Programmable modulation of ribosomal frameshifting by mRNA targeting CRISPR-Cas12a system.

Minus 1 programmed ribosomal frameshifting (-1 PRF) is a conserved translational regulation event essential for critical biological processes, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Efficient trans-modulation of the structured RNA element crucial to -1 PRF will endow the therapeutic application. Here, we demonstrate that CRISPR RNA can stimulate efficient -1 PRF. Assembled CRISPR-Cas12a, but not CRISPR-Cas9, complex further enhances -1 PRF efficiency through its higher capacity to stall translating ribosomes. We additionally perform CRISPR-Cas12a targeting to impair the SARS-CoV-2 frameshifting pseudoknot structure via a focused screening. We demonstrate that targeting CRISPR-Cas12a results in more than 70% suppression of -1 PRF in vitro and about 50% suppression in mammalian cells. Our results show the expanded function of the CRISPR-Cas12 system in modulating -1 PRF efficiency through stalling ribosomes and deforming frameshifting stimulatory signals, which could serve as a new strategy for future coronavirus pandemics.

Crystal structures of dimeric and heptameric mtHsp60 reveal the mechanism of chaperonin inactivation.

Mitochondrial Hsp60 (mtHsp60) plays a crucial role in maintaining the proper folding of proteins in the mitochondria. mtHsp60 self-assembles into a ring-shaped heptamer, which can further form a double-ring tetradecamer in the presence of ATP and mtHsp10. However, mtHsp60 tends to dissociate in vitro, unlike its prokaryotic homologue, GroEL. The molecular structure of dissociated mtHsp60 and the mechanism behind its dissociation remain unclear. In this study, we demonstrated that Epinephelus coioides mtHsp60 (EcHsp60) can form a dimeric structure with inactive ATPase activity. The crystal structure of this dimer reveals symmetrical subunit interactions and a rearranged equatorial domain. The α4 helix of each subunit extends and interacts with its adjacent subunit, leading to the disruption of the ATP-binding pocket. Furthermore, an RLK motif in the apical domain contributes to stabilizing the dimeric complex. These structural and biochemical findings provide new insights into the conformational transitions and functional regulation of this ancient chaperonin.

Optimization of 5'UTR to evade SARS-CoV-2 Nonstructural protein 1-directed inhibition of protein synthesis in cells.

Maximizing the expression level of therapeutic proteins in cells is the general goal for DNA/mRNA therapies. It is particularly challenging to achieve efficient protein expression in the cellular contexts with inhibited translation machineries, such as in the presence of cellular Nonstructural protein 1 (Nsp1) of coronaviruses (CoVs) that has been reported to inhibit overall protein synthesis of host genes and exogenously delivered mRNAs/DNAs. In this study, we thoroughly examined the sequence and structure contexts of viral and non-viral 5'UTRs that determine the protein expression levels of exogenously delivered DNAs and mRNAs in cells expressing SARS-CoV-2 Nsp1. It was found that high 5'-proximal A/U content promotes an escape from Nsp1-directed inhibition of protein synthesis and results in selective protein expression. Furthermore, 5'-proximal Cs were found to significantly enhance the protein expression in an Nsp1-dependent manner, while Gs located at a specific window close to the 5'-end counteract such enhancement. The distinct protein expression levels resulted from different 5'UTRs were found correlated to Nsp1-induced mRNA degradations. These findings ultimately enabled rational designs for optimized 5'UTRs that lead to strong expression of exogenous proteins regardless of the translationally repressive Nsp1. On the other hand, we have also identified several 5'-proximal sequences derived from host genes that are capable of mediating the escapes. These results provided novel perspectives to the optimizations of 5'UTRs for DNA/mRNA therapies and/or vaccinations, as well as shedding light on the potential host escapees from Nsp1-directed translational shutoffs. KEY POINTS: • The 5'-proximal SL1 and 5a/b derived from SARS-CoV-2 genomic RNA promote exogenous protein synthesis in cells expressing Nsp1 comparing with non-specific 5'UTRs. • Specific 5'-proximal sequence contexts are the key determinants of the escapes from Nsp1-directed translational repression and thereby enhance protein expressions. • Systematic mutagenesis identified optimized 5'UTRs that strongly enhance protein expression and promote resistance to Nsp1-induced translational repression and RNA degradation.

