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bioRxiv2018

Functional Analysis of TM6 MADS box gene in the Octoploid Strawberry by CRISPR/Cas9 directed mutagenesis

The B-class of MADS-box transcription factors has been studied in many plant species, but remain functionally uncharacterized in the Rosaceae family. APETALA3 (AP3), a member of this class, controls the identity of petals and stamens in Arabidopsis thaliana. In this work, we identified two members of the AP3 lineage in the cultivated strawberry (Fragaria x ananassa): FaAP3 and FaTM6. Interestingly, FaTM6, and not FaAP3, shows an expression pattern equivalent to that of AP3 in Arabidopsis. Genome editing using Cluster Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is becoming a robust tool for targeted and stable mutagenesis of DNA. However, whether it can be efficiently used in an octoploid species such as F. x ananassa is not known. Here we report for the first time the application of CRISPR/Cas9 in F. x ananassa to characterize the function of FaTM6 in flower development. An exhaustive analysis by high-throughput sequencing of the FaTM6 locus spanning the target sites showed a high efficiency genome editing already in the T0 generation. The phenotypic characterization of the mutant lines indicates that FaTM6 plays a key role in petal and especially in anther development in strawberry. in an octoploid species such as F. x ananassa, and offer new opportunities for engineering strawberry to improve traits of interest in breeding programs.

molecular biology↗

bioRxiv2018

Effects of Pathological Mutations on the Prion-Like Polymerisation of MyD88

A novel concept has emerged whereby the higher-order self-assembly of proteins provides a simple and robust mechanism for signal amplification. This appears to be a universal signalling mechanism within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns need to trigger a strong, binary response within cells. Previously, multiple structural studies have been limited to single domains, expressed and assembled at high protein concentrations. We therefore set out to develop new in vitro strategies to characterise the behaviour of full-length proteins at physiological levels. In this study we focus on the adaptor protein MyD88, which contains two domains with different self-assembly properties: a TIR domain that can polymerise similarly to the TIR domain of Mal, and a Death Domain that has been shown to oligomerise with helical symmetry in the Myddosome complex. To visualize the behaviour of full-length MyD88 without purification steps, we use single-molecule fluorescence coupled to eukaryotic cell-free protein expression. These experiments demonstrate that at low protein concentration, only full-length MyD88 forms prion-like polymers. We also demonstrate that the metastability of MyD88 polymerisation creates the perfect binary response required in innate signalling: the system is silenced at normal concentrations but upstream signalling creates a \"seed\" that triggers polymerisation and amplification of the response. These findings pushed us to re-interpret the role of polymerisation in MyD88-related diseases and we studied the impact of disease-associated point mutations L93P, R196C and L252P/L265P at the molecular level. We discovered that all mutations completely block the ability of MyD88 to polymerise. We also confirm that L252P, a gain-of-function mutation, allows the MyD88 mutant to form extremely stable oligomers, even when expressed at low nanomolar concentrations. Thus, our results are consistent with and greatly add to the findings on the Myddosomes digital all-or-none responses and the behaviour of the oncogenic mutation of MyD88.

molecular biology↗

bioRxiv2018

Identification and characterization of cherry (Cerasus pseudocerasus G. Don) genes in response to parthenocarpy induced by GA3 through transcriptome analysis

Fruit set after successful pollination is a key process in the production of sweet cherries, but low fruit rate is the main problem for crop production in sweet cherries. Gibberellin treatment can directly induce parthenogenesis without pollination; therefore, gibberellin treatment is a very important method to improve the fruit setting rate of sweet cherries. Exogenous gibberellin can satisfy the hormone requirement during fruit growth and development. Some related studies have mainly focused on physiological aspects, such as breeding, branching, fertilization, etc., and the molecular mechanism is not clear. In this study, we analyzed the transcriptome of Meizao sweet cherry fruit treated with gibberellin during the anthesis and hard-core period to determine the genes associated with parthenocarpic fruit set. A total of 765 and 186 differentially expressed genes (DEGs) were found at anthesis and the hard-core stage after gibberellin 3 (GA3) treatment, respectively. The differentially expressed genes between the control and GA3 treatment showed that the GA3 response mainly included parthenocarpic fruit set and cell division. Exogenous gibberellin stimulated sweet cherry parthenocarpy and enlargement, which were verified by qRT-PCR results of related genes and the parthenocarpic fruit set and fruit size. Based on our research and previous studies on Arabidopsis thaliana, we identified the key genes associated with parthenocarpic fruit set and cell division. Briefly, we found patterns in the sweet cherry fruit setting-related DEGs, especially those associated with hormone balance, cytoskeleton formation and cell wall modification. Overall, the result provides a possible molecular mechanism regulating parthenocarpic fruit set, which is important for basic research and industrial development of sweet cherries.\n\nHighlightcherry genes in response to parthenocarpy and promote to fruit setting induced by GA3.

