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Results for “Molecular Biology”

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bioRxiv2020

A novel fibrinogen gamma-chain mutation, p. γAla327Val, causes structural abnormality of D region and ultimately leads to congenital dysfibrinogenemia

Congenital dysfibrinogenemia (CD) is a coagulation disorder caused by mutations in the fibrinogen gene, which result in abnormal fibrinogen function. Many studies have confirmed that over half of dysfibrinogenemia cases are asymptomatic. In this study, we aimed to investigate the pathogenesis of CD caused by {gamma} Ala327Val heterozygous mutation, a new mutation, by studying fibrinogen function. Blood samples of patients were collected and the coagulation function, fibrinogen (Fg) aggregation test, fibrin clot lysis test, and SDS-PAGE were performed. Coagulation was monitored using a thromboelastometer, and the fibrin clot network structure was observed by scanning electron microscopy. The effect of the mutation on fibrinogen structure and function was predicted by molecular modeling. The fibrinogen activity concentration in patients with CD was significantly lower than that in healthy individuals. Thromboelastography showed that the K value of patients with CD was higher than that for healthy individuals. The Angle values were also decreased. The function of fibrinogen in patients with CD was low. Compared to fibrinogen from healthy individuals, fibrin size was different, the fiber network structure was loose, the pore size was increased, and the fiber branch nodes were increased for fibrinogen isolated from the proband. The {gamma} Ala327Val mutation led to changes in the structure of fibrinogen D region, affecting its structural stability. Ala327Val heterozygous missense mutation in exon 8 of FGG gene {gamma}-chain thus leads to abnormal fibrinogen structure and impairs the aggregation function of fibrinogen. This mutation is reported here for the first time.

molecular biology↗

bioRxiv2020

microRNA-seq of cartilage reveals an over-abundance of miR-140-3p which contains functional isomiRs

MiR-140 is selectively expressed in cartilage. Deletion of the entire miR-140 locus in mice results in growth retardation and early-onset osteoarthritis-like pathology, however the relative contribution of miR-140-5p or miR-140-3p to the phenotype remains to be determined. An unbiased small RNA sequencing approach identified miR-140-3p as significantly more abundant (>10-fold) than miR-140-5p in human cartilage. Analysis of these data identified multiple miR-140-3p isomiRs differing from the miRBase annotation at both the 5 and 3 end, with >99% having one of two seed sequences (5 bases 2-8). Canonical (miR-140-3p.2) and shifted (miR-140-3p.1) seed isomiRs were overexpressed in chondrocytes and transcriptomics performed to identify targets. miR-140-3p.1 and miR-140-3p.2 significantly down-regulated 694 and 238 genes respectively, of which only 162 genes were commonly down-regulated. IsomiR targets were validated using 3 UTR luciferase assays. miR-140-3p.1 targets were enriched within up-regulated genes in rib chondrocytes of Mir140-null mice and within down-regulated genes during human chondrogenesis. Finally, through imputing the expression of miR-140 from the expression of the host gene WWP2 in 124 previously published datasets, an inverse correlation with miR-140-3p.1 predicted targets was identified. Together these data suggest the novel seed containing isomiR miR-140-3p.1 is more functional than original consensus miR-140-3p seed containing isomiR.

molecular biology↗

bioRxiv2020

mSWI/SNF promotes distal repression by titrating polycomb dosage

The mammalian SWI/SNF, or BAF complex, has a conserved and direct role in antagonizing polycomb-mediated repression. Yet, BAF appears to also promote repression by polycomb in stem cells and cancer. How BAF both antagonizes and promotes polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-polycomb axis in embryonic stem cells on the timescale of hours. We report that rapid BAF depletion redistributes both PRC1 and PRC2 complexes from highly occupied domains, like Hox clusters, to weakly occupied sites that are normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features, and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 & PRC2 we identify both a conventional role for BAF in polycomb-mediated repression and a second mechanism acting by global redistribution of polycomb. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.

