Genetics of cystogenesis in base-edited human organoids reveal therapeutic strategies for polycystic kidney disease.

In polycystic kidney disease (PKD), microscopic tubules expand into macroscopic cysts. Among the world's most common genetic disorders, PKD is inherited via heterozygous loss-of-function mutations but is theorized to require additional loss of function. To test this, we establish human pluripotent stem cells in allelic series representing four common nonsense mutations, using CRISPR base editing. When differentiated into kidney organoids, homozygous mutants spontaneously form cysts, whereas heterozygous mutants (original or base corrected) express no phenotype. Using these, we identify eukaryotic ribosomal selective glycosides (ERSGs) as PKD therapeutics enabling ribosomal readthrough of these same nonsense mutations. Two different ERSGs not only prevent cyst initiation but also limit growth of pre-formed cysts by partially restoring polycystin expression. Furthermore, glycosides accumulate in cyst epithelia in organoids and mice. Our findings define the human polycystin threshold as a surmountable drug target for pharmacological or gene therapy interventions, with relevance for understanding disease mechanisms and future clinical trials.

CLPB Deficiency Associated Neonatal Cavitating Leukoencephalopathy: A Potential Pathomechanism Underlying Neurologic Disorder.

Caseinolytic peptidase B homolog (CLPB) is a mitochondrial protein which is highly expressed in brain. Its deficiency may be associated with severe neonatal encephalopathy. This report describes a case of fatal neonatal encephalopathy associated with biallelic stop-gain mutation in CLPB (NM_001258392.3:c.1159C>T/p.Arg387*). Neurologic disorder encompasses pre- and post-natal features including polyhydramnios, intrauterine growth restriction, respiratory insufficiency, lethargy, excessive startle reflex, generalized hypertonia, and epileptic seizures. Brain macroscopic examination demonstrates frontal severe periventricular cystic leukoencephalopathy, along with mild ex-vacuo tri-ventricular dilatation. The most striking immunohistopathologic features are striato-thalamic neurodegeneration and deep white matter loss associated with strong reactive astrogliosis. This report supports that CLPB deficiency should be considered among the neurometabolic disorders associated with severe prenatal-onset neurologic impairment that may result from cystic leukoencephalopathy.

A premature stop codon in the CYP2C19 gene may explain the unexpected sensitivity of vultures to diclofenac toxicity.

The unintended environmental exposure of vultures to diclofenac has resulted in the deaths of millions of old-world vultures on the Asian subcontinent. While toxicity has been since associated with a long half-life of elimination and zero order metabolism, the actual constraint in biotransformation is yet to be clarified. For this study we evaluated if the evident zero order metabolism could be due to defects in the CYP2C9/2C19 enzyme system. For this, using whole genome sequencing and de-novo transcriptome alignment, the vulture CYP2C19 open reading frame was identified through Splign analysis. The result sequence analysis revealed the presence of a premature stop codon on intron 7 of the identified open reading frame. Even if the stop codon was not present, amino acid residue analysis tended to suggest that the enzyme would be lower in activity than the equivalent human enzyme, with differences present at sites 105, 286 and 289. The defect was also conserved across the eight non-related vultures tested. From these results, we conclude that the sensitivity of the old-world vultures to diclofenac is due to the non-expression of a viable CYP2C19 enzyme system. This is not too dissimilar to the effects seen in certain people with a similar defective enzyme.

Characterization of an aspartate aminotransferase encoded by YPO0623 with frequent nonsense mutations in Yersinia pestis.

