Large HBV Surface Protein-Induced Unfolded Protein Response Dynamically Regulates p27 Degradation in Hepatocellular Carcinoma Progression.

Up to 50% of hepatocellular carcinoma (HCC) is caused by hepatitis B virus (HBV) infection, and the surface protein of HBV is essential for the progression of HBV-related HCC. The expression of large HBV surface antigen (LHB) is presented in HBV-associated HCC tissues and is significantly associated with the development of HCC. Gene set enrichment analysis revealed that LHB overexpression regulates the cell cycle process. Excess LHB in HCC cells induced chronic endoplasmic reticulum (ER) stress and was significantly correlated with tumor growth in vivo. Cell cycle analysis showed that cell cycle progression from G1 to S phase was greatly enhanced in vitro. We identified intensive crosstalk between ER stress and cell cycle progression in HCC. As an important regulator of the G1/S checkpoint, p27 was transcriptionally upregulated by transcription factors ATF4 and XBP1s, downstream of the unfolded protein response pathway. Moreover, LHB-induced ER stress promoted internal ribosome-entry-site-mediated selective translation of p27, and E3 ubiquitin ligase HRD1-mediated p27 ubiquitination and degradation. Ultimately, the decrease in p27 protein levels reduced G1/S arrest and promoted the progress of HCC by regulating the cell cycle.

Selective regulation in ribosome biogenesis and protein production for efficient viral translation.

As intracellular parasites, viruses depend heavily on host cell structures and their functions to complete their life cycle and produce new viral particles. Viruses utilize or modulate cellular translational machinery to achieve efficient replication; the role of ribosome biogenesis and protein synthesis in viral replication particularly highlights the importance of the ribosome quantity and/or quality in controlling viral protein synthesis. Recently reported studies have demonstrated that ribosome biogenesis factors (RBFs) and ribosomal proteins (RPs) act as multifaceted regulators in selective translation of viral transcripts. Here we summarize the recent literature on RBFs and RPs and their association with subcellular redistribution, post-translational modification, enzyme catalysis, and direct interaction with viral proteins. The advances described in this literature establish a rationale for targeting ribosome production and function in the design of the next generation of antiviral agents.

Cell-Cycle-Dependent Regulation of Translation: New Interpretations of Old Observations in Light of New Approaches.

It is a long-standing view that global translation varies during the cell cycle and is much lower in mitosis than in other cell-cycle phases. However, the central papers in the literature are not in agreement about the extent of downregulation in mitosis, ranging from a dramatic decrease to only a marginal reduction. Herein, it is argued that the discrepancy derives from technical challenges. Cell-cycle-dependent variations are most conveniently studied in synchronized cells, but the synchronization methods by themselves often evoke stress responses that, in turn, affect translation rates. Further, it is argued that previously reported cell-cycle-dependent changes in the global translation rate to a large extent reflect responses to the synchronization methods. Recent findings strongly suggest that the global translation rate is not regulated in a cell-cycle-dependent manner. Novel techniques allowing a genome-wide analysis of translational profiles suggest that the extent and importance of selective translational regulation associated with cell-cycle transitions have been underestimated. Therefore, the main question is which messenger RNAs (mRNAs) are translated, rather than whether the global translation rate is decreased.

[The mechanisms of the translation of polymerase from HBV pregenomic RNA].

Hepatitis B virus (HBV) is an important pathogen that causes different liver diseases such as viral hepatitis and liver cirrhosis. HBV pregenomic RNA (pgRNA) plays a crucial role in HBV life cycle, which is not only the translation template of core (C) and polymerase (P), but also the template of reverse transcription. The ratio of P protein to core protein is tightly regulated. Since P and core are both translated by pgRNA and the open reading frame (ORF) of P is located downstream of the ORF of core, how to initiate P protein translation is a key scientific question. Previous studies suggest that P can be translated through different mechanisms, such as leaky scanning and reinitiation. In this review, we summarized the proposed mechanisms relevant to the translation of polymerase from HBV pgRNA through literature review and derivation.

eIF2α phosphorylation and the regulation of translation.

