A luminescence-based reporter to study tau secretion reveals overlapping mechanisms for the release of healthy and pathological tau.

In Alzheimer's disease, tau pathology is thought to spread via a prion-like manner along connected neuronal networks. For this to occur, the usually cytosolic tau protein must be secreted via an unconventional mechanism prior to uptake into the connected neuron. While secretion of healthy and pathological tau has been documented, it remains under-investigated whether this occurs via overlapping or distinct processes. Here, we established a sensitive bioluminescence-based assay to assess mechanisms underlying the secretion of pseudohyperphosphorylated and wild-type tau in cultured murine hippocampal neurons. We found that under basal conditions, both wild-type and mutant tau are secreted, with mutant tau being more robustly secreted. Pharmacological stimulation of neuronal activity led to a modest increase of wild-type and mutant tau secretion, whereas inhibition of activity had no effect. Interestingly, inhibition of heparin sulfate proteoglycan (HSPG) biosynthesis drastically decreased secretion of both wild-type and mutant tau without affecting cell viability. This shows that native and pathological tau share release mechanisms; both activity-dependent and non-activity-dependent secretion of tau is facilitated by HSPGs.

Reduction in cycle time for a rapid polymerase chain reaction diagnostic test at the point of care.

Background: Rapid testing facilitates safe and effective diagnosis, but the true speed of the process is the time from collection of a sample to delivery of an accurate and reliable test result - 'end-to-end' time. Transport, unpacking and relaying of information can extend this time considerably beyond the minimum laboratory turnaround times as stipulated by PCR testing protocols. Aim/Objective: This study aimed to minimise time needed to ascertain SARS-CoV-2 status prior to treatment in a UK Dental Hospital using a novel, mobile, direct to polymerase chain reaction (PCR) workflow. Methods: Process flow analysis and PDSA (Plan, Do, Study, Act) cycles for rapid continuous improvement were employed in a service improvement programme. Primerdesign™ q16 rapid PCR instruments and PROmate® COVID-19 direct assays were used for molecular testing. Findings/Results: We showed a reduction in real-world end-to-end time for a diagnostic test from 240 min to 85 min (65% reduction) over a 4-week period. Discussion: New rapid technologies have become available that reduce analytical time to under 90 min, but the real-world clinical implementation of the test requires a fully integrated workflow from clinic to reporting.

A 5' UTR GGN repeat controls localisation and translation of a potassium leak channel mRNA through G-quadruplex formation.

RNA G-quadruplexes (G4s) are secondary structures proposed to function as regulators of post-transcriptional mRNA localisation and translation. G4s within some neuronal mRNAs are known to control distal localisation and local translation, contributing to distinct local proteomes that facilitate the synaptic remodelling attributed to normal cellular function. In this study, we characterise the G4 formation of a (GGN)13 repeat found within the 5' UTR of the potassium 2-pore domain leak channel Task3 mRNA. Biophysical analyses show that this (GGN)13 repeat forms a parallel G4 in vitro exhibiting the stereotypical potassium specificity of G4s, remaining thermostable under physiological ionic conditions. Through mouse brain tissue G4-RNA immunoprecipitation, we further confirm that Task3 mRNA forms a G4 structure in vivo. The G4 is inhibitory to translation of Task3 in vitro and is overcome through activity of a G4-specific helicase DHX36, increasing K+ leak currents and membrane hyperpolarisation in HEK293 cells. Further, we observe that this G4 is fundamental to ensuring delivery of Task3 mRNA to distal primary cortical neurites. It has been shown that aberrant Task3 expression correlates with neuronal dysfunction, we therefore posit that this G4 is important in regulated local expression of Task3 leak channels that maintain K+ leak within neurons.

Regulation of the Elongation Phase of Protein Synthesis Enhances Translation Accuracy and Modulates Lifespan.