YMLA: A comparative platform to carry out functional enrichment analysis for multiple gene lists in yeast.

Comparative analysis among multiple gene lists on their functional features is now a routine task due to the advancement of high-throughput experiments. Several enrichment analysis tools were developed in the past. However, these tools mainly focus on one gene list and contain only gene ontology or interaction features. What makes it worse, comparative investigation and customized feature set reanalysis are still unavailable. Therefore, we constructed the YMLA (Yeast Multiple List Analyzer) platform in this research. YMLA includes 39 yeast features and facilitates comparative analysis among multiple gene lists via tabular views, heatmaps, and network plots. Moreover, the customized feature set reanalysis function was implemented in YMLA to help form mechanism hypotheses based on a selected enriched feature subset. We demonstrated the biological applicability of YMLA via example lists consisting of genes with top/bottom translation efficiency values. The analysis results provided by YMLA reveal novel facts consistent with previous experiments. YMLA is available at https://cosbi7.ee.ncku.edu.tw/YMLA/.

RNA bisulfite sequencing reveals NSUN2-mediated suppression of epithelial differentiation in pancreatic cancer.

Posttranscriptional modifications in RNA have been considered to contribute to disease pathogenesis and tumor progression. NOL1/NOP2/Sun domain family member 2 (NSUN2) is an RNA methyltransferase that promotes tumor progression in several cancers. Pancreatic cancer relapse inevitably occurs even in cases where primary tumors have been successfully treated. Associations of cancer progression due to reprogramming of the cancer methyl-metabolome and the cancer genome have been noted, but the effect of base modifications, namely 5-methylcytosine (m5C), in the transcriptome remains unclear. Aberrant regulation of 5-methylcytosine turnover in cancer may affect posttranscriptional modifications in coding and noncoding RNAs in disease pathogenesis. Mutations in NSUN2 have been reported as drivers of neurodevelopmental disorders in mice, and upregulated expression of NSUN2 in tumors of the breast, bladder, and pancreas has been reported. In this study, we conducted mRNA whole transcriptomic bisulfite sequencing to categorize NSUN2 target sites in the mRNA of human pancreatic cancer cells. We identified a total of 2829 frequent m5C sites in mRNA from pancreatic cancer cells. A total of 90.9% (2572/2829) of these m5C sites were mapped to annotated genes in autosomes and sex chromosomes X and Y. Immunohistochemistry staining confirmed that the NSUN2 expression was significantly upregulated in cancer lesions in the LSL-KrasG12D/+;Trp53fl/fl;Pdx1-Cre (KPC) spontaneous pancreatic cancer mouse model induced by Pdx1-driven Cre/lox system expressing mutant KrasG12D and p53 deletion. The in vitro phenotypic analysis of NSUN2 knockdown showed mild effects on pancreatic cancer cell 2D/3D growth, morphology and gemcitabine sensitivity in the early phase of tumorigenesis, but cumulative changes after multiple cell doubling passages over time were required for these mutations to accumulate. Syngeneic transplantation of NSUN2-knockdown KPC cells via subcutaneous injection showed decreased stromal fibrosis and restored differentiation of ductal epithelium in vivo. SIGNIFICANCE: Transcriptome-wide mRNA bisulfite sequencing identified candidate m5C sites of mRNAs in human pancreatic cancer cells. NSUN2-mediated m5C mRNA metabolism was observed in a mouse model of pancreatic cancer. NSUN2 regulates cancer progression and epithelial differentiation via mRNA methylation.

Structural phylogenetic analysis reveals lineage-specific RNA repetitive structural motifs in all coronaviruses and associated variations in SARS-CoV-2.