Yersinia pestis, the causative agent of plague, is a genetically monomorphic bacterial pathogen that evolved from Yersinia pseudotuberculosis approximately 7,400 years ago. We observed unusually frequent mutations in Y. pestis YPO0623, mostly resulting in protein translation termination, which implies a strong natural selection. These mutations were found in all phylogenetic lineages of Y. pestis, and there was no apparent pattern in the spatial distribution of the mutant strains. Based on these findings, we aimed to investigate the biological function of YPO0623 and the reasons for its frequent mutation in Y. pestis. Our in vitro and in vivo assays revealed that the deletion of YPO0623 enhanced the growth of Y. pestis in nutrient-rich environments and led to increased tolerance to heat and cold shocks. With RNA-seq analysis, we also discovered that the deletion of YPO0623 resulted in the upregulation of genes associated with the type VI secretion system (T6SS) at 26°C, which probably plays a crucial role in the response of Y. pestis to environment fluctuations. Furthermore, bioinformatic analysis showed that YPO0623 has high homology with a PLP-dependent aspartate aminotransferase in Salmonella enterica, and the enzyme activity assays confirmed its aspartate aminotransferase activity. However, the enzyme activity of YPO0623 was significantly lower than that in other bacteria. These observations provide some insights into the underlying reasons for the high-frequency nonsense mutations in YPO0623, and further investigations are needed to determine the exact mechanism.

ACROSIN deficiency causes total fertilization failure in humans by preventing the sperm from penetrating the zona pellucida.

STUDY QUESTION: Does a homozygous nonsense mutation in ACR lead to total fertilization failure (TFF) resulting in male infertility in humans? SUMMARY ANSWER: A novel homozygous nonsense mutation of ACR (c.167G>A, p.Trp56X) was identified in two infertile brothers and shown to cause human TFF. WHAT IS KNOWN ALREADY: ACROSIN, encoded by ACR, is a major acrosomal enzyme expressed only in the acrosome of the sperm head. Inhibition of acrosin prevents sperm penetration of the zona pellucida (ZP) in several species, including humans. Acr-knockout in hamsters causes male infertility with completely blocked fertilization. Of note, there are no reports of ACR mutations associated with TFF in humans. STUDY DESIGN, SIZE, DURATION: Whole-exome sequencing (WES) was used for the identification of pathogenic genes for male factor TFF in eight involved couples. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data from eight infertile couples who had experienced TFF during their IVF or ICSI attempts were collected. Functional assays were used to verify the pathogenicity of the potential genetic factors identified by WES. Subzonal insemination (SUZI) and IVF assays were performed to determine the exact pathogenesis of TFF caused by deficiencies in ACROSIN. MAIN RESULTS AND THE ROLE OF CHANCE: A novel homozygous nonsense mutation in ACR, c.167G>A, p.Trp56X, was identified in two additional primary infertile brothers whose parents were first cousins. This rare mutation caused ACROSIN deficiency and acrosomal ultrastructural defects in the affected sperm. Spermatozoa lacking ACROSIN were unable to penetrate the ZP, rather than hampering sperm binding, disrupting gamete fusion, or preventing oocyte activation. These findings were supported by the fertilization success of SUZI and ICSI attempts, as well as the normal expression of ACTL7A and PLCζ in the mutant sperm, suggesting that ICSI without remedial assisted oocyte activation is an optimal treatment for ARCOSIN-deficient TFF. LIMITATIONS, REASONS FOR CAUTION: The absence of another independent pedigree to support our argument is a limitation of this study. WIDER IMPLICATIONS OF THE FINDINGS: The findings expand our understanding of the genes involved in human TFF, providing information for appropriate genetic counseling and fertility guidance for these patients. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (grant no. 82201803, 81901541, 82271639, and 32000584), University Synergy Innovation Program of Anhui Province (GXXT-2019-044), and the Nonprofit Central Research Institute Fund of the Chinese Academy of Medical Sciences (grant no. 2019PT310002). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

An Efficient Opal-Suppressor Tryptophanyl Pair Creates New Routes for Simultaneously Incorporating up to Three Distinct Noncanonical Amino Acids into Proteins in Mammalian Cells.

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA-suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual-ncAA incorporation. Using these platforms, we site-specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site-specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.

Adeno-associated viral delivery of engineered tRNA-enzyme pairs into nonsense mutation mouse models.

In this protocol, we describe how to utilize the unnatural amino acid (UAA) incorporation system to read through endogenous premature termination codons in a Duchenne muscular dystrophy mouse model. We detail how to screen and optimize tRNA-enzyme pairs for efficient UAA incorporation, deliver the system intraperitoneally or intramuscularly in pathogenic mice by an adeno-associated viral (AAV) vector, and evaluate the restoration of endogenous dystrophin and increase in muscle strength after AAV injection. For complete details on the use and execution of this protocol, please refer to Shi et al. (2021).1.