Maintaining accuracy during protein synthesis is crucial to avoid producing misfolded and/or non-functional proteins. The target of rapamycin complex 1 (TORC1) pathway and the activity of the protein synthesis machinery are known to negatively regulate lifespan in many organisms, although the precise mechanisms involved remain unclear. Mammalian TORC1 signaling accelerates the elongation stage of protein synthesis by inactivating eukaryotic elongation factor 2 kinase (eEF2K), which, when active, phosphorylates and inhibits eEF2, which mediates the movement of ribosomes along mRNAs, thereby slowing down the rate of elongation. We show that eEF2K enhances the accuracy of protein synthesis under a range of conditions and in several cell types. For example, our data reveal it links mammalian (m)TORC1 signaling to the accuracy of translation. Activation of eEF2K decreases misreading or termination readthrough errors during elongation, whereas knocking down or knocking out eEF2K increases their frequency. eEF2K also promotes the correct recognition of start codons in mRNAs. Reduced translational fidelity is known to correlate with shorter lifespan. Consistent with this, deletion of the eEF2K ortholog or other factors implicated in translation fidelity in Caenorhabditis elegans decreases lifespan, and eEF2K is required for lifespan extension induced by nutrient restriction. Our data uncover a novel mechanism linking nutrient supply, mTORC1 signaling, and the elongation stage of protein synthesis, which enhances the accuracy of protein synthesis. Our data also indicate that modulating translation elongation and its fidelity affects lifespan.

PEITC-mediated inhibition of mRNA translation is associated with both inhibition of mTORC1 and increased eIF2α phosphorylation in established cell lines and primary human leukemia cells.

Increased mRNA translation drives carcinogenesis and is an attractive target for the development of new anti-cancer drugs. In this work, we investigated effects of phenethylisothiocyanate (PEITC), a phytochemical with chemopreventive and anti-cancer activity, on mRNA translation. PEITC rapidly inhibited global mRNA translation in human breast cancer-derived MCF7 cells and mouse embryonic fibroblasts (MEFs). In addition to the known inhibitory effects of PEITC on mTORC1 activity, we demonstrate that PEITC increased eIF2α phosphorylation. PEITC also increased formation of stress granules which are typically associated with eIF2α phosphorylation and accumulation of translationally stalled mRNAs. Analysis of genetically modified MEFs demonstrated that optimal inhibition of global mRNA translation by PEITC was dependent on eIF2α phosphorylation, but not mTORC1 inhibition. We extended this study into primary leukemic B cells derived from patients with chronic lymphocytic leukaemia (CLL). CLL cells were stimulated in vitro with anti-IgM to mimic binding of antigen, a major driver of this leukemia. In CLL cells, PEITC increased eIF2α phosphorylation, inhibited anti-IgM-induced mTORC1 activation and decreased both basal and anti-IgM-induced global mRNA translation. PEITC also inhibited transcription and translation of MYC mRNA and accumulation of the MYC oncoprotein, in anti-IgM-stimulated cells. Moreover, treatment of CLL cells with PEITC and the BTK kinase inhibitor ibrutinib decreased anti-IgM-induced translation and induced cell death to a greater extent than either agent alone. Therefore, PEITC can inhibit both global and mRNA specific translation (including MYC) via effects on multiple regulatory pathways. Inhibition of mRNA translation may contribute to the chemopreventive and anti-cancer effects of PEITC.

Engagement of the B-cell receptor of chronic lymphocytic leukemia cells drives global and MYC-specific mRNA translation.

Antigenic stimulation via the B-cell receptor (BCR) is a major driver of the proliferation and survival of chronic lymphocytic leukemia (CLL) cells. However, the precise mechanisms by which BCR stimulation leads to accumulation of malignant cells remain incompletely understood. Here, we investigated the ability of BCR stimulation to increase messenger RNA (mRNA) translation, which can promote carcinogenesis by effects on both global mRNA translation and upregulated expression of specific oncoproteins. Re-analysis of gene expression profiles revealed striking upregulation of pathways linked to mRNA translation both in CLL cells derived from lymph nodes, the major site of antigen stimulation in vivo, and after BCR stimulation in vitro. Anti-IgM significantly increased mRNA translation in primary CLL cells, measured using bulk metabolic labeling and a novel flow cytometry assay to quantify responses at a single-cell level. These translational responses were suppressed by inhibitors of BTK (ibrutinib) and SYK (tamatinib). Anti-IgM-induced mRNA translation was associated with increased expression of translation initiation factors eIF4A and eIF4GI, and reduced expression of the eIF4A inhibitor, PDCD4. Anti-IgM also increased mRNA translation in normal blood B cells, but without clear modulatory effects on these factors. In addition, anti-IgM increased translation of mRNA-encoding MYC, a major driver of disease progression. mRNA translation is likely to be an important mediator of the growth-promoting effects of antigen stimulation acting, at least in part, via translational induction of MYC. Differences in mechanisms of translational regulation in CLL and normal B cells may provide opportunities for selective therapeutic attack.

