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bioRxiv2018

Functional genomics and programmed genome editing of omega-1 of the blood fluke Schistosoma mansoni

CRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 ({omega}1) of Schistosoma mansoni as a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of {omega}1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5-and 3-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within {omega}1. CRISPResso analysis of amplicons spanning the DSB revealed [~]4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding {omega}1 were reduced >80% and lysates of {omega}1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the {omega}1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of {omega}1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding {omega}1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of {omega}1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of {omega}1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

molecular biology↗

bioRxiv2018

Non-enzymatic roles of human RAD51 at stalled replication forks

The central recombination enzyme RAD51 has been implicated in replication fork processing and restart in response to replication stress. Here, we use a separation-of-function allele of RAD51 that retains DNA binding, but not strand exchange activity, to reveal mechanistic aspects of RAD51s roles in the response to replication stress. We find that cells lacking RAD51 strand exchange activity protect replication forks from MRE11-dependent degradation, as expected from previous studies. Unexpectedly we find that RAD51s strand exchange activity is not required to convert stalled forks to a form that can be degraded by DNA2. Such conversion was shown previously to require replication fork reversal, supporting a model in which fork reversal depends on a non-enzymatic function of RAD51. We also show RAD51 promotes replication restart by both strand exchange-dependent and strand exchange-independent mechanisms.

molecular biology↗

bioRxiv2018

G-quadruplex dynamics contribute to epigenetic regulation of mitochondrial function

Single-stranded DNA or RNA sequences rich in guanine (G) can adopt non-canonical structures known as G-quadruplexes (G4). Predicted G4-forming sequences in the mitochondrial genome are enriched on the heavy-strand and have been associated with formation of deletion breakpoints that cause mitochondrial disorders. However, the functional roles of G4 structures in regulating mitochondrial respiration in non-cancerous cells remain unclear. Here, we demonstrate that RHPS4, previously thought to be a nuclear G4-ligand, localizes primarily to mitochondria in live cells by mechanisms involving mitochondrial membrane potential. We find that RHPS4 exposure causes an acute inhibition of mitochondrial transcript elongation, leading to respiratory complex depletion. At higher ligand doses, RHPS4 causes mitochondrial DNA (mtDNA) replication pausing and genome depletion. Using these different levels of RHPS4 exposure, we describe discrete nuclear gene expression responses associated with mitochondrial transcription inhibition or with mtDNA depletion. Importantly, a mtDNA variant with increased anti-parallel G4-forming characteristic shows a stronger respiratory defect in response to RHPS4, supporting the conclusion that mitochondrial sensitivity to RHPS4 is G4-structure mediated. Thus, we demonstrate a direct role for G4 perturbation in mitochondrial genome replication, transcription processivity, and respiratory function in normal cells and describe the first molecule that differentially recognizes G4 structures in mtDNA.

molecular biology↗

bioRxiv2018

Multi-Method Characterisation of the Human Circulating Microbiome

The term microbiome describes the genetic material encoding the various microbial populations that inhabit our body. Whilst colonisation of various body niches (e.g. the gut) by dynamic communities of microorganisms is now universally accepted, the existence of microbial populations in other \"classically sterile\" locations, including the blood, is a relatively new concept. The presence of bacteria-specific DNA in the blood has been reported in the literature for some time, yet the true origin of this is still the subject of much deliberation. The aim of this study was to investigate the phenomenon of a \"blood microbiome\" by providing a comprehensive description of bacterially-derived nucleic acids using a range of complementary molecular and classical microbiological techniques. For this purpose we utilised a set of plasma samples from healthy subjects (n = 5) and asthmatic subjects (n = 5). DNA-level analyses involved the amplification and sequencing of the 16S rRNA gene. RNA-level analyses were based upon the de novo assembly of unmapped mRNA reads and subsequent taxonomic identification. Molecular studies were complemented by viability data from classical aerobic and anaerobic microbial culture experiments. At the phylum level, the blood microbiome was predominated by Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The key phyla detected were consistent irrespective of molecular method (DNA vs RNA), and consistent with the results of other published studies. In silico comparison of our data with that of the Human Microbiome Project revealed that members of the blood microbiome were most likely to have originated from the oral or skin communities. To our surprise, aerobic and anaerobic cultures were positive in eight of out the ten donor samples investigated, and we reflect upon their source. Our data provide further evidence of a core blood microbiome, and provide insight into the potential source of the bacterial DNA / RNA detected in the blood. Further, data reveal the importance of robust experimental procedures, and identify areas for future consideration.