molecular biology↗

bioRxiv2020

The impediment to replication at tRNA genes in S. cerevisiae does not require tRNA transcription, and is facilitated by topoisomerases and Rad18-dependent repair pathways

tRNA genes are widely studied sites of replication-fork pausing and genome instability in the budding yeast Saccharomyces cerevisiae. tRNAs are extremely highly transcribed and serve as constitutive condensin binding sites. tRNA transcription by RNA polymerase III has previously been identified as stimulating replication-fork pausing at tRNA genes, but the nature of the block to replication has not been incontrovertibly demonstrated. Here, we describe a systematic, genome-wide analysis of the contributions of candidates to replication-fork progression at tDNAs in yeast: transcription factor binding, transcription, topoisomerase activity, condensin-mediated clustering, and Rad18-dependent DNA repair. We show that an asymmetric block to replication is maintained even when tRNA transcription is abolished by depletion of one or more subunits of RNA polymerase III. By contrast, analogous depletion of the essential transcription factor TFIIIB removes the obstacle to replication. Therefore, our data suggest that the RNA polymerase III transcription complex itself represents an asymmetric obstacle to replication even in the absence of RNA synthesis. We additionally demonstrate that replication-fork progression past tRNA genes is unaffected by the global depletion of condensin from the nucleus, and can be stimulated by the removal of topoisomerases or Rad18-dependent DNA repair pathways.

molecular biology↗

bioRxiv2020

N6-methyladenosine in poly(A) tails stabilize VSG transcripts

RNA modifications are important regulators of gene expression. In Trypanosoma brucei, transcription is polycistronic and thus most regulation happens post-transcriptionally. N6-methyladenosine (m6A) has been detected in this parasite, but its function remains unknown. Here we show that [~]50% of the m6A is located in the poly(A) tail of the monoallelically expressed Variant Surface Glycoprotein (VSG) transcript. m6A residues are removed from the VSG poly(A) tail prior to deadenylation and mRNA degradation. Using genetic tools, we identified a 16-mer motif in the 3UTR of VSG that acts as a cis-acting motif required for inclusion of m6A in the poly(A) tail. Removal of this motif from the VSG 3 UTR results in poly(A) tails lacking m6A, rapid deadenylation and mRNA degradation. To our knowledge this is the first identification of an RNA modification in the poly(A) tail of any eukaryote, uncovering a novel post-transcriptional mechanism of gene regulation.

molecular biology↗

bioRxiv2020

Redox-active cysteines in TGACG-BINDING FACTOR 1 (TGA1) do not play a role in salicylic acid- or pathogen-induced expression of TGA1-regulated target genes in Arabidopsis thaliana

O_LISalicylic acid (SA) is an important signaling molecule of the plant immune system. C_LIO_LISA biosynthesis is indirectly modulated by the closely related transcription factors TGA1 (TGACG-BINDING FACTOR 1) and TGA4. They activate expression of SARD1 (SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1), the gene product of which regulates the key SA biosynthesis gene ICS1 (ISOCHORISMATE SYNTHASE 1). C_LIO_LISince TGA1 interacts with the SA receptor NPR1 (NON EXPRESSOR OF PATHOGENESIS-RELATED GENES 1) in a redox-dependent manner and since the redox state of TGA1 is altered in SA-treated plants, TGA1 was assumed to play a role in the NPR1-dependent signaling cascade. Here we identified 193 out of 2090 SA-induced genes that require TGA1/TGA4 for maximal expression after SA treatment. One robustly TGA1/TGA4-dependent gene encodes for the SA hydroxylase DLO1 (DOWNY MILDEW RESISTANT 6-LIKE OXYGENASE 1) suggesting an additional regulatory role of TGA1/TGA4 in SA catabolism. C_LIO_LIExpression of TGA1/TGA4-dependent genes in mock/SA-treated or Pseudomonas-infected plants was rescued in the tga1 tga4 double mutant after introduction of a mutant genomic TGA1 fragment encoding a TGA1 protein without any cysteines. Thus, the functional significance of the observed redox modification of TGA1 in SA-treated tissues has remained enigmatic. C_LI SIGNIFICANCE STATEMENTPrevious findings demonstrating a redox-dependent interaction between transcription factor TGA1 and NPR1 attracted considerable attention. Here we show that TGA1 can act in the NPR1- and SA-dependent signaling cascade, but that its SA-regulated redox-active cysteines do not affect its function in this process.

molecular biology↗

bioRxiv2020

An iron (II) dependent oxygenase performs the last missing step of plant lysine catabolism