Stoichiometry of the eIF2B complex is maintained by mutual stabilization of subunits.

The eukaryotic translation initiation factor eIF2B is a multi-subunit complex with a crucial role in the regulation of global protein synthesis in the cell. The complex comprises five subunits, termed α through ε in order of increasing size, arranged as a heterodecamer with two copies of each subunit. Regulation of the co-stoichiometric expression of the eIF2B subunits is crucial for the proper function and regulation of the eIF2B complex in cells. We have investigated the control of stoichiometric eIF2B complexes through mutual stabilization of eIF2B subunits. Our data show that the stable expression of the catalytic eIF2Bε subunit in human cells requires co-expression of eIF2Bγ. Similarly, stable expression of eIF2Bδ requires both eIF2Bß and eIF2Bγ+ε. The expression of these subunits decreases despite there being no change in either the levels or the translation of their mRNAs. Instead, these subunits are targeted for degradation by the ubiquitin-proteasome system. The data allow us to propose a model for the formation of stoichiometric eIF2B complexes which can ensure their stoichiometric incorporation into the holocomplex.

G-quadruplexes mediate local translation in neurons.

There has recently been a huge increase in interest in the formation of stable G-quadruplex structures in mRNAs and their functional significance. In neurons, local translation of mRNA is essential for normal neuronal behaviour. It has been discovered that local translation of specific mRNAs encoding some of the best known synaptic proteins is dependent on the presence of a G-quadruplex. The recognition of G-quadruplexes in mRNAs, their transport as repressed complexes and the control of their translation at their subcellular destinations involves a diversity of proteins, including those associated with disease pathologies. This is an exciting field, with rapid improvements to our knowledge and understanding. Here, we discuss some of the recent work on how G-quadruplexes mediate local translation in neurons.

ABC50 mutants modify translation start codon selection.

ATP-binding cassette 50 (ABC50; also known as ABCF1) binds to eukaryotic initiation factor 2 (eIF2) and is required for efficient translation initiation. An essential step of this process is accurate recognition and selection of the initiation codon. It is widely accepted that the presence and movement of eIF1, eIF1A and eIF5 are key factors in modulating the stringency of start-site selection, which normally requires an AUG codon in an appropriate sequence context. In the present study, we show that expression of ABC50 mutants, which cannot hydrolyse ATP, decreases general translation and relaxes the discrimination against the use of non-AUG codons at translation start sites. These mutants do not appear to alter the association of key initiation factors to 40S subunits. The stringency of start-site selection can be restored through overexpression of eIF1, consistent with the role of that factor in enhancing stringency. The present study indicates that interfering with the function of ABC50 influences the accuracy of initiation codon selection.

Phosphorylation of eIF4GII and 4E-BP1 in response to nocodazole treatment: a reappraisal of translation initiation during mitosis.