BrWAX3, Encoding a ß-ketoacyl-CoA Synthase, Plays an Essential Role in Cuticular Wax Biosynthesis in Chinese Cabbage.

In this study, we identified a novel glossy mutant from Chinese cabbage, named SD369, and all wax monomers longer than 26 carbons were significantly decreased. Inheritance analysis revealed that the glossy trait of SD369 was controlled by a single recessive locus, BrWAX3. We fine-mapped the BrWAX3 locus to an interval of 161.82 kb on chromosome A09. According to the annotated genome of Brassica rapa, Bra024749 (BrCER60.A09), encoding a ß-ketoacyl-CoA synthase, was identified as the candidate gene. Expression analysis showed that BrCER60.A09 was significantly downregulated in all aerial organs of glossy plants. Subcellular localization indicated that the BrCER60.A09 protein functions in the endoplasmic reticulum. A 5567-bp insertion was identified in exon 1 of BrCER60.A09 in SD369, which lead to a premature stop codon, thus causing a loss of function of the BrCER60.A09 enzyme. Moreover, comparative transcriptome analysis revealed that the 'cutin, suberine, and wax biosynthesis' pathway was significantly enriched, and genes involved in this pathway were almost upregulated in glossy plants. Further, two functional markers, BrWAX3-InDel and BrWAX3-KASP1, were developed and validated. Overall, these results provide a new information for the cuticular wax biosynthesis and provide applicable markers for marker-assisted selection (MAS)-based breeding of Brassica rapa.

Translation termination codons in protein synthesis and disease.

Fidelity of protein synthesis, a process shaped by several mechanisms involving specialized ribosome regions and external factors, ensures the precise reading of sense as well as stop codons (UGA, UAG, UAA), which are usually localized at the 3' of mRNA and drive the release of the polypeptide chain. However, either natural (NTCs) or premature (PTCs) termination codons, the latter arising from nucleotide changes, can undergo a recoding process named ribosome or translational readthrough, which insert specific amino acids (NTCs) or subset(s) depending on the stop codon type (PTCs). This process is particularly relevant for nonsense mutations, a relatively frequent cause of genetic disorders, which impair gene expression at different levels by potentially leading to mRNA degradation and/or synthesis of truncated proteins. As a matter of fact, many efforts have been made to develop efficient and safe readthrough-inducing compounds, which have been challenged in several models of human disease to provide with a therapy. In this view, the dissection of the molecular determinants shaping the outcome of readthrough, namely nucleotide and protein contexts as well as their interplay and impact on protein structure/function, is crucial to identify responsive nonsense mutations resulting in functional full-length proteins. The interpretation of experimental and mechanistic findings is also important to define a possibly clear picture of potential readthrough-favorable features useful to achieve rescue profiles compatible with therapeutic thresholds typical of each targeted disorder, which is of primary importance for the potential translatability of readthrough into a personalized and mutation-specific, and thus patient-oriented, therapeutic strategy.

Conserved FimK Truncation Coincides with Increased Expression of Type 3 Fimbriae and Cultured Bladder Epithelial Cell Association in Klebsiella quasipneumoniae.

