Role of BicDR in bristle shaft construction and support of BicD functions.

Cell polarization requires asymmetric localization of numerous mRNAs, proteins and organelles. The movement of cargo towards the minus end of microtubules mostly depends on cytoplasmic dynein motors. In the dynein-dynactin-Bicaudal-D transport machinery, Bicaudal-D (BicD) links the cargo to the motor. Here, we focus on the role of Drosophila BicD-related (BicDR, CG32137) in the development of the long bristles. Together with BicD, it contributes to the organization and stability of the actin cytoskeleton in the not-yet-chitinized bristle shaft. BicD and BicDR also support the stable expression and distribution of Rab6 and Spn-F in the bristle shaft, including the distal tip localization of Spn-F, pointing to the role of microtubule-dependent vesicle trafficking for bristle construction. BicDR supports the function of BicD, and we discuss the hypothesis whereby BicDR might transport cargo more locally, with BicD transporting cargo over long distances, such as to the distal tip. We also identified embryonic proteins that interact with BicDR and appear to be BicDR cargo. For one of them, EF1γ (also known as eEF1γ), we show that the encoding gene EF1γ interacts with BicD and BicDR in the construction of the bristles.

Functional analysis of the AUG initiator codon context reveals novel conserved sequences that disfavor mRNA translation in eukaryotes.

mRNA translation is a fundamental process for life. Selection of the translation initiation site (TIS) is crucial, as it establishes the correct open reading frame for mRNA decoding. Studies in vertebrate mRNAs discovered that a purine at -3 and a G at +4 (where A of the AUG initiator codon is numbered + 1), promote TIS recognition. However, the TIS context in other eukaryotes has been poorly experimentally analyzed. We analyzed in vitro the influence of the -3, -2, -1 and + 4 positions of the TIS context in rabbit, Drosophila, wheat, and yeast. We observed that -3A conferred the best translational efficiency across these species. However, we found variability at the + 4 position for optimal translation. In addition, the Kozak motif that was defined from mammalian cells was only weakly predictive for wheat and essentially non-predictive for yeast. We discovered eight conserved sequences that significantly disfavored translation. Due to the big differences in translational efficiency observed among weak TIS context sequences, we define a novel category that we termed 'barren AUG context sequences (BACS)', which represent sequences disfavoring translation. Analysis of mRNA-ribosomal complexes structures provided insights into the function of BACS. The gene ontology of the BACS-containing mRNAs is presented.

eIF4E as a molecular wildcard in metazoans RNA metabolism.

The evolutionary origin of eukaryotes spurred the transition from prokaryotic-like translation to a more sophisticated, eukaryotic translation. During this process, successive gene duplication of a single, primordial eIF4E gene encoding the mRNA cap-binding protein eukaryotic translation initiation factor 4E (eIF4E) gave rise to a plethora of paralog genes across eukaryotes that underwent further functional diversification in RNA metabolism. The ability to take different roles is due to eIF4E promiscuity in binding many partner proteins, rendering eIF4E a highly versatile and multifunctional player that functions as a molecular wildcard. Thus, in metazoans, eIF4E paralogs are involved in various processes, including messenger RNA (mRNA) processing, export, translation, storage, and decay. Moreover, some paralogs display differential expression in tissues and developmental stages and show variable biochemical properties. In this review, we discuss recent advances shedding light on the functional diversification of eIF4E in metazoans. We emphasise humans and two phylogenetically distant species which have become paradigms for studies on development, namely the fruit fly Drosophila melanogaster and the roundworm Caenorhabditis elegans.

Role of BicDR in bristle shaft construction, tracheal development, and support of BicD functions.