Translation mechanisms at different stages of the cell cycle have been studied for many years, resulting in the dogma that translation rates are slowed during mitosis, with cap-independent translation mechanisms favored to give expression of key regulatory proteins. However, such cell culture studies involve synchronization using harsh methods, which may in themselves stress cells and affect protein synthesis rates. One such commonly used chemical is the microtubule de-polymerization agent, nocodazole, which arrests cells in mitosis and has been used to demonstrate that translation rates are strongly reduced (down to 30% of that of asynchronous cells). Using synchronized HeLa cells released from a double thymidine block (G 1/S boundary) or the Cdk1 inhibitor, RO3306 (G 2/M boundary), we have systematically re-addressed this dogma. Using FACS analysis and pulse labeling of proteins with labeled methionine, we now show that translation rates do not slow as cells enter mitosis. This study is complemented by studies employing confocal microscopy, which show enrichment of translation initiation factors at the microtubule organizing centers, mitotic spindle, and midbody structure during the final steps of cytokinesis, suggesting that translation is maintained during mitosis. Furthermore, we show that inhibition of translation in response to extended times of exposure to nocodazole reflects increased eIF2α phosphorylation, disaggregation of polysomes, and hyperphosphorylation of selected initiation factors, including novel Cdk1-dependent N-terminal phosphorylation of eIF4GII. Our work suggests that effects on translation in nocodazole-arrested cells might be related to those of the treatment used to synchronize cells rather than cell cycle status.

SLiMPrints: conservation-based discovery of functional motif fingerprints in intrinsically disordered protein regions.

Large portions of higher eukaryotic proteomes are intrinsically disordered, and abundant evidence suggests that these unstructured regions of proteins are rich in regulatory interaction interfaces. A major class of disordered interaction interfaces are the compact and degenerate modules known as short linear motifs (SLiMs). As a result of the difficulties associated with the experimental identification and validation of SLiMs, our understanding of these modules is limited, advocating the use of computational methods to focus experimental discovery. This article evaluates the use of evolutionary conservation as a discriminatory technique for motif discovery. A statistical framework is introduced to assess the significance of relatively conserved residues, quantifying the likelihood a residue will have a particular level of conservation given the conservation of the surrounding residues. The framework is expanded to assess the significance of groupings of conserved residues, a metric that forms the basis of SLiMPrints (short linear motif fingerprints), a de novo motif discovery tool. SLiMPrints identifies relatively overconstrained proximal groupings of residues within intrinsically disordered regions, indicative of putatively functional motifs. Finally, the human proteome is analysed to create a set of highly conserved putative motif instances, including a novel site on translation initiation factor eIF2A that may regulate translation through binding of eIF4E.

Multiple isoforms of the translation initiation factor eIF4GII are generated via use of alternative promoters, splice sites and a non-canonical initiation codon.

During the initiation stage of eukaryotic mRNA translation, the eIF4G (eukaryotic initiation factor 4G) proteins act as an aggregation point for recruiting the small ribosomal subunit to an mRNA. We previously used RNAi (RNA interference) to reduce expression of endogenous eIF4GI proteins, resulting in reduced protein synthesis rates and alterations in the morphology of cells. Expression of EIF4G1 cDNAs, encoding different isoforms (f-a) which arise through selection of alternative initiation codons, rescued translation to different extents. Furthermore, overexpression of the eIF4GII paralogue in the eIF4GI-knockdown background was unable to restore translation to the same extent as eIF4GIf/e isoforms, suggesting that translation events governed by this protein are different. In the present study we show that multiple isoforms of eIF4GII exist in mammalian cells, arising from multiple promoters and alternative splicing events, and have identified a non-canonical CUG initiation codon which extends the eIF4GII N-terminus. We further show that the rescue of translation in eIF4GI/eIF4GII double-knockdown cells by our novel isoforms of eIF4GII is as robust as that observed with either eIF4GIf or eIF4GIe, and more than that observed with the original eIF4GII. As the novel eIF4GII sequence diverges from eIF4GI, these data suggest that the eIF4GII N-terminus plays an alternative role in initiation factor assembly.

Cytoplasmic mRNA: move it, use it or lose it!

Once an mRNA is synthesized and processed, the immediate translation and later destruction of the transcript is not as inevitable as the central molecular biology dogma suggests. Interest in the field of post-transcriptional control continues to grow rapidly, as regulation of these multiple steps in gene expression is implicated in diverse aspects of biology such as metabolism, neurology, reproduction and viral lifecycle regulation. Researchers who utilize various combinations of human studies, animal models, cellular, genetic, biochemical and molecular techniques were brought together at the University of Edinburgh to discuss their latest findings. In this article, we introduce the content of the related reviews presented in this issue of Biochemical Society Transactions which together illustrate a major theme of the meeting content: namely the need to understand how dynamic changes in mRNP (messenger ribonucleoprotein) complexes modulate the multifunctionality of regulatory proteins which link different post-transcriptional regulatory events.