Klebsiella spp. commonly cause both uncomplicated urinary tract infection (UTI) and recurrent UTI (rUTI). Klebsiella quasipneumoniae, a relatively newly defined species of Klebsiella, has been shown to be metabolically distinct from Klebsiella pneumoniae, but its type 1 and type 3 fimbriae have not been studied. K. pneumoniae uses both type 1 and type 3 fimbriae to attach to host epithelial cells. The type 1 fimbrial operon is well conserved between Escherichia coli and K. pneumoniae apart from fimK, which is unique to Klebsiella spp. FimK contains an N-terminal DNA binding domain and a C-terminal phosphodiesterase (PDE) domain that has been hypothesized to cross-regulate type 3 fimbriae expression via modulation of cellular levels of cyclic di-GMP. Here, we find that a conserved premature stop codon in K. quasipneumoniae fimK results in truncation of the C-terminal PDE domain and that K quasipneumoniae strain KqPF9 cultured bladder epithelial cell association and invasion are dependent on type 3 but not type 1 fimbriae. Further, we show that basal expression of both type 1 and type 3 fimbrial operons as well as cultured bladder epithelial cell association is elevated in KqPF9 relative to uropathogenic K. pneumoniae TOP52. Finally, we show that complementation of KqPF9ΔfimK with the TOP52 fimK allele reduced type 3 fimbrial expression and cultured bladder epithelial cell attachment. Taken together these data suggest that the C-terminal PDE of FimK can modulate type 3 fimbrial expression in K. pneumoniae and its absence in K. quasipneumoniae may lead to a loss of type 3 fimbrial cross-regulation. IMPORTANCE K. quasipneumoniae is often indicated as the cause of opportunistic infections, including urinary tract infection, which affects >50% of women worldwide. However, the virulence factors of K. quasipneumoniae remain uninvestigated. Prior to this work, K. quasipneumoniae and K. pneumoniae had only been distinguished phenotypically by metabolic differences. This work contributes to the understanding of K. quasipneumoniae by evaluating the contribution of type 1 and type 3 fimbriae, which are critical colonization factors encoded by all Klebsiella spp., to K. quasipneumoniae bladder epithelial cell attachment in vitro. We observe clear differences in bladder epithelial cell attachment and regulation of type 3 fimbriae between uropathogenic K. pneumoniae and K. quasipneumoniae that coincide with a structural difference in the fimbrial regulatory gene fimK.

Cytochrome P450 mono-oxygenase CYP703A2 plays a central role in sporopollenin formation and ms5ms6 fertility in cotton.

The double-recessive genic male-sterile (ms) line ms5 ms6 has been used to develop cotton (Gossypium hirsutum) hybrids for many years, but its molecular-genetic basis has remained unclear. Here, we identified the Ms5 and Ms6 loci through map-based cloning and confirmed their function in male sterility through CRISPR/Cas9 gene editing. Ms5 and Ms6 are highly expressed in stages 7-9 anthers and encode the cytochrome P450 mono-oxygenases CYP703A2-A and CYP703A2-D. The ms5 mutant carries a single-nucleotide C-to-T nonsense mutation leading to premature chain termination at amino acid 312 (GhCYP703A2-A312aa ), and ms6 carries three nonsynonymous substitutions (D98E, E168K, and G198R) and a synonymous mutation (L11L). Enzyme assays showed that GhCYP703A2 proteins hydroxylate fatty acids, and the ms5 (GhCYP703A2-A312aa ) and ms6 (GhCYP703A2-DD98E,E168K,G198R ) mutant proteins have decreased enzyme activities. Biochemical and lipidomic analyses showed that in ms5 ms6 plants, C12-C18 free fatty acid and phospholipid levels are significantly elevated in stages 7-9 anthers, while stages 8-10 anthers lack sporopollenin fluorescence around the pollen, causing microspore degradation and male sterility. Overall, our characterization uncovered functions of GhCYP703A2 in sporopollenin formation and fertility, providing guidance for creating male-sterile lines to facilitate hybrid cotton production and therefore exploit heterosis for improvement of cotton.

Ataluren suppresses a premature termination codon in an MPS I-H mouse.

