The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys isoacceptors in the P site.

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

The ABCF ATPase New1 resolves translation termination defects associated with specific tRNAArg and tRNALys isoacceptors in the P site.

The efficiency of translation termination is determined by the nature of the stop codon as well as its context. In eukaryotes, recognition of the A-site stop codon and release of the polypeptide are mediated by release factors eRF1 and eRF3, respectively. Translation termination is modulated by other factors which either directly interact with release factors or bind to the E-site and modulate the activity of the peptidyl transferase center. Previous studies suggested that the Saccharomyces cerevisiae ABCF ATPase New1 is involved in translation termination and/or ribosome recycling, however, the exact function remained unclear. Here, we have applied 5PSeq, single-particle cryo-EM and readthrough reporter assays to provide insight into the biological function of New1. We show that the lack of New1 results in ribosomal stalling at stop codons preceded by a lysine or arginine codon and that the stalling is not defined by the nature of the C-terminal amino acid but rather by the identity of the tRNA isoacceptor in the P-site. Collectively, our results suggest that translation termination is inefficient when ribosomes have specific tRNA isoacceptors in the P-site and that the recruitment of New1 rescues ribosomes at these problematic termination contexts.

Comparison of Xrn1 and Rat1 5' → 3' exoribonucleases in budding yeast supports the specific role of Xrn1 in cotranslational mRNA decay.

The yeast Saccharomyces cerevisiae and most eukaryotes carry two 5' → 3' exoribonuclease paralogs. In yeast, they are called Xrn1, which shuttles between the nucleus and the cytoplasm, and executes major cytoplasmic messenger RNA (mRNA) decay, and Rat1, which carries a strong nuclear localization sequence (NLS) and localizes to the nucleus. Xrn1 is 30% identical to Rat1 but has an extra ~500 amino acids C-terminal extension. In the cytoplasm, Xrn1 can degrade decapped mRNAs during the last round of translation by ribosomes, a process referred to as "cotranslational mRNA decay." The division of labor between the two enzymes is still enigmatic and serves as a paradigm for the subfunctionalization of many other paralogs. Here we show that Rat1 is capable of functioning in cytoplasmic mRNA decay, provided that Rat1 remains cytoplasmic due to its NLS disruption (cRat1). This indicates that the physical segregation of the two paralogs plays roles in their specific functions. However, reversing segregation is not sufficient to fully complement the Xrn1 function. Specifically, cRat1 can partially restore the cell volume, mRNA stability, the proliferation rate, and 5' → 3' decay alterations that characterize xrn1Δ cells. Nevertheless, cotranslational decay is only slightly complemented by cRat1. The use of the AlphaFold prediction for cRat1 and its subsequent docking with the ribosome complex and the sequence conservation between cRat1 and Xrn1 suggest that the tight interaction with the ribosome observed for Xrn1 is not maintained in cRat1. Adding the Xrn1 C-terminal domain to Rat1 does not improve phenotypes, which indicates that lack of the C-terminal is not responsible for partial complementation. Overall, during evolution, it appears that the two paralogs have acquired specific characteristics to make functional partitioning beneficial.

Cellular energy regulates mRNA degradation in a codon-specific manner.

Codon optimality is a major determinant of mRNA translation and degradation rates. However, whether and through which mechanisms its effects are regulated remains poorly understood. Here we show that codon optimality associates with up to 2-fold change in mRNA stability variations between human tissues, and that its effect is attenuated in tissues with high energy metabolism and amplifies with age. Mathematical modeling and perturbation data through oxygen deprivation and ATP synthesis inhibition reveal that cellular energy variations non-uniformly alter the effect of codon usage. This new mode of codon effect regulation, independent of tRNA regulation, provides a fundamental mechanistic link between cellular energy metabolism and eukaryotic gene expression.

Targeting MYC induces lipid droplet accumulation by upregulation of HILPDA in clear cell renal cell carcinoma.