molecular biology↗

bioRxiv2018

Separable recruitment of DNA Polymerase α for leading- and lagging-strand replication initiation

During eukaryotic DNA replication, DNA polymerase alpha/primase (Pol ) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol abundance, and that both processes are disrupted at similar Pol concentrations. When the replisome adaptor protein Ctf4 is absent or cannot interact with Pol , lagging-strand initiation is impaired at Pol concentrations that still support normal origin firing. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol . Using strains in which the Pol -Ctf4 interaction is disrupted, we demonstrate that this checkpoint requirement is not solely caused by reduced lagging-strand priming. Our results suggest that Pol recruitment for replication initiation and ongoing lagging-strand priming are distinctly sensitive to the presence of Ctf4. We propose that the global changes we observe in Okazaki fragment length and origin firing efficiency are consistent with distributive association of Pol at the replication fork, at least when Pol is limiting. Author summaryHalf of each eukaryotic genome is replicated continuously as the leading strand, while the other half is synthesized discontinuously as Okazaki fragments on the lagging strand. The bulk of DNA replication is completed by DNA polymerases {varepsilon} and {delta} on the leading and lagging strand respectively, while synthesis on each strand is initiated by DNA polymerase -primase (Pol ). Using the model eukaryote S. cerevisiae, we modulate cellular levels of Pol and interrogate the impact of this perturbation on both replication initiation on DNA synthesis and cellular viability. We observe that Pol can associate dynamically at the replication fork for initiation on both strands. Although the initiation of both strands is widely thought to be mechanistically similar, we determine that Ctf4, a hub that connects proteins to the replication fork, stimulates lagging-strand priming to a greater extent than leading-strand initiation. We also find that decreased leading-strand initiation results in a checkpoint response that is necessary for viability when Pol is limiting. Because the DNA replication machinery is highly conserved from budding yeast to humans, this research provides insights into how DNA replication is accomplished throughout eukaryotes.

molecular biology↗

bioRxiv2018

SmartCodes : functionalized barcodes that enable targeted retrieval of clonal lineages from a heterogeneous population

Molecular barcoding has provided means to link genotype to phenotype, to individuate cells in single-cell analyses, to enable the tracking of evolving lineages, and to facilitate the analysis of complex mixtures containing phenotypically distinct lineages. To date, all existing approaches enable retrospective associations to be made between characteristics and the lineage harbouring them, but provide no path toward isolating or manipulating those lineages within the complex mixture. Here, we describe a strategy for creating functionalized barcodes that enable straightforward manipulation of lineages within complex populations of cells, either marking and retrieval of selected lineages, or modification of their phenotype within the population, including their elimination. These \"SmartCodes\" rely on a simple CRISPR-based, molecular barcode reader that can switch measurable, or selectable markers, on or off in a binary fashion. While this approach could have broad impact, we envision initial approaches to the study of tumour heterogeneity, focused on issues of tumour progression, metastasis, and drug resistance.

molecular biology↗

bioRxiv2018

Mediator is essential for small nuclear and nucleolar RNA transcription in yeast

Eukaryotic RNA polymerase II (RNAPII) transcribes mRNA genes as well as non-protein coding RNAs (ncRNAs) including small nuclear and nucleolar RNAs (sn/snoRNAs). In metazoans, RNAPII transcription of sn/snoRNAs is facilitated by a number of specialized complexes, but no such complexes have been discovered in yeast. It has thus been proposed that yeast sn/snoRNA promoters use the same complement of factors as mRNA promoters, but the extent to which key regulators of mRNA genes act at sn/snoRNA genes in yeast is unclear. Here, we investigated a potential role for the Mediator complex, essential for mRNA gene transcription, in the transcription of sn/snoRNA genes. We found that the complete Mediator complex maps to most sn/snoRNA gene regulatory regions and that loss of Mediator function results in a robust reduction in RNAPII and TFIIB occupancy at sn/snoRNA genes. Furthermore, deletion of subunits of the activator-interacting Mediator tail module does not affect Mediator recruitment to, or transcription of, sn/snoRNAs. Taken together, our analyses indicate that Mediator promotes PIC formation and transcription at sn/snoRNA genes, expanding the role of this critical regulator beyond its known functions in mRNA gene transcription and demonstrating further mechanistic similarity between the transcription of mRNA and sn/snoRNA genes.