ABSTARCT: Suppressing translation termination at premature termination codons (PTCs), termed readthrough, is a potential therapy for genetic diseases caused by nonsense mutations. Ataluren is a compound that has shown promise for clinical use as a readthrough agent. However, some reports suggest that ataluren is ineffective at suppressing PTCs. To further evaluate the effectiveness of ataluren as a readthrough agent, we examined its ability to suppress PTCs in a variety of previously untested models. Using NanoLuc readthrough reporters expressed in two different cell types, we found that ataluren stimulated a significant level of readthrough. We also explored the ability of ataluren to suppress a nonsense mutation associated with Mucopolysaccharidosis I-Hurler (MPS I-H), a genetic disease that is caused by a deficiency of α-L-iduronidase that leads to lysosomal accumulation of glycosaminoglycans (GAGs). Using mouse embryonic fibroblasts (MEFs) derived from Idua-W402X mice, we found that ataluren partially rescued α-L-iduronidase function and significantly reduced GAG accumulation relative to controls. Two-week oral administration of ataluren to Idua-W402X mice led to significant GAG reductions in most tissues compared to controls. Together, these data reveal important details concerning the efficiency of ataluren as a readthrough agent and the mechanisms that govern its ability to suppress PTCs. KEY MESSAGES: Ataluren promotes readthrough of PTCs in a wide variety of contexts. Ataluren reduces glycosaminoglyan storage in MPS I-H cell and mouse models. Ataluren has a bell-shaped dose-response curve and a narrow effective range.

A Novel Homozygous Nonsense Mutation in ZP1 Causes Female Infertility due to Empty Follicle Syndrome.

ZP1 is a critical glycoprotein in the formation of the zona pellucida. It plays an indispensable role in the maturation of oocytes. To identify the causative gene of empty follicle syndrome (EFS) in a patient from a consanguineous family, whole-exome sequencing was performed in the proband. We identified a novel homozygous nonsense mutation c.1260C > G (p. Tyr420X) in the ZP1 gene from two primary infertile patients. Western blot showed that Y420X mutation in ZP1 gene produced a truncated protein. However, the mutation had no significant effect on subcellular localization of the mutant protein. Our findings confirmed the important role of the ZP1 gene in human female reproduction, enriched the mutation spectrums of ZP1 gene, and expanded its applications in the clinical and molecular diagnoses of EFS.

Translational Read-Through Drugs (TRIDs) Are Able to Restore Protein Expression and Ciliogenesis in Fibroblasts of Patients with Retinitis Pigmentosa Caused by a Premature Termination Codon in FAM161A.

Ataluren and Gentamicin are translational readthrough drugs (TRIDs) that induce premature termination codon (PTC) readthrough, resulting in the production of full-length proteins that usually harbor a single missense substitution. FAM161A is a ciliary protein which is expressed in photoreceptors, and pathogenic variants in this gene cause retinitis pigmentosa (RP). Applying TRIDs on fibroblasts from RP patients due to PTC in the FAM161A (p.Arg523*) gene may uncover whether TRIDs can restore expression, localization and function of this protein. Fibroblasts from six patients and five age-matched controls were starved prior to treatment with ataluren or gentamicin, and later FAM161A expression, ciliogenesis and cilia length were analyzed. In contrast to control cells, fibroblasts of patients did not express the FAM161A protein, showed a lower percentage of ciliated cells and grew shorter cilia after starvation. Ataluren and Gentamicin treatment were able to restore FAM161A expression, localization and co-localization with α-tubulin. Ciliogenesis and cilia length were restored following Ataluren treatment almost up to a level which was observed in control cells. Gentamicin was less efficient in ciliogenesis compared to Ataluren. Our results provide a proof-of-concept that PTCs in FAM161A can be effectively suppressed by Ataluren or Gentamicin, resulting in a full-length functional protein.

Renal Expression of CLC-5 and Megalin/Cubilin in Dent-1 Disease With Nonsense Mutations of CLCN5 Gene.

The study aims to explore the clinicopathological features and whether the nonsense mutations of CLCN5 gene have effect on the renal expression of CLC-5 protein and megalin/cubilin complex in children with Dent-1 disease. The clinicopathological features and genetic examination of three patients with Dent-1 disease were investigated. The expression of CLC-5 and megalin/cubilin complex in renal tissues was detected by using immunohistochemistry method. Urinary albumin, α1-microglobulin, ß2-microglobulin, retinol binding protein, and calcium levels were measured by immunonephelometry. Urinary calcium and low molecular weight proteinuria (LMWP) were enhanced in three patients, and two presented with nephrotic range proteinuria. Focal glomerular obsolescence, minor tubulointerstitial injury, and focal calcification in corticomedullary junction were found in one patient. Nonsense mutations of CLCN5 gene from their mothers were identified in all three patients with Dent-1 disease; however, the expression of CLC-5 protein was not decreased in renal tubular cells. As the receptor complex of albumin and LMWP reabsorption, the expression of megalin/cubilin in the brush border of proximal tubules was decreased in Dent-1 patients. Even if the renal CLC-5 protein is expressed normally, the reduced expression of megalin/cubilin in the brush border of renal proximal tubules may be helpful to understand the physiopathology of Dent-1 disease with nonsense mutations of CLCN5 gene.