Inhibition of mammalian target of rapamycin (mTOR) signalling in C2C12 myoblasts prevents myogenic differentiation without affecting the hyperphosphorylation of 4E-BP1.

Current accepted models suggest that hypophosphorylated 4E-binding protein (4E-BP1) binds to initiation factor 4E (eIF4E) to inhibit cap-dependent translation, a process readily reversed by its phosphorylation following activation of mammalian target of rapamycin (mTORC1) signalling. Myogenic differentiation in the C2C12 myoblast model system reflects a concerted and controlled activation of transcription and translation following the exit of cells from the cell cycle. Here we show that myogenic differentiation is associated with increased rates of translation, the up-regulation of both 4E-BP1 mRNA and protein levels and enhanced levels of eIF4E/4E-BP1 complex. Paradoxically, treatment of C2C12 myoblasts with an inhibitor of mTOR signalling (RAD001) which inhibits translation, promotes the hyperphosphorylation of 4E-BP1 on novel sites and prevents the increase in 4E-BP1 levels. In contrast, eIF4E appears to be under translational control with a significant delay between induction of mRNA and subsequent protein expression.

Canonical initiation factor requirements of the Myc family of internal ribosome entry segments.

Initiation of protein synthesis in eukaryotes requires recruitment of the ribosome to the mRNA and its translocation to the start codon. There are at least two distinct mechanisms by which this process can be achieved; the ribosome can be recruited either to the cap structure at the 5' end of the message or to an internal ribosome entry segment (IRES), a complex RNA structural element located in the 5' untranslated region (5'-UTR) of the mRNA. However, it is not well understood how cellular IRESs function to recruit the ribosome or how the 40S ribosomal subunits translocate from the initial recruitment site on the mRNA to the AUG initiation codon. We have investigated the canonical factors that are required by the IRESs found in the 5'-UTRs of c-, L-, and N-myc, using specific inhibitors and a tissue culture-based assay system, and have shown that they differ considerably in their requirements. The L-myc IRES requires the eIF4F complex and the association of PABP and eIF3 with eIF4G for activity. The minimum requirements of the N- and c-myc IRESs are the C-terminal domain of eIF4G to which eIF4A is bound and eIF3, although interestingly this protein does not appear to be recruited to the IRES RNA via eIF4G. Finally, our data show that all three IRESs require a ternary complex, although in contrast to c- and L-myc IRESs, the N-myc IRES has a lesser requirement for a ternary complex.

An alternative mechanism of eukaryotic translation initiation.

Cell stress activates signaling pathways, allowing cells to choose between survival and apoptosis. Translation plays a critical role in balancing this choice by allowing for rapid and physiologically responsive changes in de novo gene expression. The steady-state abundance of cellular inhibitor of apoptosis 2 (cIAP2) is increased in response to various cell stresses. This modular protein contains baculoviral IAP repeat (BIR) motifs and ubiquitin protein ligase (E3) activity, which allows it to bind directly to caspases and to modulate activation of the transcription factor, nuclear factor kappaB (NF-kappaB). The messenger RNA (mRNA) encoding cIAP2 is a large 5.5-kb transcript, with a highly structured 5' untranslated region (5'UTR) also containing 64 upstream initiation codons ahead of the true start codon. cIAP2 employs an unusual cap-dependent mechanism of ribosome shunting to bypass the majority of the inhibitory elements in the 5'UTR, a mechanism first described for plant pararetroviruses. Furthermore, in mammalian cells, this poorly understood mechanism of translation for cIAP2 is enhanced during mild stress in the absence of pararetrovirus-encoded proteins known to be essential for this process in plant cells. Here, we discuss how cIAP2 might utilize the stress-mediated shunt process in the absence of viral proteins, which suggests a more widespread role for canonical initiation factors, internal ribosome entry sequence-specific trans-acting factors, and mRNA structure in translational control during stress.

Functional analysis of individual binding activities of the scaffold protein eIF4G.