molecular biology↗

bioRxiv2018

Structural rearrangement of TFIIS- and TFIIF/TFIIE-like subunits in RNA polymerase I transcription complexes

RNA polymerase (Pol) I is a 14-subunit enzyme that solely transcribes pre-ribosomal RNA. Cryo-EM structures of Pol I initiation and elongation complexes have given first insights into the molecular mechanisms of Pol I transcription. Here, we present cryo-electron microscopy structures of yeast Pol I elongation complexes (ECs) bound to the nucleotide analog GMPCPP at 3.2 to 3.4 [A] resolution that provide additional insight into the functional interplay between the TFIIE/TFIIF-like A49-A34.5 heterodimer and the TFIIS-like subunit A12.2 present in Pol I. Strikingly, most of the nucleotide-bound ECs lack the A49-A34.5 heterodimer and adopt a Pol II-like conformation, in which the A12.2 C-terminal domain is bound in a previously unobserved position at the A135 surface. Our work suggests a regulatory mechanism of Pol I transcription where the association of the A49-A34.5 heterodimer to Pol I is regulated by subunit A12.2, thereby explaining in vitro biochemical and kinetic data.

molecular biology↗

bioRxiv2018

Molecular basis for a novel systemic form of human hereditary apoA-I amyloidosis with vision loss

Hereditary apolipoprotein A-I (apoA-I) amyloidosis (AApoAI) is a life-threatening incurable genetic disorder whose molecular underpinnings and the full spectrum of afflicted organs are unclear. We report a new form of AApoAI with amyloid deposition in multiple organs, including an unprecedented retinal amyloidosis. Genetic and proteomic analyses identified Glu34Lys apoA-I as the fibrillar protein causing the clinical manifestations. A life-saving combined hepatorenal transplantation was performed for one Glu34Lys carrier. To elucidate structural underpinnings for amyloidogenic properties of Glu34Lys, we generated its recombinant globular domain and compared the conformation and dynamics of its lipid-free form with those of two other naturally occurring apoA-I variants, Phe71Tyr (amyloidogenic) and Leu159Arg (non-amyloidogenic). All variants showed reduced stability and altered aromatic residue packing. Molecular dynamics simulations revealed local helical unfolding and suggested that transient opening of Trp72 induced mutation-dependent structural perturbations in a sensitive region, including the major amyloid hotspot residues 14-22. We posit that a shift from the \"closed\" to an \"open\" orientation of Trp72 modulates structural protection of amyloid hotspots, suggesting a previously unknown early step in protein misfolding.

molecular biology↗

bioRxiv2018

Mammalian CST averts replication failure by preventing G-quadruplex accumulation

Human CST (CTC1-STN1-TEN1) is an RPA-like complex that associates with G-rich single-strand DNA and helps resolve replication problems both at telomeres and genome-wide. We previously showed that CST binds and disrupts G-quadruplex (G4) DNA in vitro, suggesting that CST may prevent in vivo blocks to replication by resolving G4 structures. Here, we demonstrate that CST binds and unfolds G4 with similar efficiency to RPA. In cells, CST is recruited to telomeric and non-telomeric chromatin upon G4 stabilization. STN1 depletion increases G4 accumulation and slows bulk genomic DNA replication. At telomeres, combined STN1 depletion and G4 stabilization causes multi-telomere FISH signals and telomere loss, hallmarks of deficient telomere duplex replication. Strand-specific telomere FISH indicates preferential loss of C-strand DNA while analysis of BrdU uptake during leading and lagging-strand telomere replication shows preferential under-replication of lagging telomeres. Together these results indicate a block to Okazaki fragment synthesis. Overall, our findings indicate a novel role for CST in maintaining genome integrity through resolution of G4 structures both ahead of the replication fork and on the lagging strand template.

molecular biology↗

bioRxiv2018

5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5'-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

molecular biology↗

bioRxiv2018

Brr2 is a splicing fidelity factor

Many spliceosomal DExD/H box helicases act as fidelity factors during pre-mRNA splicing, promoting on-pathway interactions while simultaneously minimizing errors. Mutations linked to Retinitis Pigmentosa (RP), a form of heritable blindness, map to key domains of spliceosomal helicase Brr2 (SNRNP200 in humans). Previous data show that such mutations negatively impact spliceosome activation, likely due to defects in brr2-RP RNA binding, helicase, and ATPase activities. Furthermore, data from human reporter constructs suggest that brr2-RP might impact 5' splice site selection. Here we undertake a systematic analysis of brr2-RP effects on splicing fidelity. We show that a subset of brr2-RP mutants exhibit intron retention in vivo. Furthermore, brr2-RP mutants display hyperaccurate and/or error-prone splicing of a variety of splicing reporters. Branch-site fidelity is particularly impacted in this reporter assay. In addition, multiple brr2-RP alleles genetically interact with prp16 alleles known to impact the fidelity of branch site selection. Together these data implicate Brr2 in the fidelity of branch-site selection, and suggest that RP results not just from defects in spliceosome activation, but also from fidelity defects arising throughout the splicing cycle and in splicing fidelity.