Metabolic reprogramming is critical during clear cell renal cell carcinoma (ccRCC) tumorigenesis, manifested by accumulation of lipid droplets (LDs), organelles that have emerged as new hallmarks of cancer. Yet, regulation of their biogenesis is still poorly understood. Here, we demonstrate that MYC inhibition in ccRCC cells lacking the von Hippel Lindau (VHL) gene leads to increased triglyceride content potentiating LD formation in a glutamine-dependent manner. Importantly, the concurrent inhibition of MYC signaling and glutamine metabolism prevented LD accumulation and reduced tumor burden in vivo. Furthermore, we identified the hypoxia-inducible lipid droplet-associated protein (HILPDA) as the key driver for induction of MYC-driven LD accumulation and demonstrated that conversely, proliferation, LD formation, and tumor growth are impaired upon its downregulation. Finally, analysis of ccRCC tissue as well as healthy renal control samples postulated HILPDA as a specific ccRCC biomarker. Together, these results provide an attractive approach for development of alternative therapeutic interventions for the treatment of this type of renal cancer.

The association of the RSC remodeler complex with chromatin is influenced by the prefoldin-like Bud27 and determines nucleosome positioning and polyadenylation sites usage in Saccharomyces cerevisiae.

The tripartite interaction between the chromatin remodeler complex RSC, RNA polymerase subunit Rpb5 and prefoldin-like Bud27 is necessary for proper RNA pol II elongation. Indeed lack of Bud27 alters this association and affects transcription elongation. This work investigates the consequences of lack of Bud27 on the chromatin association of RSC and RNA pol II, and on nucleosome positioning. Our results demonstrate that RSC binds chromatin in gene bodies and lack of Bud27 alters this association, mainly around polyA sites. This alteration impacts chromatin organization and leads to the accumulation of RNA pol II molecules around polyA sites, likely due to pausing or arrest. Our data suggest that RSC is necessary to maintain chromatin organization around those sites, and any alteration of this organization results in the widespread use of alternative polyA sites. Finally, we also find a similar molecular phenotype that occurs upon TOR inhibition with rapamycin, which suggests that alternative polyadenylation observed upon TOR inhibition is likely Bud27-dependent.

Digital assay for rapid electronic quantification of clinical pathogens using DNA nanoballs.

Fast and accurate detection of nucleic acids is key for pathogen identification. Methods for DNA detection generally rely on fluorescent or colorimetric readout. The development of label-free assays decreases costs and test complexity. We present a novel method combining a one-pot isothermal generation of DNA nanoballs with their detection by electrical impedance. We modified loop-mediated isothermal amplification by using compaction oligonucleotides that self-assemble the amplified target into nanoballs. Next, we use capillary-driven flow to passively pass these nanoballs through a microfluidic impedance cytometer, thus enabling a fully compact system with no moving parts. The movement of individual nanoballs is detected by a change in impedance providing a quantized readout. This approach is flexible for the detection of DNA/RNA of numerous targets (severe acute respiratory syndrome coronavirus 2, HIV, ß-lactamase gene, etc.), and we anticipate that its integration into a standalone device would provide an inexpensive (<$5), sensitive (10 target copies), and rapid test (<1 hour).

Atlas of mRNA translation and decay for bacteria.

Regulation of messenger RNA stability is pivotal for programmed gene expression in bacteria and is achieved by a myriad of molecular mechanisms. By bulk sequencing of 5' monophosphorylated mRNA decay intermediates (5'P), we show that cotranslational mRNA degradation is conserved among both Gram-positive and -negative bacteria. We demonstrate that, in species with 5'-3' exonucleases, the exoribonuclease RNase J tracks the trailing ribosome to produce an in vivo single-nucleotide toeprint of the 5' position of the ribosome. In other species lacking 5'-3' exonucleases, ribosome positioning alters endonucleolytic cleavage sites. Using our metadegradome (5'P degradome) sequencing approach, we characterize 5'P mRNA decay intermediates in 96 species including Bacillus subtilis, Escherichia coli, Synechocystis spp. and Prevotella copri and identify codon- and gene-level ribosome stalling responses to stress and drug treatment. We also apply 5'P sequencing to complex clinical and environmental microbiomes and demonstrate that metadegradome sequencing provides fast, species-specific posttranscriptional characterization of responses to drug or environmental perturbations. Finally we produce a degradome atlas for 96 species to enable analysis of mechanisms of RNA degradation in bacteria. Our work paves the way for the application of metadegradome sequencing to investigation of posttranscriptional regulation in unculturable species and complex microbial communities.

Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events.

BACKGROUND: The Ccr4-Not complex is mostly known as the major eukaryotic deadenylase. However, several studies have uncovered roles of the complex, in particular of the Not subunits, unrelated to deadenylation and relevant for translation. In particular, the existence of Not condensates that regulate translation elongation dynamics has been reported. Typical studies that evaluate translation efficiency rely on soluble extracts obtained after the disruption of cells and ribosome profiling. Yet cellular mRNAs in condensates can be actively translated and may not be present in such extracts. RESULTS: In this work, by analyzing soluble and insoluble mRNA decay intermediates in yeast, we determine that insoluble mRNAs are enriched for ribosomes dwelling at non-optimal codons compared to soluble mRNAs. mRNA decay is higher for soluble RNAs, but the proportion of co-translational degradation relative to the overall mRNA decay is higher for insoluble mRNAs. We show that depletion of Not1 and Not4 inversely impacts mRNA solubilities and, for soluble mRNAs, ribosome dwelling according to codon optimality. Depletion of Not4 solubilizes mRNAs with lower non-optimal codon content and higher expression that are rendered insoluble by Not1 depletion. By contrast, depletion of Not1 solubilizes mitochondrial mRNAs, which are rendered insoluble upon Not4 depletion. CONCLUSIONS: Our results reveal that mRNA solubility defines the dynamics of co-translation events and is oppositely regulated by Not1 and Not4, a mechanism that we additionally determine may already be set by Not1 promoter association in the nucleus.

A palmitate-rich metastatic niche enables metastasis growth via p65 acetylation resulting in pro-metastatic NF-κB signaling.

Metabolic rewiring is often considered an adaptive pressure limiting metastasis formation; however, some nutrients available at distant organs may inherently promote metastatic growth. We find that the lung and liver are lipid-rich environments. Moreover, we observe that pre-metastatic niche formation increases palmitate availability only in the lung, whereas a high-fat diet increases it in both organs. In line with this, targeting palmitate processing inhibits breast cancer-derived lung metastasis formation. Mechanistically, breast cancer cells use palmitate to synthesize acetyl-CoA in a carnitine palmitoyltransferase 1a-dependent manner. Concomitantly, lysine acetyltransferase 2a expression is promoted by palmitate, linking the available acetyl-CoA to the acetylation of the nuclear factor-kappaB subunit p65. Deletion of lysine acetyltransferase 2a or carnitine palmitoyltransferase 1a reduces metastasis formation in lean and high-fat diet mice, and lung and liver metastases from patients with breast cancer show coexpression of both proteins. In conclusion, palmitate-rich environments foster metastases growth by increasing p65 acetylation, resulting in a pro-metastatic nuclear factor-kappaB signaling.

Differential regulation of mRNA stability modulates transcriptional memory and facilitates environmental adaptation.

Transcriptional memory, by which cells respond faster to repeated stimuli, is key for cellular adaptation and organism survival. Chromatin organization has been shown to play a role in the faster response of primed cells. However, the contribution of post-transcriptional regulation is not yet explored. Here we perform a genome-wide screen to identify novel factors modulating transcriptional memory in S. cerevisiae in response to galactose. We find that depletion of the nuclear RNA exosome increases GAL1 expression in primed cells. Our work shows that gene-specific differences in intrinsic nuclear surveillance factor association can enhance both gene induction and repression in primed cells. Finally, we show that primed cells present altered levels of RNA degradation machinery and that both nuclear and cytoplasmic mRNA decay modulate transcriptional memory. Our results demonstrate that mRNA post-transcriptional regulation, and not only transcription regulation, should be considered when investigating gene expression memory.

RNA Pol II Assembly Affects ncRNA Expression.