We discuss novel insight into the role and consequences of the phosphorylation of the translation initiation factor eIF2α in the context of stress responses and cell-cycle regulation. eIF2α is centrally located to regulate translation and its phosphorylation in response to different environmental challenges is one of the best characterized stress-response pathways. In addition to its role in stress management, eIF2α phosphorylation is also linked to cell-cycle progression and memory consolidation in the nervous system. The best known consequences of eIF2α phosphorylation are downregulation of global translation and stimulation of translation of some mRNAs. However, recent evidence shows that (i) eIF2α phosphorylation is not always required for the downregulation of global translation after exposure to stress and (ii) eIF2α phosphorylation does not necessarily lead to the downregulation of global translation. These results suggest that the textbook view of eIF2α phosphorylation needs to be revised and that there must be additional regulatory mechanisms at play.

Regulation of Translation in the Protozoan Parasite Leishmania.

Leishmaniasis represents a serious health problem worldwide and drug resistance is a growing concern. Leishmania parasites use unusual mechanisms to control their gene expression. In contrast to many other species, they do not have transcriptional regulation. The lack of transcriptional control is mainly compensated by post-transcriptional mechanisms, including tight translational control and regulation of mRNA stability/translatability by RNA-binding proteins. Modulation of translation plays a major role in parasite survival and adaptation to dramatically different environments during change of host; however, our knowledge of fine molecular mechanisms of translation in Leishmania remains limited. Here, we review the current progress in our understanding of how changes in the translational machinery promote parasite differentiation during transmission from a sand fly to a mammalian host, and discuss how translational reprogramming can contribute to the development of drug resistance.

A checkpoint-independent mechanism delays entry into mitosis after UV irradiation.

When cells are exposed to stress they delay entry into mitosis. The most extensively studied mechanism behind this delay is the DNA-damage-induced G2/M checkpoint. Here, we show the existence of an additional stress-response pathway in Schizosaccharomyces pombe that is independent of the classic ATR/Rad3-dependent checkpoint. This novel mechanism delays entry mitosis independently of the spindle assembly checkpoint and the mitotic kinases Fin1, Ark1 and Plo1. The pathway delays activation of the mitotic cyclin-dependent kinase (CDK) Cdc2 after UV irradiation. Furthermore, we demonstrate that translation of the mitotic cyclin Cdc13 is selectively downregulated after UV irradiation, and we propose that this downregulation of Cdc13 contributes to the delayed activation of Cdc2 and the delayed mitosis.

The role of IRES trans-acting factors in carcinogenesis.

Regulation of protein expression through RNA metabolism is a key aspect of cellular homeostasis. Upon specific cellular stresses, distinct transcripts are selectively controlled to modify protein output in order to quickly and appropriately respond to stress. Reprogramming of the translation machinery is one node of this strict control that typically consists of an attenuation of the global, cap-dependent translation and accompanying switch to alternative mechanisms of translation initiation, such as internal ribosome entry site (IRES)-mediated initiation. In cancer, many aspects of the RNA metabolism are frequently misregulated to provide cancer cells with a growth and survival advantage. This includes changes in the expression and function of RNA binding proteins termed IRES trans-acting factors (ITAFs) that are central to IRES translation. In this review, we will examine select emerging, as well as established, ITAFs with important roles in cancer initiation and progression, and in particular their role in IRES-mediated translation. This article is part of a Special Issue entitled: Translation and Cancer.

Customization of the translational complex regulates mRNA-specific translation to control CNS regeneration.

In the adult mammalian central nervous system (CNS), axons fail to regenerate spontaneously after injury because of a combination of extrinsic and intrinsic factors. Despite recent advances targeting the intrinsic regenerative properties of adult neurons, the molecular mechanisms underlying axon regeneration are not fully understood. Here, we uncover a regulatory mechanism that controls the expression of key proteins involved in regeneration at the translational level. Our results show that mRNA-specific translation is critical for promoting axon regeneration. Indeed, we demonstrate that specific ribosome-interacting proteins, such as the protein Huntingtin (HTT), selectively control the translation of a specific subset of mRNAs. Moreover, modulating the expression of these translationally regulated mRNAs is crucial for promoting axon regeneration. Altogether, our findings highlight that selective translation through the customization of the translational complex is a key mechanism of axon regeneration with major implications in the development of therapeutic strategies for CNS repair.