molecular biology↗

bioRxiv2018

A total synthetic approach to CRISPR/Cas9 genome editing and homology directed repair

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We compared synthetic, chemically modified sgRNAs to in vitro transcribed sgRNAs and demonstrate the increased activity of synthetic sgRNAs in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to optimize the design of synthetic DNA templates to promote HDR. Utilizing these principles, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic sgRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.

molecular biology↗

bioRxiv2018

Intracellular calcium dysregulation precedes muscle decline in C. elegans modeling Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by loss of the protein dystrophin. In humans, DMD has early onset, causes developmental delays, muscle necrosis, loss of ambulation, and early death. Current animal models have been challenged by their inability to model the early onset and severity of the disease. Thus it remains unresolved if increased sarcoplasmic calcium observed in dystrophic muscles follows or leads the mechanical insults caused by the muscles disrupted contractile machinery. This knowledge has important applications for patients, as potential physiotherapeutic treatments may either help or exacerbate symptoms, depending on how dystrophic muscles differ from healthy ones. Recently we showed how burrowing dystrophic (dys-1) C. elegans recapitulate many salient phenotypes of DMD, including loss of mobility and muscle necrosis. Here we report dys-1 worms display early pathogenesis, including dysregulated sarcoplasmic calcium, and increased lethality. Sarcoplasmic calcium dysregulation in dys-1 worms precedes overt structural phenotypes (e.g. mitochondrial, and contractile machinery damage) and can be mitigated by silencing calmodulin expression. To learn how dystrophic musculature responds to altered physical activity, we cultivated dys-1 animals in environments requiring high amplitude, or high frequency of muscle exertion during locomotion. We find that several muscular parameters (such as size) improve with increased activity. However, longevity in dystrophic animals was negatively associated with muscular exertion, regardless of the duration of the effort. The high degree of phenotypic conservation between dystrophic worms and humans provides a unique opportunity to gain insights into the etiology of the disease, as well as the initial assessment of potential treatment strategies.\n\nSIGNIFICANCEDuchenne muscular dystrophy is a degenerative disease affecting tens of thousands of people in the US alone. Much remains unknown about the disease, including the chain of events that links the loss of dystrophin to muscle death, or the extent to which exercise might be able to protect degenerating muscles. We used the nematode C. elegans to show that sarcoplasmic calcium dysregulation takes place in dystrophic muscles long before other overt signs of damage manifest. When placed in assays that altered muscular activity by increasing either contraction frequency or amplitude, we observed several metrics associated with muscular repair increase. However, no treatment positively affected the life expectancy of dystrophic animals.

molecular biology↗

bioRxiv2018

Role of Cnot6l in maternal mRNA turnover

Removal of poly(A) tail is an important mechanism controlling eukaryotic mRNA turnover. The major eukaryotic deadenylase complex CCR4-NOT contains two deadenylase components, CCR4 and CAF1 for which mammalian CCR4 is encoded by Cnot6 or Cnot6l paralogs. We show that Cnot6l apparently supplies the majority of CCR4 in the maternal CCR4-NOT complex in mouse, hamster, and bovine oocytes. Deletion of Cnot6l yielded viable mice but Cnot6l-/- females exhibited ~40% smaller litter size. The main onset of the phenotype was post-zygotic: fertilized Cnot6l-/- eggs developed slower and arrested more frequently than Cnot6l+/- eggs suggesting that maternal CNOT6L is necessary for accurate oocyte-to-embryo transition (OET). Transcriptome analysis revealed major transcriptome changes in Cnot6l-/- ovulated eggs and 1-cell zygotes. In contrast, minimal transcriptome changes in preovulatory Cnot6l-/- oocytes were consistent with reported Cnot6l mRNA dormancy. A minimal overlap between transcripts sensitive to decapping inhibition and Cnot6l loss suggests that decapping and CNOT6L-mediated deadenylation selectively target distinct subsets of mRNAs during OET in mouse.