molecular biology↗

bioRxiv2018

The exon junction complex undergoes a compositional switch that alters mRNP structure and nonsense-mediated mRNA decay activity

The exon junction complex (EJC) deposited upstream of mRNA exon junctions shapes structure, composition and fate of spliced mRNA ribonucleoprotein particles (mRNPs). To achieve this, the EJC core nucleates assembly of a dynamic shell of peripheral proteins that function in diverse post-transcriptional processes. To illuminate consequences of EJC composition change, we purified EJCs from human cells via peripheral proteins RNPS1 and CASC3. We show that EJC originates as an SR-rich mega-dalton sized RNP that contains RNPS1 but lacks CASC3. After mRNP export to the cytoplasm and before translation, the EJC undergoes a remarkable compositional and structural remodeling into an SR-devoid monomeric complex that contains CASC3. Surprisingly, RNPS1 is important for nonsense-mediated mRNA decay (NMD) in general whereas CASC3 is needed for NMD of only select mRNAs. The promotion of switch to CASC3-EJC slows down NMD. Overall, the EJC compositional switch dramatically alters mRNP structure and specifies two distinct phases of EJC-dependent NMD.

molecular biology↗

bioRxiv2018

Kinetic basis for DNA target specificity of CRISPR-Cas12a

Class II CRISPR-Cas nucleases are programmable via a single guide RNA, enabling genome editing applications in nearly all organisms. However, DNA cleavage at off-target sites that resemble the target sequence is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically-separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite the functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence, implying substantial reversibility during R-loop formation -a late transition state- and the absence of a seed region. Our results provide a quantitative underpinning for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity of Cas12a than the Cas9 nuclease.

molecular biology↗

bioRxiv2018

Gut bacterial tyrosine decarboxylases restrict the bioavailability of levodopa, the primary treatment in Parkinson’s disease

Human gut bacteria play a critical role in the regulation of immune and metabolic systems, as well as in the function of the nervous system. The microbiota senses its environment and responds by releasing metabolites, some of which are key regulators of human health and disease. In this study, we identify and characterize gut-associated bacteria in their ability to decarboxylate L-DOPA (also known as Levodopa or L-3,4-dihydroxyphenylalanine) to dopamine via the tyrosine decarboxylases, which are mainly present in the class Bacilli. Although the bacterial tyrosine decarboxylases have a higher affinity for tyrosine compared to L-DOPA, this does not affect their ability to decarboxylate L-DOPA, nor does any inhibitor of the human decarboxylase. This study indicates that in situ bioavailability of L-DOPA is compromised by the gut bacterial tyrosine decarboxylase abundance in Parkinsons patients. Finally, we show that the tyrosine decarboxylase abundance in the microbiota at the site of L-DOPA absorption, the proximal small intestine, significantly influences L-DOPA bioavailability in the plasma of rats. Our results highlight the role of microbial metabolism in drug bioavailability, and specifically, that small intestinal abundance of bacterial tyrosine decarboxylase can explain the highly variable L-DOPA dosage regimens required in the treatment of individual Parkinsons patients.\n\nHighlightsO_LISmall intestinal bacteria is able to convert L-DOPA to dopamine\nC_LIO_LIL-DOPA metabolism by gut bacteria reduce the bioavailability of L-DOPA in the body, thus is a significant explanatory factor of the highly variable L-DOPA dosage regimens required in the treatment of individual Parkinsons patients.\nC_LIO_LIInhibitors of the human DOPA decarboxylase are not potent inhibitors for bacterial tyrosine decarboxylases\nC_LI

molecular biology↗

bioRxiv2018

Dynamic m6A methylation facilitates mRNA triaging to stress granules

Summary blurbm6A-modification in the 5 vicinity of the coding sequence of transcripts provides a selective mechanism for triaging mRNAs to stress granules and is mediated by YTHDF3 reader protein.\n\nAbstractReversible post-transcriptional modifications on messenger RNA emerge as prevalent phenomena in RNA metabolism. The most abundant among them is N6-methyladenosine (m6A) which is pivotal for RNA metabolism and function, its role in stress response remains elusive. We have discovered that in response to oxidative stress, transcripts are additionally m6A-modified in their 5 vicinity. Distinct from that of the translationally-active mRNAs, this methylation pattern provides a selective mechanism for triaging mRNAs from the translatable pool to stress-induced stress granules. These stress-induced newly methylated sites are selectively recognized by the YTH domain family 3 (YTHDF3) reader protein, thereby revealing a new role for YTHDF3 in shaping the selectivity of stress response. Our findings describe a previously unappreciated function for RNA m6A modification in the oxidative-stress response and expand the breadth of physiological roles of m6A.