Strategies of Influenza A Virus to Ensure the Translation of Viral mRNAs.

Viruses are obligatorily intracellular pathogens. To generate progeny virus particles, influenza A viruses (IAVs) have to divert the cellular machinery to ensure sufficient translation of viral mRNAs. To this end, several strategies have been exploited by IAVs, such as host gene shutoff, suppression of host innate immune responses, and selective translation of viral mRNAs. Various IAV proteins are responsible for host gene shutoff, e.g., NS1, PA-X, and RdRp, through inhibition of cellular gene transcription, suppression of cellular RNA processing, degradation of cellular RNAs, and blockage of cellular mRNA export from the nucleus. Host shutoff should suppress the innate immune responses and also increase the translation of viral mRNAs indirectly due to the reduced competition from cellular mRNAs for cellular translational machinery. However, many other mechanisms are also responsible for the suppression of innate immune responses by IAV, such as prevention of the detection of the viral RNAs by the RLRs, inhibition of the activities of proteins involved in signaling events of interferon production, and inhibition of the activities of interferon-stimulated genes, mainly through viral NS1, PB1-F2, and PA-X proteins. IAV mRNAs may be selectively translated in favor of cellular mRNAs through interacting with viral and/or cellular proteins, such as NS1, PABPI, and/or IFIT2, in the 5'-UTR of viral mRNAs. This review briefly summarizes the strategies utilized by IAVs to ensure sufficient translation of viral mRNAs focusing on recent developments.

Negative charge in the RACK1 loop broadens the translational capacity of the human ribosome.

Although the roles of initiation factors, RNA binding proteins, and RNA elements in regulating translation are well defined, how the ribosome functionally diversifies remains poorly understood. In their human hosts, poxviruses phosphorylate serine 278 (S278) at the tip of a loop domain in the small subunit ribosomal protein RACK1, thereby mimicking negatively charged residues in the RACK1 loops of dicot plants and protists to stimulate translation of transcripts with 5' poly(A) leaders. However, how a negatively charged RACK1 loop affects ribosome structure and its broader translational output is not known. Here, we show that although ribotoxin-induced stress signaling and stalling on poly(A) sequences are unaffected, negative charge in the RACK1 loop alters the swivel motion of the 40S head domain in a manner similar to several internal ribosome entry sites (IRESs), confers resistance to various protein synthesis inhibitors, and broadly supports noncanonical modes of translation.

Fluorescent Polysome Profiling in Caenorhabditis elegans.

An important but often overlooked aspect of gene regulation occurs at the level of protein translation. Many genes are regulated not only by transcription but by their propensity to be recruited to actively translating ribosomes (polysomes). Polysome profiling allows for the separation of unbound 40S and 60S subunits, 80S monosomes, and actively translating mRNA bound by two or more ribosomes. Thus, this technique allows for actively translated mRNA to be isolated. Transcript abundance can then be compared between actively translated mRNA and all mRNA present in a sample to identify instances of post-transcriptional regulation. Additionally, polysome profiling can be used as a readout of global translation rates by quantifying the proportion of actively translating ribosomes within a sample. Previously established protocols for polysome profiling rely on the absorbance of RNA to visualize the presence of polysomes within the fractions. However, with the advent of flow cells capable of detecting fluorescence, the association of fluorescently tagged proteins with polysomes can be detected and quantified in addition to the absorbance of RNA. This protocol provides detailed instructions on how to perform fluorescent polysome profiling in C. elegans to collect actively translated mRNA, to quantify changes in global translation, and to detect ribosomal binding partners.

Selective Translation of Low Abundance and Upregulated Transcripts in Halobacterium salinarum.