molecular biology↗

bioRxiv2018

Therapeutic gene editing in hematopoietic progenitor cells from a mouse model of Fanconi anemia

The promising ability to genetically modify hematopoietic stem and progenitor cells (HSPCs) by precise gene editing remains challenging due to their sensitivity and poor permissiveness. This represents the first evidence of implementing a gene editing strategy in a murine safe harbor locus that phenotypically corrects primary cells derived from a mouse model of Fanconi anemia (FA).\n\nBy co-delivering TALENs and a donor therapeutic FANCA cassette template to the Mbs85 locus (ortholog of the hAAVS1 safe harbor locus), we achieved efficient gene targeting (23%) in FA mouse embryonic fibroblasts (MEFs). This resulted in the phenotypic correction of these cells, as revealed by the improvement of their hypersensitivity to mitomycinC. Moreover, robust evidence of targeted integration was observed in murine WT and FA-A hematopoietic progenitor cells (HPC) reaching mean targeted integration values of 20.98% and 16.33% respectively, with phenotypic correction of FA HPCs. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy in a murine safe harbor locus, such as the Mbs85 gene, of MEFs and murine HPC from a FA mouse model.

molecular biology↗

bioRxiv2018

CRISPR-Cas12b-assisted nucleic acid detection platform

Rapid molecular diagnostic technology is very useful in many areas, including public health, environmental testing and criminal investigation. We recently showed that Cas12a had trans-cleavage activity upon collateral single-stranded DNA (ssDNA), with which the HOLMES platform (one-HOur Low-cost Multipurpose highly Efficient System) was developed. Here, we combine the thermophilic Cas12b, which also has the ssDNA trans-cleavage activity, with Loop-Mediated Isothermal Amplification (LAMP), and create HOLMESv2. In HOLMESv2, LAMP amplification and Cas12b trans-cleavage can be integrated into a one-step system with a constant temperature, which therefore brings much convenience in nucleic acid detection. Moreover, we also simplify the RNA detection procedures in HOLMESv2, using an RNA-dependent DNA polymerase for amplification and therefore omitting an extra reverse transcription step.\n\nOne Sentence SummaryWe combine LAMP and Cas12b to develop HOLMESv2 for conveniently detecting target nucleic acid in a one-step approach.

molecular biology↗

bioRxiv2018

Rab1b and ARF5 are novel RNA-binding proteins involved in IRES-driven RNA localization

Internal ribosome entry site (IRES) elements are organized in domains that guide internal initiation of translation. Here we have combined proteomic and imaging analysis to study novel IRES interactors recognizing specific RNA structural subdomains. Besides known IRES-binding proteins, we identified novel factors belonging to networks involved in RNA and protein transport. Among those, Rab1b and ARF5, two components of the ER-Golgi, revealed direct binding to IRES transcripts. However, these proteins exert different effects on translation. While a dominant-negative mutant of Rab1b decreased IRES function, ARF5 silencing stimulated IRES activity. RNA FISH studies revealed novel features of the IRES element. First, IRES-RNA formed clusters within the cell cytoplasm, whereas cap-RNA displayed disperse punctuated distribution. Second, the IRES-driven RNA colocalized with ARF5 and Rab1b, but not with the dominant-negative of Rab1b. Thus, our data suggest a role for domain 3 of the IRES in RNA localization around ER-Golgi, a ribosome-rich cellular compartment.

molecular biology↗

bioRxiv2018

Personalized expression of bitter ‘taste’ receptors in human skin

The integumentary (i.e., skin) and gustatory systems both function to protect the human body and are a first point of contact with poisons and pathogens. These systems may share a similar protective mechanism because both human taste and skin cells express mRNA for bitter taste receptors (TAS2Rs). Here, we used gene-specific methods to measure mRNA from all known bitter receptor genes in adult human skin from freshly biopsied samples and from samples collected at autopsy from the Genotype-Tissue Expression project. Human skin expressed some but not all TAS2Rs, and for those that were expressed, the relative amounts differed markedly among individuals. For some TAS2Rs, mRNA abundance was related to sun exposure (TAS2R14, TAS2R30, TAS2R42, and TAS2R60), sex (TAS2R3, TAS2R4, TAS2R8, TAS2R9, TAS2R14, and TAS2R60), and age (TAS2R5), although these effects were not large. These findings contribute to our understanding of extraoral expression of chemosensory receptors.

molecular biology↗

bioRxiv2018

Single-cell isoform RNA sequencing (ScISOr-Seq) across thousands of cells reveals isoforms of cerebellar cell types.

