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bioRxiv2018

A rapid microwave method for isolation of genomic DNA and identification of white rot fungi.

White rot fungi (WRF) produce lignolytic enzymes comprised by laccases and peroxidases responsible for mineralization of recalcitrant lignin. Because of the so-called lignin modifying enzymes(LMEs), these fungi have potential applications in biodegradation and bioremediation processes. Increased demand for lignolytic enzymes to exploit their various applications has sparked interest in identifying and characterizing new novel strains of WRF. Despite this undisputed biotechnological significance, molecular identification of WRF, remains a daunting task for researchers as genomic DNA isolation is a tedious process, unsuccessful many a times because of their rigid and resistant cell walls. A rapid, effective and efficient method to identify the innumerable fungal strains within no time is the need of the hour. The fungal mycelia of various unknown as well as know isolates of WRF, after alternative washing with TE buffer and sterile water were suspended in TE buffer. Fungi in solution were then exposed to microwave. The crude extract contained genomic DNA which was extracted and amplified using ITS primers for further identification. Based on sequencing results the identity of known cultures was confirmed, while the unknown cultures were identified as Clitopilus scyphoides (AGUM004, BankIt2098576 MH172163); Ganoderma rasinaceum (AGUM007, BankIt2098576 MH172163); Schizophyllum sp (KONA001 BankIt2098576 MH172164; AGUM011 BankIt2098576 MH172165and AGUM021 BankIt2098576 MH172166respectively), Coprinellus disseminatus (BANG001, BankIt2098576 MH172167) and Lentinus squarrosulus (TAMI004, BankIt2098576 MH172167). The microwave method described for isolating quality DNA of WRF without further purification steps proved a novel method requiring less than ten minutes and minimized the chances of the presence of PCR inhibitors.\n\nIMPORTANCEWhite rot fungi which decay wood, possess selective lignin degrading enzymes responsible for degrading a wide variety of environmental pollutants, xenobiotic compounds in addition to mineralizing chemicals that are insoluble and recalcitrant. Lignolytic enzymes hold potential towards replacing conventional chemical processes and their increased demand in the market has ignited interest in identifying and characterizing new strains of WRF. A rapid, efficient method capable of quickly identifying fungal isolates is a constraint. The microwave method is a novel quick method for isolating superior quality DNA. Its adoption circumvents the initial purification steps and /or interference of PCR inhibitors, which are encompassed in the use of conventional methods. The microwave method thus permits the thorough amplification of the ITS region thereby aiding in the easy identification of unknown species. Use of the microwave method will permit researchers to obtain DNA from fungi very quickly for further application in molecular studies.

molecular biology↗

bioRxiv2018

Genetic analysis of RNA polymerase I unveils new role of the Rpa12 subunit during transcription

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6{Delta} and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that wild-type RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.\n\nAuthor summaryThe nuclear genome of eukaryotic cells is transcribed by three RNA polymerases. RNA polymerase I (Pol I) is a multimeric enzyme specialized in the synthesis of ribosomal RNA. Deregulation of the Pol I function is linked to the etiology of a broad range of human diseases. Understanding the Pol I activity and regulation represents therefore a major challenge. We chose the budding yeast Saccharomyces cerevisiae as a model, because Pol I transcription apparatus is genetically amenable in this organism. Analyses of phenotypic consequences of deletion/truncation of Pol I subunits-coding genes in yeast indeed provided insights into the activity and regulation of the enzyme. Here, we characterized mutations in Pol I that can alleviate the growth defect caused by the absence of Rpa49, one of the subunits composing this multi-protein enzyme. We mapped these mutations on the Pol I structure and found that they all cluster in a well-described structural element, the jaw-lobe module. Combining genetic and biochemical approaches, we showed that Pol I bearing one of these mutations in the Rpa135 subunit is able to produce more ribosomal RNA in vivo and in vitro. We propose that this super-activity is explained by structural rearrangement of the Pol I jaw/lobe interface.

molecular biology↗

bioRxiv2018

M1A within cytoplasmic mRNAs at single nucleotide resolution: A reconciled transcriptome-wide map

Following synthesis, RNA can be modified with over 100 chemically distinct modifications, and in recent years it was shown that processing, localization, stability and translation of mRNAs can be impacted by an increasing number of these modifications. A modification that recently gained attention is N1-methyladenosine (m1A), which is present across all three domains of life. Recently, two studies - one of them ours - developed conceptually similar approaches to map m1A in a transcriptome-wide manner and at single nucleotide resolution. Surprisingly, the two studies diverged quite substantially in terms of their estimates of the abundance, whereabouts and stoichiometry of this modification within internal sites in cytosolic mRNAs: One study reported it to be a very rare modification, present at very low stoichiometries, and invariably catalyzed by TRMT6/61A. The other found it to be present at >470 sites, in dozens of which at relatively high levels, and in the vast majority of cases these sites were highly unlikely to be substrates of TRMT6/61A, suggesting that additional methyltransferases are active on cytosolic mRNAs. Here we aim to reconcile the contradictions between the two studies, primarily by reanalyzing and re-annotating the set of sites identified in the latter study. We find that the vast majority of sites detected in this study originate from duplications, misannotations, mismapping, SNPs, sequencing errors, and a set of sites originating from the very first transcribed base ( TSS sites). We raise concerns as to whether the TSS sites truly reflect m1A originating from the first transcribed base. We find that only 53 of the sites detected in this study likely reflect bona-fide internal modifications of cytoplasmically encoded mRNA molecules. The vast majority of these are likely to be TRMT6/TRMT61A substrates, and are typically modified at low to undetectable levels. We conclude that within cytosolic mRNAs, m1A is a rare internal modification where it is typically catalyzed at ultra-low stoichiometries via TRMT6/TRMT61A. Our findings offer a clear and consistent view on the abundance and whereabouts of this modification, and lays out key directions for future studies in the field.