In many single-stranded (ss) RNA viruses, the cis-acting packaging signal that confers selectivity genome packaging usually encompasses short structured RNA repeats. These structural units, termed repetitive structural motifs (RSMs), potentially mediate capsid assembly by specific RNA-protein interactions. However, general knowledge of the conservation and/or the diversity of RSMs in the positive-sense ssRNA coronaviruses (CoVs) is limited. By performing structural phylogenetic analysis, we identified a variety of RSMs in nearly all CoV genomic RNAs, which are exclusively located in the 5'-untranslated regions (UTRs) and/or in the inter-domain regions of poly-protein 1ab coding sequences in a lineage-specific manner. In all alpha- and beta-CoVs, except for Embecovirus spp, two to four copies of 5'-gUUYCGUc-3' RSMs displaying conserved hexa-loop sequences were generally identified in Stem-loop 5 (SL5) located in the 5'-UTRs of genomic RNAs. In Embecovirus spp., however, two to eight copies of 5'-agc-3'/guAAu RSMs were found in the coding regions of non-structural protein (NSP) 3 and/or NSP15 in open reading frame (ORF) 1ab. In gamma- and delta-CoVs, other types of RSMs were found in several clustered structural elements in 5'-UTRs and/or ORF1ab. The identification of RSM-encompassing structural elements in all CoVs suggests that these RNA elements play fundamental roles in the life cycle of CoVs. In the recently emerged SARS-CoV-2, beta-CoV-specific RSMs are also found in its SL5, displaying two copies of 5'-gUUUCGUc-3' motifs. However, multiple sequence alignment reveals that the majority of SARS-CoV-2 possesses a variant RSM harboring SL5b C241U, and intriguingly, several variations in the coding sequences of viral proteins, such as Nsp12 P323L, S protein D614G, and N protein R203K-G204R, are concurrently found with such variant RSM. In conclusion, the comprehensive exploration for RSMs reveals phylogenetic insights into the RNA structural elements in CoVs as a whole and provides a new perspective on variations currently found in SARS-CoV-2.

eRF1 mediates codon usage effects on mRNA translation efficiency through premature termination at rare codons.

Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and plays an important role in regulating gene expression levels. A major role of codon usage is thought to regulate protein expression levels by affecting mRNA translation efficiency, but the underlying mechanism is unclear. By analyzing ribosome profiling results, here we showed that codon usage regulates translation elongation rate and that rare codons are decoded more slowly than common codons in all codon families in Neurospora. Rare codons resulted in ribosome stalling in manners both dependent and independent of protein sequence context and caused premature translation termination. This mechanism was shown to be conserved in Drosophila cells. In both Neurospora and Drosophila cells, codon usage plays an important role in regulating mRNA translation efficiency. We found that the rare codon-dependent premature termination is mediated by the translation termination factor eRF1, which recognizes ribosomes stalled on rare sense codons. Silencing of eRF1 expression resulted in codon usage-dependent changes in protein expression. Together, these results establish a mechanism for how codon usage regulates mRNA translation efficiency.

Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells.

Codon usage biases are found in all eukaryotic and prokaryotic genomes and have been proposed to regulate different aspects of translation process. Codon optimality has been shown to regulate translation elongation speed in fungal systems, but its effect on translation elongation speed in animal systems is not clear. In this study, we used a Drosophila cell-free translation system to directly compare the velocity of mRNA translation elongation. Our results demonstrate that optimal synonymous codons speed up translation elongation while non-optimal codons slow down translation. In addition, codon usage regulates ribosome movement and stalling on mRNA during translation. Finally, we show that codon usage affects protein structure and function in vitro and in Drosophila cells. Together, these results suggest that the effect of codon usage on translation elongation speed is a conserved mechanism from fungi to animals that can affect protein folding in eukaryotic organisms.

Codon usage is an important determinant of gene expression levels largely through its effects on transcription.