Due to low abundance in many staple food crops, the essential amino acid lysine must be produced industrially to meet global food supply needs. Despite intensive study, manipulation, and agricultural importance, the steps of plant lysine catabolism beyond the 2-oxoadipate (2OA) intermediate remain undescribed. Recently we described a missing step in the D-lysine catabolic pathway of the bacterium Pseudomonas putida in which 2OA is converted to D-2-hydroxyglutarate (D2HG) via hydroxyglutarate synthase (HglS), an enzyme belonging to the previously uncharacterized DUF1338 protein family. Here we solve the structure of HglS to 1.1[A] resolution in the substrate-free form and in complex with 2OA. Structural similarity to hydroxymandelate synthase suggested a successive decarboxylation and intramolecular hydroxylation mechanism forming 2HG in a Fe(II)- and O2-dependent manner, which is validated experimentally. 2OA specificity was mediated by a single arginine (R74), highly conserved across nearly all DUF1338 family proteins, including in 76% of plant enzymes. In Arabidopsis thaliana, a DUF1338 homolog is coexpressed with known lysine catabolism enzymes, and mutants show significant germination rate defects consistent with disrupted lysine catabolism. Structural and biochemical analysis of the Oryza sativa homolog FLO7 revealed identical activity to HglS despite low sequence identity. Our results suggest that nearly all DUF1338 containing enzymes likely catalyze the same biochemical reaction, exerting the same physiological function across bacteria and eukaryotes. SignificanceTo meet human demands, millions of tons of lysine are produced by bacterial fermentation annually due to its low abundance in staple crops. Here, we show the last missing step of lysine catabolism in nearly all plant endosperms is likely catalyzed by an iron-dependant DUF1338-containing enzyme homologous to the bacterial hydroxyglutarate synthase. Structural and bioninformatic analyses of DUF1338-containing enzymes showed high conservation of critical catalytic and specificity-conferring residues across multiple domains of life despite low sequence identity. These results suggest that the DUF1338 family evolved a specific physiological function within lysine catabolism across multiple domains of life.

molecular biology↗

bioRxiv2020

Elucidation of novel miRNA candidates and their role in unraveling the pathology of Non-Alcoholic Fatty Liver Disease

Non-Alcoholic Fatty Liver Disease (NAFLD) is a chronic liver disease which is observed in people who do not abuse alcohol. Main cause of NAFLD is Non-Alcoholic Steatohepatitis (NASH) where there is accumulation of fats such as triglyceride in liver and the disease progression ranges from simple steatosis to fibrosis and cirrhosis. MicroRNAs are critical players in post-transcriptional gene regulation of diseases with complex etiology. In this study, we have elucidated the role of microRNAs (miRNAs) in the pathophysiology of NAFLD/NASH and unravelled molecular markers for diagnosis of NAFLD. A subset of genes (n=10) responsible for NAFLD/NASH were selected and detailed in silico analysis carried out using multiple tools. miRDB and DIANA-microT were used to find putative miRNA binding sites followed by analysis using miRTarBase which is an experimentally validated database of miRNA-target interactions. The study elucidated a number of statistically significant predictions for both miRDB (scores >80) and DIANA-microT (values >0.90) and also strong experimental validation in miRTarBase. The analysis revealed that certain miRNAs like miR-7 & miR-548 family members are found in both the programmes, miRDB and DIANA-microT, targeting genes involved in liver function. They were also identified in the experimental validation database miRTarBase. These miRNAs probably play an important role in the pathophysiology of this disease. They can also be used as prognostic/diagnostic markers for assessment of NAFLD.

molecular biology↗

bioRxiv2020

Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies

Calcium homeostasis modulator (CALHM) family proteins are Ca2+-regulated ATP-release channels involved in neural functions including neurotransmission in gustation. Here we present the cryo-EM structures of killifish CALHM1, human CALHM2, and C. elegans CLHM-1 at resolutions of 2.66, 3.51, and 3.60 [A], respectively. The CALHM1 octamer structure reveals that the N-terminal helix forms the constriction site at the channel pore in the open state, and modulates the ATP conductance. The CALHM2 undecamer and CLHM-1 nonomer structures show the different oligomeric stoichiometries among CALHM homologs. We further report the cryo-EM structures of the chimeric construct, revealing that the inter-subunit interactions at the transmembrane domain define the oligomeric stoichiometry. These findings advance our understanding of the ATP conduction and oligomerization mechanisms of CALHM channels. One Sentence SummaryCryo-EM structures reveal the ATP conduction and oligomeric assembly mechanisms of CALHM channels.

molecular biology↗

bioRxiv2020

The stress specific impact of ALKBH1 on tRNA cleavage and tiRNA generation

tiRNAs are small non-coding RNAs produced when tRNA is cleaved under stress. tRNA methylation modifications has emerged in recent years as important regulators for tRNA structural stability and sensitivity to cleavage and tiRNA generation during stress, however, the specificity and higher regulation of such a process is not fully understood. Alkbh1 is a m1A demethylase that leads to destabilization of tRNA and enhanced tRNA cleavage. We examined the impact of Alkbh1 targeting via gene knockdown or overexpression on B35 rat neuroblastoma cell line fate following stresses and on tRNA cleavage. We show that Alkbh1 impact on cell fate and tRNA cleavage is a stress specific process that is impacted by the demethylating capacity of the cellular stress in question. We also show that not all tRNAs are cleaved equally following Alkbh1 manipulation and stress, and that Alkbh1 KD fails to rescue tRNAs from cleavage following demethylating stresses. These findings shed a light on the specificity and higher regulation of tRNA cleavage and should act as a guide for future work exploring the utility of Alkbh1 as a therapeutic target for cancers or ischemic insult.