Cell polarization requires asymmetric localization of numerous mRNAs, proteins, and organelles. The movement of cargo towards the minus end of microtubules mostly depends on cytoplasmic dynein motors, which function as multiprotein complexes. In the dynein/dynactin/Bicaudal-D (DDB) transport machinery, Bicaudal-D (BicD) links the cargo to the motor. Here we focus on the role of BicD-related (BicDR) and its contribution to microtubule-dependent transport processes. Drosophila BicDR is required for the normal development of bristles and dorsal trunk tracheae. Together with BicD, it contributes to the organization and stability of the actin cytoskeleton in the not-yet-chitinized bristle shaft and the localization of Spn-F and Rab6 at the distal tip. We show that BicDR supports the function of BicD in bristle development and our results suggest that BicDR transports cargo more locally whereas BicD is more responsible for delivering functional cargo over the long distance to the distal tip. We identified the proteins that interact with BicDR and appear to be BicDR cargo in embryonic tissues. For one of them, EF1γ, we showed that EF1γ genetically interacts with BicD and BicDR in the construction of the bristles.

The dark side of mRNA translation and the translation machinery in glioblastoma.

Among the different types of cancer affecting the central nervous system (CNS), glioblastoma (GB) is classified by the World Health Organization (WHO) as the most common and aggressive CNS cancer in adults. GB incidence is more frequent among persons aged 45-55 years old. GB treatments are based on tumor resection, radiation, and chemotherapies. The current development of novel molecular biomarkers (MB) has led to a more accurate prediction of GB progression. Moreover, clinical, epidemiological, and experimental studies have established genetic variants consistently associated with the risk of suffering GB. However, despite the advances in these fields, the survival expectancy of GB patients is still shorter than 2 years. Thus, fundamental processes inducing tumor onset and progression remain to be elucidated. In recent years, mRNA translation has been in the spotlight, as its dysregulation is emerging as a key cause of GB. In particular, the initiation phase of translation is most involved in this process. Among the crucial events, the machinery performing this phase undergoes a reconfiguration under the hypoxic conditions in the tumor microenvironment. In addition, ribosomal proteins (RPs) have been reported to play translation-independent roles in GB development. This review focuses on the research elucidating the tight relationship between translation initiation, the translation machinery, and GB. We also summarize the state-of-the-art drugs targeting the translation machinery to improve patients' survival. Overall, the recent advances in this field are shedding new light on the dark side of translation in GB.

Eukaryotic translation initiation factor eIF4E-5 is required for spermiogenesis in Drosophila melanogaster.

Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5' mRNA cap. In addition to canonical eIF4E-1, Drosophila has multiple eIF4E paralogs, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Among these, only eIF4E-3 has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, and eIF4E-5 mutants exhibit defects during post-meiotic stages, including a mild defect in spermatid cyst polarization. eIF4E-5 mutants also have a fully penetrant defect in individualization, resulting in failure to produce mature sperm. Indeed, our data indicate that eIF4E-5 regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis.

Drosophila Me31B is a Dual eIF4E-Interacting Protein.

Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). In this study, we show that Drosophila eIF4E-1 and eIF4E-3 occur in PBs along the DEAD-box RNA helicase Me31B. We show that Me31B interacts with eIF4E-1 and eIF4E-3 by means of yeast two-hybrid system, FRET in D. melanogaster S2 cells and coimmunoprecipitation in testis. Truncation and point mutations of Me31B proteins show two eIF4E-binding sites located in different protein domains. Residues Y401-L407 (at the carboxy-terminus) are essential for interaction with eIF4E-1, whereas residues F63-L70 (at the amino-terminus) are critical for interaction with eIF4E-3. The residue W117 in eIF4E-1 and the homolog position F103 in eIF4E-3 are necessary for Me31B-eIF4E interaction suggesting that the change of tryptophan to phenylalanine provides specificity. Me31B represents a novel type of eIF4E-interacting protein with dual and specific interaction domains that might be recognized by different eIF4E isoforms in different tissues, adding complexity to the control of gene expression in eukaryotes.

Schrödinger and the Possible Existence of Different Types of Life.