Eukaryotic initiation factor (eIF) 4G is an integral member of the translation initiation machinery. The molecule serves as a scaffold for several other initiation factors, including eIF4E, eIF4AI, the eIF3 complex, and poly(A)-binding protein (PABP). Previous work indicates that complexes between these proteins exhibit enhanced mRNA cap-binding and RNA helicase activities relative to the respective individual proteins, eIF4E and eIF4A. The eIF4G-PABP interaction has been implicated in enhancing the formation of 48 S and 80 S initiation complexes and ribosome recycling through mRNA circularization. The eIF3-eIF4GI interaction is believed to forge the link between the 40 S subunit and the mRNA. Here we have investigated the behavior in vitro and in intact cells of eIF4GIf molecules lacking either the PABP-binding site, the eIF3-binding site, the middle domain eIF4A-binding site, or the C-terminal segment that includes the second eIF4A-binding site. Although in some cases the mutant forms were recruited more slowly, all of these eIF4G variants could form complexes with eIF4E, enter 48 S complexes and polysomes in vivo and in vitro, and partially rescue translation in cells targeted with eIF4GI short interfering RNA. In the reticulocyte lysate, eIF4G unable to interact directly with PABP showed little impairment in its ability to support translation, whereas loss of either of the eIF4A-binding sites or the eIF3-binding site resulted in a marked decrease in activity. We conclude that there is considerable redundancy in the mechanisms forming initiation complexes in mammalian cells, such that many individual interactions have regulatory rather than essential roles.

Specific isoforms of translation initiation factor 4GI show differences in translational activity.

The eukaryotic initiation factor (eIF) 4GI gene locus (eIF4GI) contains three identified promoters, generating alternately spliced mRNAs, yielding a total of five eIF4GI protein isoforms. Although eIF4GI plays a critical role in mRNA recruitment to the ribosomes, little is known about the functions of the different isoforms, their partner binding capacities, or the role of the homolog, eIF4GII, in translation initiation. To directly address this, we have used short interfering RNAs (siRNAs) expressed from DNA vectors to silence the expression of eIF4GI in HeLa cells. Here we show that reduced levels of specific mRNA and eIF4GI isoforms in HeLa cells promoted aberrant morphology and a partial inhibition of translation. The latter reflected dephosphorylation of 4E-BP1 and decreased eIF4F complex levels, with no change in eIF2alpha phosphorylation. Expression of siRNA-resistant Myc-tagged eIF4GI isoforms has allowed us to show that the different isoforms exhibit significant differences in their ability to restore translation rates. Here we quantify the efficiency of eIF4GI promoter usage in mammalian cells and demonstrate that even though the longest isoform of eIF4GI (eIF4GIf) was relatively poorly expressed when reintroduced, it was more efficient at promoting the translation of cellular mRNAs than the more highly expressed shorter isoforms used in previous functional studies.

Expression of fragments of translation initiation factor eIF4GI reveals a nuclear localisation signal within the N-terminal apoptotic cleavage fragment N-FAG.

The eukaryotic initiation factor eIF4GI plays a central role in the assembly of a competent initiation complex at the 5' end of an mRNA. Five isoforms of eIF4G exist in cells, arising from alternative translation initiation. During picornaviral infection or apoptosis, eIF4GI is cleaved proteolytically to yield distinct fragments. Using HeLa cells, we have examined the fate of these proteins in the cell. We have found that while endogenous eIF4GI is predominantly cytoplasmic, a population can also be visualised in the nucleus. Furthermore, eIF4GI is localised primarily at the nuclear periphery in the vicinity of eIF4E and PABP1. Transient transfection of HeLa cells with different myc-tagged isoforms of eIF4GI did not result in any obvious differences in their localisation. However, expression of discrete fragments of eIF4GI corresponding to those generated after apoptosis or picornaviral infection generated a distinctive, but intricate localisation pattern. Our work shows that the N-terminal apoptotic cleavage fragment N-FAG contains a sequence of basic amino acids that can act as a nuclear localisation signal. In addition, the presence or absence of the sequence flanking and including the eIF4E binding site (residues 533-682) confers a distinct cellular distribution pattern for the central domain of eIF4GI.