Exogenous PDE5 Expression Rescues Photoreceptors in RD1 Mice.

BACKGROUND: Catalytic hydrolysis of cyclic guanosine monophosphate (cGMP) by phosphodiesterase 6 (PDE6) is critical in phototransduction signalling in photoreceptors. Mutations in the genes encoding any of the three PDE6 subunits are associated with retinitis pigmentosa, the most common form of inherited retinal diseases. The RD1 mouse carries a naturally occurring nonsense mutation in the Pde6b gene. The RD1 mouse retina rapidly degenerates and fails to form rod photoreceptor outer segments due to the elevated cGMP level and subsequent excessive Ca2+ influx. In this study, we aim to test whether the PDE5 expression, a non-photoreceptor-specific member of the PDE superfamily, rescues photoreceptors in the RD1 retina. METHODS: Electroporation used the PDE5 expression plasmid to transfect neonatal RD1 mice. The mouse retina degeneration was assessed by retinal sections' stains with DAPI. The expression and localization of phototransduction proteins in photoreceptors were analysed by immunostaining. The expression of proteins in cultured cells was analysed by immunoblotting. RESULTS: The exogenous PDE5 expression, a non-photoreceptor-specific member of the PDE superfamily, prevents photoreceptor degeneration in RD1 mice. Unlike endogenous photoreceptor-specific PDE6 localised in the outer segments of photoreceptors, ectopically- expressed PDE5 was distributed in inner segments and synaptic terminals. PDE5 also promoted the development of the outer segments in RD1 mice. PDE5 co-expression with rhodopsin in cultured cells showed enhanced rhodopsin expression. CONCLUSION: Lowering the cGMP level in photoreceptors by PDE5 is sufficient to rescue photoreceptors in RD1 retinas. cGMP may also play a role in rhodopsin expression regulation in photoreceptors.

5' preS1 Mutations To Prevent Large Envelope Protein Expression from Hepatitis B Virus Genotype A or Genotype D Markedly Increase Polymerase-Envelope Fusion Protein.

Hepatitis B virus (HBV) large (L) envelope protein is translated from 2.4-kb RNA. It contains preS1, preS2, and S domains and is detected in Western blotting as p39 and gp42. The 3.5-kb pregenomic RNA produces core and polymerase (P) proteins. We generated L-minus mutants of a genotype A clone and a genotype D clone from 1.1-mer or 1.3-mer construct, with the former overproducing pregenomic RNA. Surprisingly, mutating a preS1 ATG codon(s) or introducing a nonsense mutation soon afterwards switched secreted p39/gp42 to a p41/p44 doublet, with its amount further increased by a nonsense mutation in the core gene. A further-downstream preS1 nonsense mutation prevented p41/p44 production. Tunicamycin treatment confirmed p44 as the glycosylated form of p41. In this regard, splicing of 3.5-kb RNA to generate a junction at nucleotides (nt) 2447 to 2902 for genotype D enables translation of p43, with the N-terminal 47 residues of P protein fused to the C-terminal 371 residues of L protein. Indeed p41/p44 were detectable by an antibody against the N terminus of P protein and eliminated by a nonsense mutation at the 5' P gene or a point mutation to prevent that splicing. Therefore, lost L (and core) protein expression from the 1.1-mer or 1.3-mer construct markedly increased p41/p44 (p43), the P-L fusion protein. Cotransfection with an expression construct for L/M proteins reversed high extracellular p41/p44 associated with L-minus mutants, suggesting that L protein retains p43 in wild-type HBV to promote its intracellular degradation. Considering that p43 lacks N-terminal preS1 sequence critical for receptor binding, its physiological significance during natural infection and therapeutic potential warrant further investigation. IMPORTANCE The large (L) envelope protein of hepatitis B virus (HBV) is translated from 2.4-kb RNA and detected in Western blotting as p39 and gp42. Polymerase (P) protein is expressed at a low level from 3.5-kb RNA. The major spliced form of 3.5-kb RNA will produce a fusion protein between the first 47 residues of P protein and a short irrelevant sequence, although also at a low level. Another spliced form has the same P protein sequence fused to L protein missing its first 18 residues. We found that some point mutations to eliminate L and core protein expression from overlength HBV DNA constructs converted p39/gp42 to p41/gp44, which turned out to be the P-L fusion protein. Thus, the P-L fusion protein can be expressed at extremely high level when L protein expression is prevented. The underlying mechanism and functional significance of this variant form of L protein warrant further investigation.