RNA pol II assembly occurs in the cytoplasm before translocation of the enzyme to the nucleus. Affecting this assembly influences mRNA transcription in the nucleus and mRNA decay in the cytoplasm. However, very little is known about the consequences on ncRNA synthesis. In this work, we show that impairment of RNA pol II assembly leads to a decrease in cryptic non-coding RNAs (preferentially CUTs and SUTs). This alteration is partially restored upon overcoming the assembly defect. Notably, this drop in ncRNAs is only partially dependent on the nuclear exosome, which suggests a major specific effect of enzyme assembly. Our data also point out a defect in transcription termination, which leads us to propose that CTD phosphatase Rtr1 could be involved in this process.

OPUSeq simplifies detection of low-frequency DNA variants and uncovers fragmentase-associated artifacts.

Detection of low-frequency DNA variants (below 1%) is becoming increasingly important in biomedical research and clinical practice, but is challenging to do with standard sequencing approaches due to high error rates. The use of double-stranded unique molecular identifiers (dsUMIs) allows correction of errors by comparing reads arising from the same original DNA duplex. However, the implementation of such approaches is still challenging. Here, we present a novel method, one-pot dsUMI sequencing (OPUSeq), which allows incorporation of dsUMIs in the same reaction as the library PCR. This obviates the need for adapter pre-synthesis or additional enzymatic steps. OPUSeq can be incorporated into standard DNA library preparation approaches and coupled with hybridization target capture. We demonstrate successful error correction and detection of variants down to allele frequency of 0.01%. Using OPUSeq, we also show that the use of enzymatic fragmentation can lead to the appearance of spurious double-stranded variants, interfering with detection of variant fractions below 0.1%.

Structural basis for PoxtA-mediated resistance to phenicol and oxazolidinone antibiotics.

PoxtA and OptrA are ATP binding cassette (ABC) proteins of the F subtype (ABCF). They confer resistance to oxazolidinone and phenicol antibiotics, such as linezolid and chloramphenicol, which stall translating ribosomes when certain amino acids are present at a defined position in the nascent polypeptide chain. These proteins are often encoded on mobile genetic elements, facilitating their rapid spread amongst Gram-positive bacteria, and are thought to confer resistance by binding to the ribosome and dislodging the bound antibiotic. However, the mechanistic basis of this resistance remains unclear. Here we refine the PoxtA spectrum of action, demonstrate alleviation of linezolid-induced context-dependent translational stalling, and present cryo-electron microscopy structures of PoxtA in complex with the Enterococcus faecalis 70S ribosome. PoxtA perturbs the CCA-end of the P-site tRNA, causing it to shift by ∼4 Šout of the ribosome, corresponding to a register shift of approximately one amino acid for an attached nascent polypeptide chain. We postulate that the perturbation of the P-site tRNA by PoxtA thereby alters the conformation of the attached nascent chain to disrupt the drug binding site.

Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A.

In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1. Not5 resides in punctate loci, co-purifies with ribosomes and Rli1, but not with eIF5A, and limits mRNA solubility. Overexpression of wild-type or non-complementing Rli1 and loss of Rps7A ubiquitination enable Not4 E3 ligase-dependent translation of polyarginine stretches. We propose that Not4 and Not5 modulate translation elongation dynamics to produce a soluble proteome by Rps7A ubiquitination, dynamic condensates that limit mRNA solubility and exclude eIF5A, and a moonlighting function of Rli1.

Simultaneous detection of SARS-CoV-2 and pandemic (H1N1) 2009 virus with real-time isothermal platform.