When organisms encounter an unfavorable environment, they transition to a physiologically distinct, quiescent state wherein abundant transcripts from the previous active growth state continue to persist, albeit their active transcription is downregulated. In order to generate proteins for the new quiescent physiological state, we hypothesized that the translation machinery must selectively translate upregulated transcripts in an intracellular milieu crowded with considerably higher abundance transcripts from the previous active growth state. Here, we have analyzed genome-wide changes in the transcriptome (RNA sequencing [RNA-seq]), changes in translational regulation and efficiency by ribosome profiling across all transcripts (ribosome profiling [Ribo-seq]), and protein level changes in assembled ribosomal proteins (sequential window acquisition of all theoretical mass spectra [SWATH-MS]) to investigate the interplay of transcriptional and translational regulation in Halobacterium salinarum as it transitions from active growth to quiescence. We have discovered that interplay of regulatory processes at different levels of information processing generates condition-specific ribosomal complexes to translate preferentially pools of low abundance and upregulated transcripts. Through analysis of the gene regulatory network architecture of H. salinarum, Escherichia coli, and Saccharomyces cerevisiae, we demonstrate that this conditional, modular organization of regulatory programs governing translational systems is a generalized feature across all domains of life.IMPORTANCE Our findings demonstrate conclusively that low abundance and upregulated transcripts are preferentially translated, potentially by environment-specific translation systems with distinct ribosomal protein composition. We show that a complex interplay of transcriptional and posttranscriptional regulation underlies the conditional and modular regulatory programs that generate ribosomes of distinct protein composition. The modular regulation of ribosomal proteins with other transcription, translation, and metabolic genes is generalizable to bacterial and eukaryotic microbes. These findings are relevant to how microorganisms adapt to unfavorable environments when they transition from active growth to quiescence by generating proteins from upregulated transcripts that are in considerably lower abundance relative to transcripts associated with the previous physiological state. Selective translation of transcripts by distinct ribosomes could form the basis for adaptive evolution to new environments through a modular regulation of the translational systems.

Alterations of the translation apparatus during aging and stress response.

Aging is a biological process characterized by the irreversible and time-dependent deterioration of cell functions, tissues, and organs. Accumulating studies in a wide range of species from yeast to human revealed changes associated with the aging process to be conserved throughout evolution. The main characteristics of aging are (i) genomic instability, (ii) loss of telomere function, (iii) epigenetic changes,(iv) increased cellular senescence, (v) depletion of the stem cell pool, (vi) altered intercellular communication and (vii) loss of protein homeostasis. Among the multiple molecular mechanisms underlying aging, alterations of the translation machinery affecting the rate and selectivity of protein biosynthesis seem to play a central role. At the very heart of translation is the ribosome, a multifaceted and universally conserved RNA-protein particle responsible for accurate polypeptide synthesis and co-translational protein folding. Here we summarize and discuss recent developments on the contribution of altered translation and age-dependent modifications on the ribosome structure to aging and cellular senescence.

Elevated levels of ribosomal proteins eL36 and eL42 control expression of Hsp90 in rhabdomyosarcoma.

Mammalian 90 kDa heat shock protein (Hsp90) is a ubiquitous molecular chaperone whose expression is selectively upregulated during stress, although the precise control mechanism of this increase is yet to be fully elucidated. We used polysome profiling to show that Hsp90α mRNA is selectively translated, while global translation is inhibited during heat stress. Furthermore, we have identified 2 ribosomal proteins, eL36 and eL42 that modulate Hsp90α expression under both normal and heat shock conditions. Importantly, we noted that expression of eL36 and eL42 is elevated in a panel of human rhabdomyosarcomas where it drives high expression of Hsp90 and modulates sensitivity of these cells to an Hsp90 inhibitor 17-AAG.

Could the eIF2α-Independent Translation Be the Achilles Heel of Cancer?

Eukaryotic initiation factor eIF2 is a key component of the ternary complex whose role is to deliver initiator tRNA into the ribosome. A variety of stimuli, both physiologic and pathophysiologic activate eIF2 kinases that phosphorylate the α subunit of eIF2, preventing it from forming the ternary complex, thus attenuating cellular protein synthesis. Paradoxically, in cancer cells, the phosphorylation of eIF2α is associated with activation of survival pathways. This review explores the recently emerged novel mechanism of eIF2α-independent translation initiation. This mechanism, which appears to be shared by some RNA viruses and Internal Ribosome Entry Site-containing cellular mRNAs and utilizes auxiliary proteins, such as eIF5B, eIF2D, and MCT-1, is responsible for the selective translation of cancer-associated genes and could represent a weak point amenable to specific targeting for the treatment of cancer.