Full-length isoform sequencing has advanced our knowledge of isoform biology1-11. However, apart from applying full-length isoform sequencing to very few single cells12,13, isoform sequencing has been limited to bulk tissue, cell lines, or sorted cells. Single splicing events have been described for <=200 single cells with great statistical success14,15, but these methods do not describe full-length mRNAs. Single cell short-read 3 sequencing has allowed identification of many cell sub-types16-23, but full-length isoforms for these cell types have not been profiled. Using our new method of single-cell-isoform-RNA-sequencing (ScISOr-Seq) we determine isoform-expression in thousands of individual cells from a heterogeneous bulk tissue (cerebellum), without specific antibody-fluorescence activated cell sorting. We elucidate isoform usage in high-level cell types such as neurons, astrocytes and microglia and finer sub-types, such as Purkinje cells and Granule cells, including the combination patterns of distant splice sites6-9,24,25, which for individual molecules requires long reads. We produce an enhanced genome annotation revealing cell-type specific expression of known and 16,872 novel (with respect to mouse Gencode version 10) isoforms (see isoformatlas.com).\n\nScISOr-Seq describes isoforms from >1,000 single cells from bulk tissue without cell sorting by leveraging two technologies in three steps: In step one, we employ microfluidics to produce amplified full-length cDNAs barcoded for their cell of origin. This cDNA is split into two pools: one pool for 3 sequencing to measure gene expression (step 2) and another pool for long-read sequencing and isoform expression (step 3). In step two, short-read 3-sequencing provides molecular counts for each gene and cell, which allows clustering cells and assigning a cell type using cell-type specific markers. In step three, an aliquot of the same cDNAs (each barcoded for the individual cell of origin) is sequenced using Pacific Biosciences (\"PacBio\")1,2,4,5,26 or Oxford Nanopore3. Since these long reads carry the single-cell barcodes identified in step two, one can determine the individual cell from which each long read originates. Since most single cells are assigned to a named cluster, we can also assign the cells cluster name (e.g. \"Purkinje cell\" or \"astrocyte\") to the long read in question (Fig 1A) - without losing the cell of origin of each long read.\n\nO_FIG O_LINKSMALLFIG WIDTH=180 HEIGHT=200 SRC=\"FIGDIR/small/364950_fig1.gif\" ALT=\"Figure 1\">\nView larger version (66K):\norg.highwire.dtl.DTLVardef@4df0cdorg.highwire.dtl.DTLVardef@fc4beborg.highwire.dtl.DTLVardef@1dc485forg.highwire.dtl.DTLVardef@1138a3e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1:C_FLOATNO (A) Outline of our ScISOr-Seq approach. (B) TSNE-plot depicting cell clusters, marker genes and names given to clusters, including: Bergman glia (BG), External granule cell layer neurons (EGL), Internal granule cell layer and other neurons in the interior of the cerebellum (IGL), two clusters of Purkinje cell layer neurons (PCL), oligodendrocyte progenitor cells (OPCs), Atoh1+ neuronal progenitors, Ptf1a+ neuronal progenitors and other neuronal progenitors (NPCs) (C) In-situ hybridization images from the Allen Brain Atlas depicting expression of marker genes in specific layers. (D) Expression patterns of selected marker genes across cell types.\n\nC_FIG

molecular biology↗

bioRxiv2018

Drosophila ZDHHC8 palmitoylates scribble and Ras64B and controls growth and viability