Codon usage biases are found in all eukaryotic and prokaryotic genomes, and preferred codons are more frequently used in highly expressed genes. The effects of codon usage on gene expression were previously thought to be mainly mediated by its impacts on translation. Here, we show that codon usage strongly correlates with both protein and mRNA levels genome-wide in the filamentous fungus Neurospora Gene codon optimization also results in strong up-regulation of protein and RNA levels, suggesting that codon usage is an important determinant of gene expression. Surprisingly, we found that the impact of codon usage on gene expression results mainly from effects on transcription and is largely independent of mRNA translation and mRNA stability. Furthermore, we show that histone H3 lysine 9 trimethylation is one of the mechanisms responsible for the codon usage-mediated transcriptional silencing of some genes with nonoptimal codons. Together, these results uncovered an unexpected important role of codon usage in ORF sequences in determining transcription levels and suggest that codon biases are an adaptation of protein coding sequences to both transcription and translation machineries. Therefore, synonymous codons not only specify protein sequences and translation dynamics, but also help determine gene expression levels.

Comparison of the surface roughness of gypsum models constructed using various impression materials and gypsum products.

BACKGROUND/PURPOSE: This study compared the surface roughness of gypsum models constructed using various impression materials, gypsum products, and storage times before repouring. MATERIALS AND METHODS: Three alginate impression materials, four commercial silicone impression materials, and three types of gypsum product (MG crystal rock, Super hard stone, and MS plaster) were used. Impression materials were mixed and poured into five plastic rings (20 mm in diameter and 2 mm high) for each group, and the surfaces of the set gypsum product models of 63 groups, which were poured immediately, and 1 hour and 24 hours later, were assessed using a surface roughness tester. One-way ANOVA and Bonferroni's comparison tests were used for the statistical analyses. RESULTS: The surface roughness: (1) was greater for most specimens constructed from alginate impression material (2.72 ± 0.45-7.42 ± 0.66 µm) than from silicone impression materials (1.86 ± 0.19-2.75 ± 0.44 µm); (2) differed with the type of gypsum product when using alginate impression materials (surface roughness of Super hard stone > MG crystal rock > MS plaster), but differed little for silicone impression materials; and (3) differed very little with the storage time before repouring. CONCLUSION: The surface roughness of stone models was mainly determined by the type of alginate impression material, and was less affected by the type of silicone rubber impression material or gypsum product, or the storage time before repouring.

Codon Usage Influences the Local Rate of Translation Elongation to Regulate Co-translational Protein Folding.

Codon usage bias is a universal feature of eukaryotic and prokaryotic genomes and has been proposed to regulate translation efficiency, accuracy, and protein folding based on the assumption that codon usage affects translation dynamics. The roles of codon usage in translation, however, are not clear and have been challenged by recent ribosome profiling studies. Here we used a Neurospora cell-free translation system to directly monitor the velocity of mRNA translation. We demonstrated that the preferred codons enhance the rate of translation elongation, whereas non-optimal codons slow elongation. Codon usage also controls ribosome traffic on mRNA. These conclusions were supported by ribosome profiling results in vitro and in vivo with template mRNAs designed to increase the signal-to-noise ratio. Finally, we demonstrate that codon usage regulates protein function by affecting co-translational protein folding. These results resolve a long-standing fundamental question and suggest the existence of a codon usage code for protein folding.

Monitoring ribosomal frameshifting as a platform to screen anti-riboswitch drug candidates.

Riboswitches are regions within mRNAs that can regulate downstream expression of genes through metabolite-induced alteration of their secondary structures. Due to the significant association of bacterial essential or virulence genes, bacterial riboswitches have become promising targets for development of putative antibacterial drugs. However, most of the screening systems to date are based on in vitro or bacterial systems, lacking the possibility to preobserve the adverse effects to the host's translation machinery. This chapter describes a novel screening method based on monitoring the riboswitch-induced -1 ribosomal frameshifting (-1 FS) efficiency in a mammalian cell-free lysate system using preQ1 class-I (preQ1-I) riboswitches as model target.

The Aß peptide forms non-amyloid fibrils in the presence of carbon nanotubes.