Eighty years ago, Nobel Prize-winner physicist Erwin Schrödinger gave three lectures in Dublin's Trinity College, titled What is Life? The physical aspect of the living cell to explain life in terms of the chemistry and physics laws. Life definitions rely on the cellular theory, which poses in the first place that life is made up of cells. The recent discovery of giant viruses, along with the development of synthetic cells at the beginning of century 21st, has challenged the current idea of what life is. Thus, rather than having arrived at a close answer to Schrödinger's question, modern biology has touched down at a novel scenario in which several types of life-as opposed to only one-actually might exist on Earth and possibly the Universe. Eighty years after the Dublin lectures, the Schrödinger question could be: "What are lives"?

Female meiosis II and pronuclear fusion require the microtubule transport factor Bicaudal D.

Bicaudal D (BicD) is a dynein adaptor that transports different cargoes along microtubules. Reducing the activity of BicD specifically in freshly laid Drosophila eggs by acute protein degradation revealed that BicD is needed to produce normal female meiosis II products, to prevent female meiotic products from re-entering the cell cycle, and for pronuclear fusion. Given that BicD is required to localize the spindle assembly checkpoint (SAC) components Mad2 and BubR1 to the female meiotic products, it appears that BicD functions to localize these components to control metaphase arrest of polar bodies. BicD interacts with Clathrin heavy chain (Chc), and both proteins localize to centrosomes, mitotic spindles and the tandem spindles during female meiosis II. Furthermore, BicD is required to localize clathrin and the microtubule-stabilizing factors transforming acidic coiled-coil protein (D-TACC/Tacc) and Mini spindles (Msps) correctly to the meiosis II spindles, suggesting that failure to localize these proteins may perturb SAC function. Furthermore, immediately after the establishment of the female pronucleus, D-TACC and Caenorhabditis elegans BicD, tacc and Chc are also needed for pronuclear fusion, suggesting that the underlying mechanism might be more widely used across species.

The versatile relationships between eIF4E and eIF4E-interacting proteins.

RNA metabolism and gene expression lie at the core of cellular life. eIF4E has emerged as a central interface in both processes as it plays critical roles in mRNA processing, transport, translation, and storage. Crucially, eIF4E depends on its association with a universe of proteins to form biologically meaningful complexes.

Interplay between SERCA, 4E-BP, and eIF4E in the Drosophila heart.

Appropriate cardiac performance depends on a tightly controlled handling of Ca2+ in a broad range of species, from invertebrates to mammals. The role of the Ca2+ ATPase, SERCA, in Ca2+ handling is pivotal, and its activity is regulated, inter alia, by interacting with distinct proteins. Herein, we give evidence that 4E binding protein (4E-BP) is a novel regulator of SERCA activity in Drosophila melanogaster during cardiac function. Flies over-expressing 4E-BP showed improved cardiac performance in young individuals associated with incremented SERCA activity. Moreover, we demonstrate that SERCA interacts with translation initiation factors eIF4E-1, eIF4E-2 and eIF4E-4 in a yeast two-hybrid assay. The specific identification of eIF4E-4 in cardiac tissue leads us to propose that the interaction of elF4E-4 with SERCA may be the basis of the cardiac effects observed in 4E-BP over-expressing flies associated with incremented SERCA activity.

New insights into the interactions of HPV-16 E6*I and E6*II with p53 isoforms and induction of apoptosis in cancer-derived cell lines.