Genetic diversity of Staphylococcus aureus wall teichoic acid glycosyltransferases affects immune recognition.

Staphylococcus aureus is a leading cause of skin and soft tissue infections and systemic infections. Wall teichoic acids (WTAs) are cell wall-anchored glycopolymers that are important for S. aureus nasal colonization, phage-mediated horizontal gene transfer, and antibiotic resistance. WTAs consist of a polymerized ribitol phosphate (RboP) chain that can be glycosylated with N-acetylglucosamine (GlcNAc) by three glycosyltransferases: TarS, TarM, and TarP. TarS and TarP modify WTA with ß-linked GlcNAc at the C-4 (ß1,4-GlcNAc) and the C-3 position (ß1,3-GlcNAc) of the RboP subunit, respectively, whereas TarM modifies WTA with α-linked GlcNAc at the C-4 position (α1,4-GlcNAc). Importantly, these WTA glycosylation patterns impact immune recognition and clearance of S. aureus. Previous studies suggest that tarS is near-universally present within the S. aureus population, whereas a smaller proportion co-contain either tarM or tarP. To gain more insight into the presence and genetic variation of tarS, tarM and tarP in the S. aureus population, we analysed a collection of 25 652 S. aureus genomes within the PubMLST database. Over 99 % of isolates contained tarS. Co-presence of tarS/tarM or tarS/tarP occurred in 37 and 7 % of isolates, respectively, and was associated with specific S. aureus clonal complexes. We also identified 26 isolates (0.1 %) that contained all three glycosyltransferase genes. At sequence level, we identified tar alleles with amino acid substitutions in critical enzymatic residues or with premature stop codons. Several tar variants were expressed in a S. aureus tar-negative strain. Analysis using specific monoclonal antibodies and human langerin showed that WTA glycosylation was severely attenuated or absent. Overall, our data provide a broad overview of the genetic diversity of the three WTA glycosyltransferases in the S. aureus population and the functional consequences for immune recognition.

A small molecule that induces translational readthrough of CFTR nonsense mutations by eRF1 depletion.

Premature termination codons (PTCs) prevent translation of a full-length protein and trigger nonsense-mediated mRNA decay (NMD). Nonsense suppression (also termed readthrough) therapy restores protein function by selectively suppressing translation termination at PTCs. Poor efficacy of current readthrough agents prompted us to search for better compounds. An NMD-sensitive NanoLuc readthrough reporter was used to screen 771,345 compounds. Among the 180 compounds identified with readthrough activity, SRI-37240 and its more potent derivative SRI-41315, induce a prolonged pause at stop codons and suppress PTCs associated with cystic fibrosis in immortalized and primary human bronchial epithelial cells, restoring CFTR expression and function. SRI-41315 suppresses PTCs by reducing the abundance of the termination factor eRF1. SRI-41315 also potentiates aminoglycoside-mediated readthrough, leading to synergistic increases in CFTR activity. Combining readthrough agents that target distinct components of the translation machinery is a promising treatment strategy for diseases caused by PTCs.

Small molecule Y-320 stimulates ribosome biogenesis, protein synthesis, and aminoglycoside-induced premature termination codon readthrough.

Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding center can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD, and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labeling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 up-regulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1, and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.