molecular biology↗

bioRxiv2018

Slow Normal Modes of Proteins are Accurately Reproduced across Different Platforms

The Protein Data Bank (PDB) [1] contains the atomic structures of over 105 biomolecules with better than 2.8[A] resolution. The listing of the identities and coordinates of the atoms comprising each macromolecule permits an analysis of the slow-time vibrational response of these large systems to minor perturbations. 3D video animations of individual modes of oscillation demonstrate how regions interdigitate to create cohesive collective motions, providing a comprehensive framework for and familiarity with the overall 3D architecture. Furthermore, the isolation and representation of the softest, slowest deformation coordinates provide opportunities for the development of mechanical models of enzyme function. The eigenvector decomposition, therefore, must be accurate, reliable as well as rapid to be generally reported upon. We obtain the eigenmodes of a 1.2[A] 34kDa PDB entry using either exclusively heavy atoms or partly or fully reduced atomic sets; Cartesian or internal coordinates; interatomic force fields derived either from a full Cartesian potential, a reduced atomic potential or a Gaussian distance-dependent potential; and independently developed software. These varied technologies are similar in that each maintains proper stereochemistry either by use of dihedral degrees of freedom which freezes bond lengths and bond angles, or by use of a full atomic potential that includes realistic bond length and angle restraints. We find that the shapes of the slowest eigenvectors are nearly identical, not merely similar.

molecular biology↗

bioRxiv2018

3’ Branch Ligation: A Novel Method to Ligate Non-Complementary DNA to Recessed or Internal 3’OH Ends in DNA or RNA

Nucleic acid ligases are crucial enzymes that repair breaks in DNA or RNA during synthesis, repair and recombination. Various molecular tools have been developed using the diverse activities of DNA/RNA ligases. Herein, we demonstrate a non-conventional ability of T4 DNA ligase to join 5 phosphorylated blunt-end double-stranded DNA to DNA breaks at 3 recessive ends, gaps, or nicks to form a 3 branch structure. Therefore, this base pairing-independent ligation is termed 3 branch ligation (3BL). In an extensive study of optimal ligation conditions, similar to blunt-end ligation, the presence of 10% PEG-8000 in the ligation buffer significantly increased ligation efficiency. A low level of nucleotide preference was observed at the junction sites using different synthetic DNAs. Furthermore, we discovered that T4 DNA ligase efficiently ligated DNA to the 3 recessed end of RNA, not to that of DNA, in a DNA/RNA hybrid, whereas RNA ligases are less efficient in this reaction. These novel properties of T4 DNA ligase can be utilized as a broad molecular technique in many important applications. We performed a proof-of-concept study of a new directional tagmentation protocol for next generation sequencing (NGS) library construction that eliminates inverted adapters and allows sample barcode insertion adjacent to genomic DNA. 3BL after single transposon tagmentation can theoretically achieve 100% usable template, and our empirical data demonstrate that the new approach produced higher yield compared with traditional double transposon or Y transposon tagmentation. We further explore the potential use of 3BL for preparing targeted RNA NGS libraries with mitigated structure-based bias and adapter dimer problems.

molecular biology↗

bioRxiv2018

Experimental determination of codon usage-dependent selective pressure on high copy-number genes in Saccharomyces cerevisiae

One of the central hypotheses in the theory of codon usage evolution is that in highly expressed genes particular codon usage patterns arise because they facilitate efficient gene expression and are thus selected for in evolution. Here we use plasmid copy number assays and growth rate measurements to explore details of the relationship between codon usage, gene expression level, and selective pressure in Saccharomyces cerevisiae. We find that when high expression levels are required optimal codon usage is beneficial and provides a fitness advantage, consistent with evolutionary theory. However, when high expression levels are not required, optimal codon usage is surprisingly and strongly selected against. We show that this selection acts at the level of protein synthesis, and we exclude a number of molecular mechanisms as the source for this negative selective pressure including nutrient and ribosome limitations and proteotoxicity effects. These findings inform our understanding of the evolution of codon usage bias, as well as the design of recombinant protein expression systems.

molecular biology↗