An important characteristic of cancers associated with high-risk human papillomaviruses (HR-HPV) is the inability of p53 to activate apoptosis due to the effect of the oncoprotein E6. However, the effect of HPV-16 E6 splice variant isoforms (namely E6*I and E6*II), their interaction with the existing p53 isoforms, and their influence on apoptosis is unclear. Here, we report the outcome of ectopic expression of HPV-16 E6, E6*I, and E6*II on the relative levels of p53 and p53 isoforms Δ40p53 and Δ133p53 and their interactions with these proteins. Additionally, we evaluated the effect of ectopic expression of p53, Δ40p53, and Δ133p53 on apoptosis in a p53 null pulmonary cell line (H1299) co-transfected with E6 isoforms and p53+/+ cell lines with HR-HPV (SiHa and HeLa), transfected with p53 isoforms and treated with cisplatin, a conventional drug used to treat cervical cancer. Our results show that E6 and E6*II induced a significant decrease in p53, but only E6 triggered a Δ40p53 decrease and that E6*II interacts with p53 but not with Δ40p53 and Δ133p53. On the other hand, E6*I did not show any effect or interaction with the p53 isoforms. We found that apoptosis was elevated in H1299 cells transfected with p53 (p = 0.0001) and Δ40p53 (p = 0.0001). A weak apoptotic effect was observed when Δ133p53 was ectopically expressed (p = 0.0195). We observed that both p53 (p = 0.0006) and Δ40p53 (p = 0.0014) induced apoptosis in cisplatin-treated SiHa cells; however in cisplatin-treated HeLa cells, only p53 induced apoptosis (p = 0.0029). No significant differences in apoptosis were observed upon ectopic expression of p53, Δ40p53, and Δ133p53 in SiHa and HeLa cells. Our findings suggest a possible therapeutic application for the combining of p53 or Δ40p53 with cisplatin to induce an increased apoptosis of cancer cells expressing E6 isoforms from HPV-16.

High-risk human papillomavirus-18 uses an mRNA sequence to synthesize oncoprotein E6 in tumors.

Cervical cancer is the fourth most common cause of cancer in women worldwide in terms of both incidence and mortality. Persistent infection with high-risk types of human papillomavirus (HPV), namely 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68, constitute a necessary cause for the development of cervical cancer. Viral oncoproteins E6 and E7 play central roles in the carcinogenic process by virtue of their interactions with cell master proteins such as p53, retinoblastoma (Rb), mammalian target of rapamycin (mTOR), and c-MYC. For the synthesis of E6 and E7, HPVs use a bicistronic messenger RNA (mRNA) that has been studied in cultured cells. Here, we report that in cervical tumors, HPV-18, -39, and -45 transcribe E6/E7 mRNAs with extremely short 5' untranslated regions (UTRs) or even lacking a 5' UTR (i.e., zero to three nucleotides long) to express E6. We show that the translation of HPV-18 E6 cistron is regulated by the motif ACCaugGCGCG(C/A)UUU surrounding the AUG start codon, which we term Translation Initiation of Leaderless mRNAs (TILM). This motif is conserved in all HPV types of the phylogenetically coherent group forming genus alpha, species 7, which infect mucosal epithelia. We further show that the translation of HPV-18 E6 largely relies on the cap structure and eIF4E and eIF4AI, two key translation initiation factors linking translation and cancer but does not involve scanning. Our results support the notion that E6 forms the center of the positive oncogenic feedback loop node involving eIF4E, the mTOR cascade, and p53.

Translational control in the naked mole-rat as a model highly resistant to cancer.

Dysregulation of mRNA translation is involved in the onset and progression of different types of cancer. To gain insight into novel genetic strategies to avoid this malady, we reviewed the available genomic, transcriptomic, and proteomic data about the translational machinery from the naked-mole rat (NMR) Heterocephalus glaber, a new model of study that exhibits high resistance to cancer. The principal features that might confer cancer resistance are 28S rRNA fragmentation, RPL26 and eIF4G overexpression, global downregulation of mTOR pathway, specific amino acid residues in RAPTOR (P908) and RICTOR (V1695), and the absence of 4E-BP3. These features are not only associated with cancer but also might couple longevity and adaptation to hypoxia. We propose that the regulation of translation is among the strategies endowing NMR cancer resistance.

Unorthodox Mechanisms to Initiate Translation Open Novel Paths for Gene Expression.