Carbon nanotubes have specific properties that make them potentially useful in biomedicine and biotechnology. However, carbon nanotubes may themselves be toxic, making it imperative to understand how carbon nanotubes interact with biomolecules such as proteins. Here, we used NMR, CD, and ThT/fluorescence spectroscopy together with AFM imaging to study pH-dependent molecular interactions between single walled carbon nanotubes (SWNTs) and the amyloid-beta (Aß) peptide. The aggregation of the Aß peptide, first into oligomers and later into amyloid fibrils, is considered to be the toxic mechanism behind Alzheimer's disease. We found that SWNTs direct the Aß peptides to form a new class of ß-sheet-rich yet non-amyloid fibrils.

Stimulation of ribosomal frameshifting by RNA G-quadruplex structures.

Guanine-rich sequences can fold into four-stranded structures of stacked guanine-tetrads, so-called G-quadruplexes (G4). These unique motifs have been extensively studied on the DNA level; however, exploration of the biological roles of G4s at the RNA level is just emerging. Here we show that G4 RNA when introduced within coding regions are capable of stimulating -1 ribosomal frameshifting (-1 FS) in vitro and in cultured cells. Systematic manipulation of the loop length between each G-tract revealed that the -1 FS efficiency positively correlates with G4 stability. Addition of a G4-stabilizing ligand, PhenDC3, resulted in higher -1 FS. Further, we demonstrated that the G4s can stimulate +1 FS and stop codon readthrough as well. These results suggest a potentially novel translational gene regulation mechanism mediated by G4 RNA.

Inhibiting and reversing amyloid-ß peptide (1-40) fibril formation with gramicidin S and engineered analogues.

In Alzheimer's disease, amyloid-ß (Aß) peptides aggregate into extracellular fibrillar deposits. Although these deposits may not be the prime cause of the neurodegeneration that characterizes this disease, inhibition or dissolution of amyloid fibril formation by Aß peptides is likely to affect its development. ThT fluorescence measurements and AFM images showed that the natural antibiotic gramicidin S significantly inhibited Aß amyloid formation in vitro and could dissolve amyloids that had formed in the absence of the antibiotic. In silico docking suggested that gramicidin S, a cyclic decapeptide that adopts a ß-sheet conformation, binds to the Aß peptide hairpin-stacked fibril through ß-sheet interactions. This may explain why gramicidin S reduces fibril formation. Analogues of gramicidin S were also tested. An analogue with a potency that was four-times higher than that of the natural product was identified.

Cellular polyamines promote amyloid-beta (Aß) peptide fibrillation and modulate the aggregation pathways.

The cellular polyamines spermine, spermidine, and their metabolic precursor putrescine, have long been associated with cell-growth, tumor-related gene regulations, and Alzheimer's disease. Here, we show by in vitro spectroscopy and AFM imaging, that these molecules promote aggregation of amyloid-beta (Aß) peptides into fibrils and modulate the aggregation pathways. NMR measurements showed that the three polyamines share a similar binding mode to monomeric Aß(1-40) peptide. Kinetic ThT studies showed that already very low polyamine concentrations promote amyloid formation: addition of 10 µM spermine (normal intracellular concentration is ~1 mM) significantly decreased the lag and transition times of the aggregation process. Spermidine and putrescine additions yielded similar but weaker effects. CD measurements demonstrated that the three polyamines induce different aggregation pathways, involving different forms of induced secondary structure. This is supported by AFM images showing that the three polyamines induce Aß(1-40) aggregates with different morphologies. The results reinforce the notion that designing suitable ligands which modulate the aggregation of Aß peptides toward minimally toxic pathways may be a possible therapeutic strategy for Alzheimer's disease.

Exploiting preQ(1) riboswitches to regulate ribosomal frameshifting.