SummaryPalmitoylation is an important posttranslational modification regulating diverse cellular functions. Consequently, aberrant palmitoylation can lead to diseases such as neuronal disorders or cancer. In humans there are roughly one hundred times more palmitoylated proteins than enzymes catalyzing palmitoylation (palmitoyltransferases). Therefore, it is an important challenge to establish the links between palmitoyltransferases and their targets. From publicly available data, we find that expression of human ZDHHC8 correlates significantly with cancer survival. To elucidate the organismal function of ZDHHC8, we study the Drosophila ortholog of hZDHHC8, CG34449/dZDHHC8. Knockdown of dZDHHC8 causes tissue overgrowth while dZDHHC8 mutants are larval lethal. We provide a list of 159 palmitoylated proteins in Drosophila and present data suggesting that scribble and Ras64B are targets of dZDHHC8.

molecular biology↗

bioRxiv2018

Modulation of ADARs mRNA expression in congenital heart defect patients

Adenosine (A) to inosine (I) RNA editing, is a hydrolytic deamination reaction catalyzed by adenosine deaminase (ADAR) acting on RNA enzymes. RNA editing is a molecular process that involves the post-transcriptional modification of RNA transcripts. Interestingly, few studies have been carried out to determine the role of RNA editing in vascular disease. The current study found that in blood samples positive for congenital heart disease (CHD) ADAR1 and ADAR2 expression change at RNA level was opposite to each other. That is, an increase of ADAR1 mRNA was noticed in human CHD cases, whereas ADAR2 mRNA was vastly down-regulated. The increase in ADAR1 may be explained by the stress induced by CHD. The dramatic decrease in ADAR2 in CHD cases was unexpected and prompted further investigation into its effects on the heart. Therefore we performed expression analysis on a microarray data encompassing ischemic and non-Ischemic cardiomyopathy patient myocardial tissues. A strong down-regulation of ADAR2 was observed in both ischemic and especially non-ischemic cases. However, ADAR1 showed a mild increase in the case of non-ischemic myocardial tissues. To further explore the role of ADAR2 with respect to heart physiology. We selected a protein coding gene filamin B (FLNB). FLNB is known to play an important role in heart development. Although there were no observable changes in its expression, the editing levels of FLNB dropped dramatically in ADAR2-/- mice. We also performed miRNA profiling from ADAR2 -/- mice heart tissue revealed a decrease in expression of miRNAs. It is established that aberrant expression of these miRNAs is often associated with cardiac defects. This study proposes that sufficient amounts of ADAR2 might play a vital role in preventing cardiovascular defects.

molecular biology↗

bioRxiv2018

RepB C-terminus mutation of an ori pRi vector affects plasmid copy number in Agrobacterium and transgene copy number in plants

A native repABC replication origin, ori pRi, was previously reported as a single copy plasmid in Agrobacterium tumefaciens and can improve the production of transgenic plants with a single copy insertion of transgenes when it is used in binary vectors for Agrobacterium-mediated transformation. A high copy ori pRi variant plasmid, pTF::Ri, which does not improve the frequency of single copy transgenic plants, has been reported in the literature. Sequencing the high copy pTF::Ri repABC operon revealed the presence of two mutations: one silent mutation and one missense mutation that changes a tyrosine to a histidine (Y299H) in a highly conserved area of the C-terminus of the RepB protein (RepBY299H). Reproducing these mutations in the wild-type oriRi binary vector showed that Agrobacterium cells with the RepBY299H mutation grow faster on both solidified and in liquid medium, and have higher plasmid copy number as determined by ddPCR. In order to investigate the impact of the RepBY299H mutation on transformation and quality plant production, the RepBY299H mutated ori pRi binary vector was compared with the original wild-type ori pRi binary vector and a multi-copy oriV binary vector in canola transformation. Molecular analyses of the canola transgenic plants demonstrated that the multi-copy ori pRi with the RepBY299H mutation in Agrobacterium cells lost the advantage of generating high frequency single copy, backbone-free transgenic plants compared to using the single copy wild-type ori pRi binary vector.