molecular biology↗

bioRxiv2020

Xpo7 negatively regulates Hedgehog signaling by exporting Gli2 from the nucleus

Dynamic bidirectional transport between the nucleus and the cytoplasm is critical for the regulation of many transcription factors, whose levels inside the nucleus must be tightly controlled. Efficient shuttling across the nuclear membrane is especially crucial with regard to the Hedgehog (Hh) pathway, where the transcriptional signal depends on the fine balance between the amounts of Gli protein activator and repressor forms in the nucleus. The nuclear export machinery prevents the unchecked nuclear accumulation of Gli proteins, but the mechanistic insight into this process is limited. We show that the atypical exportin Xpo7 functions as a major nuclear export receptor that actively excludes Gli2 from the nucleus and controls the outcome of Hh signaling. We show that Xpo7 interacts with several domains of Gli2 and that this interaction is dependent on SuFu, a key negative regulator of Hh signaling. Our data pave the way for a more complete understanding of the nuclear shuttling of Gli proteins and the regulation of their transcriptional activity.

molecular biology↗

bioRxiv2020

Widespread divergent transcription from prokaryotic promoters

Promoters are DNA sequences that stimulate the initiation of transcription. In all prokaryotes, promoters are believed to drive transcription in a single direction. Here we show that prokaryotic promoters are frequently bidirectional and drive divergent transcription. Mechanistically, this occurs because key promoter elements have inherent symmetry and often coincide on opposite DNA strands. Reciprocal stimulation between divergent transcription start sites also contributes. Horizontally acquired DNA is enriched for bidirectional promoters suggesting that they represent an early step in prokaryotic promoter evolution.

molecular biology↗

bioRxiv2020

Single-molecule imaging of telomerase RNA reveals a Recruitment-Retention model for telomere elongation

Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatio-temporal coordination of telomerase and telomeres, and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combined CRISPR genome editing and MS2 RNA-tagging to image single-molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, then base-pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life-cycle of telomerase RNA and provides a framework to understand how cancer-associated mutations mechanistically drive defects in telomere homeostasis.

molecular biology↗

bioRxiv2020

Telomere relocalization to the nuclear pore complex in response to replication stress

Mutations in the telomere binding protein, POT1 are associated with solid tumors and leukemias. POT1 alterations cause rapid telomere elongation, ATR kinase activation, telomere fragility, and accelerated tumor development. Here, we investigated the impact of mutant POT1 alleles through complementary genetic and proteomic approaches based on CRISPR-interference and biotin-based proximity labelling, respectively. These screens revealed that replication stress is a major vulnerability in cells expressing mutant POT1 and manifest in increased mitotic DNA synthesis (MiDAS) at telomeres. Our study also unveiled a role for the nuclear pore complex (NPC) in resolving replication defects at telomeres. Depletion of NPC subunits in the context of POT1 dysfunction increased DNA damage signaling and telomere fragility. Furthermore, we observed telomere repositioning to the nuclear periphery driven by nuclear F-actin polymerization in cells with POT1 mutations. In conclusion, our study establishes that relocalization of dysfunctional telomeres to the nuclear periphery is critical to preserve telomere repeat integrity.

molecular biology↗

bioRxiv2020

Nuclear sensing of mitochondrial DNA breaks enhances immune surveillance

Mitochondrial double-strand breaks (mtDSBs) are toxic lesions that compromise mitochondrial function. Mito-nuclear communication is essential to maintain cellular homeostasis, however, the nuclear response to mtDSBs remains unknown. Using mitochondrial-targeted TALENs, we show that mtDSBs activate a type I interferon response evidenced by phosphorylation of STAT1 and activation of interferon stimulated genes (ISG). Following mtDNA break formation, BAK-BAX mediated herniation releases mitochondrial RNA to the cytoplasm and trigger a RIG-I/MAVS-dependent immune response. In an independent set of experiments, we investigate the role of mtDSBs in interferon signaling due to genotoxic stress. Our data reveal that activation of ISGs is greatly diminished when cells lacking mtDNA are exposed to ionizing radiation. Furthermore, we show that mtDNA breaks synergize with nuclear DNA damage to mount a robust interferon response. In conclusion, cytoplasmic accumulation of mitochondrial RNA is as an intrinsic immune surveillance mechanism for cells to cope with mtDSBs, including ones inflicted by genotoxic agents.

molecular biology↗

bioRxiv2020

LncRNA GAS5 attenuates fibroblast activation through inhiting Smad3-mediated myofibroblast marker gene expression.

