/RNA‐binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA‐ and RNA‐dependent events is determined by its localization in the cell. Distribution of YB‐1 between the nucleus and the cytoplasm is known to be dependent on nuclear targeting and cytoplasmic retention signals located within the C‐terminal portion of YB‐1. Here, we report that YB‐1 undergoes a specific proteolytic cleavage by the 20S proteasome, which splits off the C‐terminal 105‐amino‐acid‐long YB‐1 fragment containing a cytoplasmic retention signal. Cleavage of YB‐1 by the 20S proteasome in vitro appears to be ubiquitin‐ and ATP‐independent, and is abolished by the association of YB‐1 with messenger RNA. We also found that genotoxic stress triggers a proteasome‐mediated cleavage of YB‐1 in vivo and leads to accumulation of the truncated protein in nuclei of stressed cells. Endoproteolytic activity of the proteasome may therefore play an important role in regulating YB‐1 functioning, especially under certain stress conditions.

Y-box binding protein 1: providing a new angle on translational regulation.

Current models of translational regulation are mostly focused on how translational factors engage a messenger mRNA to the ribosome to initiate translation. Since the majority of mRNAs in eukaryotes are translated in a cap-dependent manner, the mRNA 5’ cap-binding protein eIF4E was characterized as a key player responsible for the recruitment of mRNAs to the initiation complex. The availability of eIF4E is believed to be especially critical for translational activation of mRNAs with extensive secondary structures in their 5’UTRs, many of which code for labile regulatory proteins essential for cell growth or viability. Surprisingly, little attention is paid to the other side of translational control, e.g., to define mechanisms responsible for translational silencing and storage of the above messages. In this review, we discuss the possibility that eIF4E per se may not be sufficient to release mRNAs from translational block. We found that many growth- and stress-related mRNAs are associated with the translational repressor YB-1, which can compete with the eIF4E-driven translation initiation complex for binding to the capped 5’ mRNA terminus. Moreover, the cap-dependent repressor activity of YB-1 appears to be negatively regulated via Akt-mediated phosphorylation of the Ser-102 residue of YB-1. Taken together with recent evidence suggesting that translational activation of growth-related messages is a primary cellular response to activation of Ras-Erk and PI3K-Akt signaling pathways, our data suggest that differential expression of specific mRNA subsets is regulated by the PI3K-Akt pathway and achieved via coordinated activation of the components of translational machinery and inactivation of general translational repressors such as YB-1.

YB-1 protein: functions and regulation.

The Y‐box binding protein 1 (YB‐1, YBX1) is a member of the family of DNA‐ and RNA‐binding proteins with an evolutionarily ancient and conserved cold shock domain. It falls into a group of intrinsically disordered proteins that do not follow the classical rule ‘one protein–one function’ but introduce a novel principle stating that a disordered structure suggests many functions. YB‐1 participates in a wide variety of DNA/RNA‐dependent events, including DNA reparation, pre‐mRNA transcription and splicing, mRNA packaging, and regulation of mRNA stability and translation. At the cell level, the multiple activities of YB‐1 are manifested as its involvement in cell proliferation and differentiation, stress response, and malignant cell transformation. WIREs RNA 2014, 5:95–110. doi: 10.1002/wrna.1200