Background: mTORC1 plays an important role in the regulation of TOP mRNA translation. Results: LARP1 is a target of mTORC1 that associates with TOP mRNAs via their 5ЈTOP motif to repress their translation. Conclusion: LARP1 represses TOP mRNA translation downstream of mTORC1. Significance: We elucidate an important novel signaling pathway downstream of mTORC1 that controls the production of ribosomes and translation factors in eukaryotic cells.

Highlights RT-ddPCR is more sensitive to inhibitors than RT-qPCR for primary clarified sludge Primary clarified sludge has elevated frequency of SARS-CoV-2 RNA detection Primary clarified sludge allows detection of RNA during low COVID-19 incidence PMMV normalization of RNA data reduces noise and increases precision PMMV normalization of RNA shows strongest correlation to epidemiological metrics

In the absence of an effective vaccine to prevent COVID-19 it is important to be able to track community infections to inform public health interventions aimed at reducing the spread and therefore reduce pressures on health-care units, improve health outcomes and reduce economic uncertainty. Wastewater surveillance has rapidly emerged as a potential tool to effectively monitor community infections for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), through measuring trends of viral RNA signal in wastewater systems. In this study SARS-CoV-2 viral RNA N1 and N2 genes are quantified in solids collected from influent post grit solids (PGS) and primary clarified sludge (PCS) in two water resource recovery facilities (WRRF) serving Canada's national capital region, i.e., the City of Ottawa, ON (pop. = 1.1M) and the City of Gatineau, QC (pop. = 280K). PCS samples show signal inhibition using RT-ddPCR compared to RT-qPCR, with PGS samples showing similar quantifiable concentrations of RNA using both assays. RT-qPCR shows higher frequency of detection of N1 and N2 genes in PCS (92.7, 90.6%) as compared to PGS samples (79.2, 82.3%). Sampling of PCS may therefore be an effective approach for SARS-CoV-2 viral quantification, especially during periods of declining and low COVID-19 incidence in the community. The pepper mild mottle virus (PMMV) is determined to have a less variable RNA signal in PCS over a three month period for two WRRFs, regardless of environmental conditions, compared to Bacteroides 16S rRNA or human eukaryotic 18S rRNA, making PMMV a potentially useful biomarker for normalization of SARS-CoV-2 signal. PMMV-normalized PCS RNA signal from WRRFs of two cities correlated with the regional public health epidemiological metrics, identifying PCS normalized to a fecal indicator (PMMV) as a potentially effective tool for monitoring trends during decreasing and low-incidence of infection of SARS-Cov-2 in communities.

Some forms of synaptic plasticity require rapid, local activation of protein synthesis. Although this is thought to reflect recruitment of mRNAs to free ribosomes, this would limit the speed and magnitude of translational activation. Here we provide compelling in situ evidence supporting an alternative model in which synaptic mRNAs are transported as stably paused polyribosomes. Remarkably, we show that metabotropic glutamate receptor activation allows the synthesis of proteins that lead to a functional long-term depression phenotype even when translation initiation has been greatly reduced. Thus, neurons evolved a unique mechanism to swiftly translate synaptic mRNAs into functional protein upon synaptic signaling using stalled polyribosomes to bypass the ratelimiting step of translation initiation. Because dysregulated plasticity is implicated in neurodevelopmental and psychiatric disorders such as fragile X syndrome, this work uncovers a unique translational target for therapies.M ost studies of translational control focus on initiation, the process where mRNAs recruit ribosomes and catalyze the first step of translation (1). This highly regulated and normally rate-limiting step of translation is followed by elongation and termination, resulting in completed proteins. Although multiple ribosomes on a given mRNA (a polyribosome) imply active peptide synthesis, we and others identified neuronal RNA granulesmotile aggregates of nontranslating ribosomes (2, 3). These electron-dense bodies contain single copies of synaptic mRNAs that are translationally silenced during their transport from soma to synapse (1, 4).Many models assume that neuronally transported mRNAs are translationally paused before completion of the initiation step of translation during transport. An appropriate synaptic signal would then activate translation (initiation/elongation/termination) of the granule mRNA. However, it is not clear how many free ribosomal subunits are present at synapses to support translation initiation. Further, at a typical translation elongation rate of six amino acids per s (5, 6), synthesis of larger synaptic proteins (e.g., microtubule-associated protein 1b; MAP1b) would take over 5 min even if initiation were immediate. These two factors constrain the speed and magnitude of synaptic translation and, thus, plasticity. As some forms of synaptic plasticity require rapid (<10 min) and localized activation of protein synthesis, an alternative model is wanting (7-9).We have previously proposed the concept of a neuronal RNA granule as a stalled polyribosome (10, 11). Ribosomal stalling has been shown to occur in lysates from a mouse neuroblastoma cell line and in an in vitro rabbit reticulocyte lysate translation assay programmed with brain homogenate (12). Whether neuronal ribosome stalling occurs in vivo is uncertain. We hypothesized that neuronal RNA granules contain paused ribosomes with incomplete proteins initiated in the soma before their packaging and transport to dendrites, where translation can be rapidly and loca...