Transforming Growth Factor {beta} (TGF-{beta})-induced fibroblast activation is a key pathological event during tissue fibrosis. Long noncoding RNA (lncRNA) is a class of versatile gene regulators participating in various cellular and molecular processes. However, the function of lncRNA in fibroblast activation is still poorly understood. In this study, we identified growth arrest-specific transcript 5 (GAS5) as a novel regulator for TGF-{beta}-induced fibroblast activation. GAS5 expression was downregulated in cultured fibroblasts by TGF-{beta} and in resident fibroblasts from bleomycin-treated skin tissues. Overexpression of GAS5 suppressed TGF-{beta}-induced fibroblast to myofibroblast differentiation. Mechanistically, GAS5 directly bound Smad3 and promoted Smad3 binding to PPM1A, a Smad3 dephosphatase, and thus accelerated Smad3 dephosphorylation in TGF-{beta}-treated fibroblasts. In addition, GAS5 inhibited fibroblast proliferation. Importantly, local delivery of GAS5 via adenoviral vector suppressed bleomycin-induced skin fibrosis in mice. Collectively, our data revealed that GAS5 suppresses fibroblast activation and fibrogenesis through inhibiting TGF-{beta}/Smad3 signaling, which provides a rationale for an lncRNA-based therapy to treat fibrotic diseases.

molecular biology↗

bioRxiv2020

Detecting strawberry cultivar misidentification in the Philippines using single nucleotide polymorphism markers from the anthocyanin reductase gene

BACKGROUNDCommercial strawberry production in the Philippines is done by small-holder farmers in La Trinidad, Benguet, where the climate is conducive for optimal growth of this temperate crop. However, these farmers are not cognizant of the importance of proper cultivar identification, particularly during runner propagation, distribution, and transplanting. Thus, there is a high likelihood that misidentification of commonly grown cultivars has taken place. OBJECTIVEThe study aimed to develop single nucleotide polymorphism markers and use them to detect possible misidentification among strawberry cultivars. METHODSLeaf samples from several cultivars were obtained from farmers and the germplasm collection of a local university in La Trinidad, Benguet, Philippines. Expressed sequence tags from the ANR gene were screened for putative SNPs. Eleven SNP markers were developed and used to discriminate among the collected samples. RESULTSThe SNP markers grouped the cultivars into five genotypic clusters with seven distinct genotypic identities. Clustering analysis revealed inconsistencies between the farmers identification and the molecular classification. Sweet Charlie samples were assigned to four genotypic clusters and Strawberry Festival samples were grouped into three separate clusters. CONCLUSIONThere is a high probability that cultivar misidentification has indeed occurred. The molecular markers developed in this study could assist in future cultivar verification efforts, germplasm management, and breeding programs.

molecular biology↗

bioRxiv2020

Gut lymph purification regulates monocyte activity in rats with ischemia-reperfusion injury-induced sepsis

ObjectiveTo confirm that gut lymph purification (GLP) based on oXiris regulates monocyte activity by targeting the removal of ischemia-reperfusion injury (IRI)-induced intestinal toxic substances (ITSs) in rats. MethodsSepsis was induced by intestinal IRI in 24 adult male Sprague-Dawley rats that were randomly divided into the control, intestinal IRI, and IRI+GLP groups. The gut lymph fluid (GLF) was drained for 180 minutes. The ITSs levels and the proliferation, apoptosis and positive expression rates of MHC-II molecules of monocytes coincubated with the GLF were detected. ResultsEndotoxin, TNF-, IL-4, IL-6 and IL-10 levels in the lymph and plasma of the IRI group were significantly higher than those of the control group (p < 0.01). Compared with the IRI group, GLP treatment significantly decreased the ITS levels (p < 0.05). Monocyte proliferation and the positive expression rate of MHC-{square} molecules were significantly reduced after co-culturing with GLF upon IRI (p < 0.01), and the apoptotic rate was significantly increased (p < 0.01). However, culturing monocytes with GLP significantly enhanced the monocyte proliferation, increased the positive expression rate of MHC-{square} monocytes (p < 0.01), and reduced the apoptotic rate (p < 0.01). ConclusionsGLP therapy based on oXiris effectively removed ITSs from the GLF after IRI, thereby blocking the main process of multiple organ dysfunction syndrome by regulating monocyte activity.

molecular biology↗