Translation in eukaryotes is dependent on the activity of translation initiation factor (eIF) 4G family of proteins, a scaffold protein that, during the initiation step, coordinates the activity of other eIFs to recruit the 40S ribosomal subunit to the mRNA. Three decades of research on protein synthesis and its regulation has provided a wealth of evidence supporting the crucial role of cap-dependent translation initiation, which involves eIF4G. However, the recent discovery of a surprising variety of alternative mechanisms to initiate translation in the absence of eIF4G has stirred the orthodox view of how protein synthesis is performed. These mechanisms involve novel interactions among known eIFs, or between known eIFs and other proteins not previously linked to translation. Thus, a new picture is emerging in which the unorthodox translation initiation complexes contribute to the diversity of mechanisms that regulate gene expression in eukaryotes.

Autophagy Regulation by the Translation Machinery and Its Implications in Cancer.

Various metabolic pathways and molecular processes in the cell act intertwined, and dysregulating the interplay between some of them may lead to cancer. It is only recently that defects in the translation process, i.e., the synthesis of proteins by the ribosome using a messenger (m)RNA as a template and translation factors, have begun to gain strong attention as a cause of autophagy dysregulation with effects in different maladies, including cancer. Autophagy is an evolutionarily conserved catabolic process that degrades cytoplasmic elements in lysosomes. It maintains cellular homeostasis and preserves cell viability under various stress conditions, which is crucial for all eukaryotic cells. In this review, we discuss recent advances shedding light on the crosstalk between the translation and the autophagy machineries and its impact on tumorigenesis. We also summarize how this interaction is being the target for novel therapies to treat cancer.

A ribosomal protein S5 isoform is essential for oogenesis and interacts with distinct RNAs in Drosophila melanogaster.

In Drosophila melanogaster there are two genes encoding ribosomal protein S5, RpS5a and RpS5b. Here, we demonstrate that RpS5b is required for oogenesis. Females lacking RpS5b produce ovaries with numerous developmental defects that undergo widespread apoptosis in mid-oogenesis. Females lacking germline RpS5a are fully fertile, but germline expression of interfering RNA targeting germline RpS5a in an RpS5b mutant background worsened the RpS5b phenotype and blocked oogenesis before egg chambers form. A broad spectrum of mRNAs co-purified in immunoprecipitations with RpS5a, while RpS5b-associated mRNAs were specifically enriched for GO terms related to mitochondrial electron transport and cellular metabolic processes. Consistent with this, RpS5b mitochondrial fractions are depleted for proteins linked to oxidative phosphorylation and mitochondrial respiration, and RpS5b mitochondria tended to form large clusters and had more heterogeneous morphology than those from controls. We conclude that RpS5b-containing ribosomes preferentially associate with particular mRNAs and serve an essential function in oogenesis.

The Diverse Roles of RNA-Binding Proteins in Glioma Development.

Post-transcriptional regulation of gene expression is fundamental for all forms of life, as it critically contributes to the composition and quantity of a cell's proteome. These processes encompass splicing, polyadenylation, mRNA decay, mRNA editing and modification and translation and are modulated by a variety of RNA-binding proteins (RBPs). Alterations affecting RBP expression and activity contribute to the development of different types of cancer. In this chapter, we discuss current research shedding light on the role of different RBPs in gliomas. These studies place RBPs as modulators of critical signaling pathways, establish their relevance as prognostic markers and open doors for new therapeutic strategies.

Conservation and Variability of the AUG Initiation Codon Context in Eukaryotes.

Selection of the translation initiation site (TIS) is a crucial step during translation. In the 1980s Marylin Kozak performed key studies on vertebrate mRNAs to characterize the optimal TIS consensus sequence, the Kozak motif. Within this motif, conservation of nucleotides in crucial positions, namely a purine at -3 and a G at +4 (where the A of the AUG is numbered +1), is essential for TIS recognition. Ever since its characterization the Kozak motif has been regarded as the optimal sequence to initiate translation in all eukaryotes. We revisit here published in silico data on TIS consensus sequences, as well as experimental studies from diverse eukaryotic lineages, and propose that, while the -3A/G position is universally conserved, the remaining variability of the consensus sequences enables their classification as optimal, strong, and moderate TIS sequences.