molecular biology↗

bioRxiv2018

Thy1 (CD90) expression is regulated by DNA methylation during adipogenesis

The obesity epidemic is developing into the most costly health problem facing the world. Obesity, characterized by excessive adipogenesis and enlarged adipocytes, promotes morbidities such as diabetes, cardiovascular disease and cancer. Regulation of adipogenesis is critical to our understanding of how fat cell formation causes obesity and associated health problems. Thy1 (also called CD90), a widely used stem cell marker, blocks adipogenesis and reduces lipid accumulation. Thy1 knockout-mice are prone to diet-induced obesity. While the importance of Thy1 in adipogenesis and obesity is now evident, how its expression is regulated is not. We hypothesized that DNA methylation plays a role in promoting adipogenesis and affects Thy1 expression. Using the methylation inhibitor 5-aza-2-deoxycytidine (5-aza-dC), we investigated whether DNA methylation alters Thy1 expression during adipogenesis in both mouse 3T3-L1 pre-adipocytes and mouse mesenchymal stem cells. Thy1 protein and mRNA levels were decreased dramatically during adipogenesis. However, 5-aza-dC treatment prevented this phenomenon. Pyrosequencing analysis shows that the CpG sites at the Thy1 locus are methylated during adipogenesis. These new findings highlight the potential role of Thy1 and DNA methylation in adipogenesis and obesity.

molecular biology↗

bioRxiv2018

Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error

Mutations in LRP2, a transmembrane receptor, cause ocular enlargement and high-myopia. LRP2 is expressed by the RPE and eye ciliary epithelia, binding many extracellular ligands, including Bmp4 and Shh. Signaling mediated by LRP2 is very context-dependent, and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled, sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia (proper refraction). Using zebrafish, we demonstrate that BACE sheddase-dependent LRP2 cleavage produces a soluble domain that binds BMP4, inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand-binding receptor forms available: a soluble BMP trap, and a membrane-bound RPE signaling facilitator. By modulating LRP2 cleavage, cells can fine-tune and coordinate multiple signaling pathways, as well as growth and turnover of the extracellular matrix, control of which is important to maintain proper eye size. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth.\n\nAuthor SummaryFor proper focusing and normal vision, the axial length of the eye needs to match the refractive power of the lens. This is achieved by fine-tuning multiple signaling pathways to regulate the shape of the eye primarily by remodeling of the sclera, the outermost layer of the eye. This process is termed emmetropization. Emmetropization cues are initiated by visual input, but how signals are transduced from the photoreceptors across the retinal pigment epithelium to the sclera is incompletely understood. Here we show that cleavage of Lrp2, a large receptor expressed on RPE cells in the eye, alters BMP signaling, which contributes to proper eye size control. Dysregulation of BMP signaling by a) absence of Lrp2 in mutant zebrafish or b) overexpression of BMP antagonists from the RPE both cause eye enlargement and myopia. Understanding how regulated cleavage of Lrp2 affects paracrine signaling provides critical insight to emmetropization, raising the possibility for development of therapeutic agents to combat the epidemic incidence of refractive error.

molecular biology↗

bioRxiv2018

Multi-protein Bridging Factor 1(Mbf1), Rps3 and Asc1 prevent stalled ribosomes from frameshifting

Stalled ribosomes in bacteria frameshift, but stalled ribosomes in eukaryotes do not frameshift and abort translation, suggesting that eukaryote-specific mechanisms might prevent frameshifting. We show that the conserved eukaryotic/archaeal protein Mbf1 acts with ribosomal proteins Rps3/uS3 and eukaryotic Asc1/RACK1 to prevent frameshifting at inhibitory CGA-CGA codon pairs in Saccharomyces cerevisiae. Mutations in RPS3 that allow frameshifting implicate eukaryotic conserved residues near the mRNA entry site. Mbf1 and Rps3 cooperate to maintain the reading frame of stalled ribosomes, while Asc1 mediates distinct events that result in aborted translation. Frameshifting occurs through a +1 shift with a CGA codon in the P site and involves competition between codons entering the A site, implying that the wobble interaction of the P site codon destabilizes translation elongation. Thus, eukaryotes have evolved unique mechanisms involving both a universally conserved ribosome component and two eukaryotic-specific proteins to maintain the reading frame at ribosome stalls.

molecular biology↗