The recent ongoing outbreak of novel coronavirus SARS-CoV-2 (known as COVID-19) is a severe threat to human health worldwide. By press time, more than 3.3 million people have died from COVID-19, with many countries experiencing peaks in infections and hospitalizations. The main symptoms of infection with SARS-CoV-2 include fever, chills, coughing, shortness of breath or difficulty breathing, fatigue, muscle or body aches and pains. While the symptoms of the pandemic (H1N1) 2009 virus have many similarities to the signs and transmission routes of the novel coronavirus, e.g., fever, cough, sore throat, body aches, headache, chills and fatigue. And a few cases of serious illness, rapid progress, can appear viral pneumonia, combined with respiratory failure, multiple organ function damage, serious people can die. Therefore, there is an urgent need to develop a rapid and accurate field diagnostic method to effectively identify the two viruses and treat these early infections on time, thus helping to control the spread of the disease. Among molecular detection methods, RT-LAMP (real-time reverse transcription-loop-mediated isothermal amplification) has some advantages in pathogen detection due to its rapid, accurate and effective detection characteristics. Here, we combined the primers of the two viruses with the fluorescent probes on the RT-LAMP detection platform to detect the two viruses simultaneously. Firstly, RT-LAMP method was used respectively to detect the two viruses at different concentrations to determine the effectiveness and sensitivity of probe primers to the RNA samples. And then, the two virus samples were detected simultaneously in the same reaction tube to validate if testing for the two viruses together had an impact on the results compared to detecting alone. We verified the detection efficiency of three highly active BST variants during RT-LAMP assay. We expect that this assay can effectively and accurately distinguish COVID-19 from the pandemic (H1N1) 2009, so that these two diseases with similar symptoms can be appropriately differentiated and treated.

Using TIF-Seq2 to investigate association between 5´ and 3´mRNA ends.

The development of high-throughput technologies has revealed pervasive transcription in all genomes that have been investigated so far. This has uncovered a highly interleaved transcriptome organization involving thousands of overlapping coding and non-coding RNA isoforms that challenge our traditional definitions of genes and functional regions of the genome. In this chapter, we discuss the application of an improved Transcript Isoform Sequencing approach (TIF-Seq2) able to concurrently determine the start and end sites of individual RNA molecules. We exemplify its use for the investigation of the human transcriptome and show how it is especially well suited to discriminate between overlapping molecules and accurately define their boundaries.

Sample-to-answer COVID-19 nucleic acid testing using a low-cost centrifugal microfluidic platform with bead-based signal enhancement and smartphone read-out.

With its origin estimated around December 2019 in Wuhan, China, the ongoing SARS-CoV-2 pandemic is a major global health challenge. The demand for scalable, rapid and sensitive viral diagnostics is thus particularly pressing at present to help contain the rapid spread of infection and prevent overwhelming the capacity of health systems. While high-income countries have managed to rapidly expand diagnostic capacities, such is not the case in resource-limited settings of low- to medium-income countries. Aiming at developing cost-effective viral load detection systems for point-of-care COVID-19 diagnostics in resource-limited and resource-rich settings alike, we report the development of an integrated modular centrifugal microfluidic platform to perform loop-mediated isothermal amplification (LAMP) of viral RNA directly from heat-inactivated nasopharyngeal swab samples. The discs were pre-packed with dried n-benzyl-n-methylethanolamine modified agarose beads used to selectively remove primer dimers, inactivate the reaction post-amplification and allowing enhanced fluorescence detection via a smartphone camera. Sample-to-answer analysis within 1 hour from sample collection and a detection limit of approximately 100 RNA copies in 10 µL reaction volume were achieved. The platform was validated with a panel of 162 nasopharyngeal swab samples collected from patients with COVID-19 symptoms, providing a sensitivity of 96.6% (82.2-99.9%, 95% CI) for samples with Ct values below 26 and a specificity of 100% (90-100%, 95% CI), thus being fit-for-purpose to diagnose patients with a high risk of viral transmission. These results show significant promise towards bringing routine point-of-care COVID-19 diagnostics to resource-limited settings.

Application of high-throughput 5'P sequencing for the study of co-translational mRNA decay.

mRNA degradation is connected to the translation process up to the degree that 5'-3' mRNA degradation follows the last translating ribosome. To study 5'-3'co-translational mRNA decay and the associated ribosome dynamics, here we present an improved high-throughput 5'P degradome RNA sequencing protocol (HT-5Pseq). We exemplify its application in Saccharomyces cerevisiae, but in principle, it could be applied to any other eukaryotic organism. HT-5Pseq is easy, scalable, and uses affordable duplex-specific nuclease-based rRNA depletion. For complete details on the use and execution of this protocol, please refer to Zhang and Pelechano (2021).