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DAP5/p97 is a member of the eIF4G family of translation initiation factors that has been suggested to play an important role in the translation of select messenger RNA molecules. We have shown previously that the caspase-cleaved form of DAP5/p97, termed p86, is required for the induction of the endoplasmic reticulum (ER)-stress-responsive internal ribosome entry site (IRES) of the caspase inhibitor HIAP2. We show here that expression of DAP5/p97 is enhanced during ER stress by selective recruitment of DAP5/p97 mRNA into polysomes via the DAP5/p97 IRES. Importantly, enhanced translation mediated by the DAP5/p97 IRES is dependent on DAP5/p97 itself, thus providing a positive feedback loop. In addition, we show that activation of DAP5/p97 and HIAP2 IRES during ER stress requires DAP5/p97. Significantly, the induction of DAP5/p97 during ER stress is caspase-independent, whereas the induction of HIAP2 requires proteolytic processing of DAP5/p97. Thus, DAP5/p97 is a translational activator that selectively modulates translation of specific mRNAs during conditions of cellular stress in both a caspase-dependent and caspase-independent manner.

Mechanistic target of rapamcyin (mTOR) is a central player in cell growth throughout the organism. However, mTOR takes on an additional, more specialized role in the developed neuron, where it regulates the protein synthesis-dependent, plastic changes underlying learning and memory. mTOR is sequestered in two multiprotein complexes (mTORC1 and mTORC2) that have different substrate specificities, thus allowing for distinct functions at synapses. We will examine how learning activates the mTOR complexes, survey the critical effectors of this pathway in the context of synaptic plasticity, and assess whether mTOR plays an instructive or permissive role in generating molecular memory traces.

Expression of the cellular inhibitor of apoptosis protein 1 (cIAP1) is unexpectedly repressed at the level of translation under normal physiological conditions in many cell lines. We have previously shown that the 5′ untranslated region of cIAP1 mRNA contains a stress-inducible internal ribosome entry site (IRES) that governs expression of cIAP1 protein. Although inactive in unstressed cells, the IRES supports cap-independent translation of cIAP1 in response to endoplasmic reticulum stress. To gain an insight into the mechanism of cIAP1 IRES function, we empirically derived the minimal free energy secondary structure of the cIAP1 IRES using enzymatic cleavage mapping. We subsequently used RNA affinity chromatography to identify several cellular proteins, including nuclear factor 45 (NF45) as cIAP1 IRES binding proteins. In this report we show that NF45 is a novel RNA binding protein that enhances IRES-dependent translation of endogenous cIAP1. Further, we show that NF45 is required for IRES-mediated induction of cIAP1 protein during the unfolded protein response. The data presented are consistent with a model in which translation of cIAP1 is governed, at least in part, by NF45, a novel cellular IRES trans-acting factor.