Knowing the molecular details of the interaction between riboswitch aptamers and their corresponding metabolites is important to understand gene expression. Here we report on a novel in vitro assay to study preQ(1) riboswitch aptamers upon binding of 7-aminomethyl-7-deazaguanine (preQ(1)). The assay is based on the ability of the preQ(1) aptamer to fold, upon ligand binding, into a pseudoknotted structure that is capable of stimulating -1 ribosomal frameshifting (-1 FS). Aptamers from three different species were found to induce between 7% and 20% of -1 FS in response to increasing preQ(1) levels, whereas preQ(1) analogues were 100-1000-fold less efficient. In depth mutational analysis of the Fusobacterium nucleatum aptamer recapitulates most of the structural details previously identified for preQ(1) aptamers from other bacteria by crystallography and/or NMR spectroscopy. In addition to providing insight into the role of individual nucleotides of the preQ(1) riboswitch aptamer in ligand binding, the presented system provides a valuable tool to screen small molecules against bacterial riboswitches in a eukaryotic background.

Dental and medical students' perspectives on early exposure to PBL in Taiwan.

A hybrid problem-based learning (PBL) curriculum adopted in 2002 for medical students at China Medical University, Taiwan, was extended to dental students in 2007. Before that, PBL workshops were conducted for all students. Two PBL cases on basic biomedical issues were used for second-year medical students and second-year dental students to explore the feasibility of adopting PBL as part of the dental curriculum. This study compared the medical and dental students' attitudes toward the PBL tutorials and PBL curricula. Upon completion of the PBL component, an eighteen-item questionnaire asked students to assess (on a ten-point scale with 10 as the most positive response) their perceptions of the learning process in the PBL tutorials. Forty-six dental students from a cohort of fifty (92 percent) and 107 medical students from a cohort of 119 (90 percent) completed the questionnaires (fifty-three females and 100 males). The importance of all items was rated above 6.00. The medical students' mean score (7.29) was higher than the dental students' mean score (7.10). Of the eighteen attributes of the PBL process, the students indicated being generally comfortable with fourteen. No statistical significance was found between the dental and medical students' scores, but there was a significant difference (p=0.006) in their perception of PBL curricula. Overall, the medical students expressed a more positive outlook toward the PBL learning process than the dental students and were more willing to accept PBL as a pedagogy.

Stem-loop structures can effectively substitute for an RNA pseudoknot in -1 ribosomal frameshifting.

-1 Programmed ribosomal frameshifting (PRF) in synthesizing the gag-pro precursor polyprotein of Simian retrovirus type-1 (SRV-1) is stimulated by a classical H-type pseudoknot which forms an extended triple helix involving base-base and base-sugar interactions between loop and stem nucleotides. Recently, we showed that mutation of bases involved in triple helix formation affected frameshifting, again emphasizing the role of the triple helix in -1 PRF. Here, we investigated the efficiency of hairpins of similar base pair composition as the SRV-1 gag-pro pseudoknot. Although not capable of triple helix formation they proved worthy stimulators of frameshifting. Subsequent investigation of ∼30 different hairpin constructs revealed that next to thermodynamic stability, loop size and composition and stem irregularities can influence frameshifting. Interestingly, hairpins carrying the stable GAAA tetraloop were significantly less shifty than other hairpins, including those with a UUCG motif. The data are discussed in relation to natural shifty hairpins.

Stimulation of ribosomal frameshifting by antisense LNA.

Programmed ribosomal frameshifting is a translational recoding mechanism commonly used by RNA viruses to express two or more proteins from a single mRNA at a fixed ratio. An essential element in this process is the presence of an RNA secondary structure, such as a pseudoknot or a hairpin, located downstream of the slippery sequence. Here, we have tested the efficiency of RNA oligonucleotides annealing downstream of the slippery sequence to induce frameshifting in vitro. Maximal frameshifting was observed with oligonucleotides of 12-18 nt. Antisense oligonucleotides bearing locked nucleic acid (LNA) modifications also proved to be efficient frameshift-stimulators in contrast to DNA oligonucleotides. The number, sequence and location of LNA bases in an otherwise DNA oligonucleotide have to be carefully manipulated to obtain optimal levels of frameshifting. Our data favor a model in which RNA stability at the entrance of the ribosomal tunnel is the major determinant of stimulating slippage rather than a specific three-dimensional structure of the stimulating RNA element.