molecular biology↗

bioRxiv2018

Fungal Bioaerosols at Five Dairy Farms: A Novel Approach to Describe Workers Exposure

Occupational exposure to harmful bioaerosols in industrial environments is a real treat to the workers. In particular, dairy-farm workers are exposed to high levels of fungal bioaerosols on a daily basis. Associating bioaerosol exposure and health problems is challenging and adequate exposure monitoring is a top priority for aerosol scientists. Using only culture-based tools do not express the overall microbial diversity and underestimate the large spectrum of microbes in bioaerosols and therefore the identification of new airborne etiological agents. The aim of this study was to provide an in-depth characterization of fungal exposure at Eastern Canadian dairy farms using qPCR and next-generation sequencing methods. Concentrations of Penicillium/Aspergillus ranged from 4.6 x 106 to 9.4 x 106 gene copies/m3 and from 1 x 104 gene copies/m3 to 4.8 x 105 gene copies/m3 for Aspergillus fumigatus. Differences in the diversity profiles of the five dairy farms support the idea that the novel approach identifies a large number of fungal taxa. These variations may be explained by the presence of different and multiple sources of fungal bioaerosols at dairy farms. The presence of a diverse portrait of fungi in air may represent a health risk for workers who are exposed on a daily basis. In some cases, the allergen/infective activity of the fungi may not be known and can increase the risks to workers. The broad spectrum of fungi detected in this study includes many known pathogens and proves that adequate monitoring of bioaerosol exposure is necessary to evaluate and minimize risks.\n\nImportanceWhile bioaerosols are a major concern for public health, accurately assessing human exposure is challenging. Highly contaminated environments, such as agricultural facilities, contain a broad diversity of aerosolized fungi that may impact human health. Effective bioaerosol monitoring is increasingly recognized as a strategic approach for achieving occupational exposure description. Workers exposure to diverse fungal communities is certain, as fungi are ubiquitous in the environments and the presence of potential sources increase their presence in the air. Applying new molecular approaches to describe occupational exposure is a necessary work around the traditional culture approaches and the biases they introduce to such studies. The importance of the newly developed approach can help to prevent workers health problems.

molecular biology↗

bioRxiv2018

Regulation of transcription factor SP1 by β-catenin destruction complex modulates Wnt response

The ubiquitous transcription factor Specificity protein 1 (SP1) is heavily modified post-translationally. These modifications are critical for switching its functions and modulation of its transcriptional activity, DNA-binding and stability. However the mechanism governing the stability of SP1 by cellular signaling pathways is not well understood. Here, we provide biochemical and functional evidences that SP1 is an integral part of the Wnt signaling pathway. We identified a phosphodegron motif in SP1 that is specific to mammals. In absence of Wnt signaling, GSK3{beta} kinase mediated phosphorylation and {beta}-TrCP E3 ubiquitin ligase mediated ubiquitination is required to induce SP1 degradation. When Wnt signaling is on, SP1 is stabilized in {beta}-catenin-dependent manner. SP1 directly interacts with {beta}-catenin and Wnt signaling induces the stabilization of SP1 by impeding its interaction with {beta}-TrCP and AXIN1, components of the destruction complex. Wnt signaling suppresses ubiquitination and subsequent proteosomal degradation of SP1. Furthermore, SP1 regulates Wnt-dependent stability of {beta}-catenin and their mutual stabilization is critical for target gene expression, suggesting a feedback mechanism. Upon stabilization SP1 and {beta}-catenin co-occupy the promoters of TCFL2/{beta}-catenin target genes. Collectively, this study uncovers a direct link between SP1 and {beta}-catenin in Wnt signaling pathway.

molecular biology↗

bioRxiv2018

HDAC11 Deficiency Prevents High-Fat Diet-Induced Obesity and Metabolic Syndrome

Significance StatementObesity is a serious and widespread health problem which has become a growing concern in many societies. Most currently available weight-loss medications do not work for everyone, and the effects decline over time. Thus, there is an urgent need to identify new molecular targets to improve drug development for the treatment of obesity and obesity-related diseases. In this study, we discovered that the histone deacetylase 11 (HDAC11) enzyme is a key regulator of metabolism and obesity, and the absence of HDAC11 prevents obesity in mice. Our findings will facilitate the development of novel therapeutics to treat obesity by targeting HDAC11.\n\nAbstractObesity and its associated metabolic syndromes are the consequence of susceptible genes and obesogenic environments. We report here that histone deacetylase 11 (HDAC11) plays a critical role in the development of obesity and in metabolic homeostasis. HDAC11 knockout mice display resistance to high-fat diet-induced obesity and associated syndromes by enhancing glucose tolerance and insulin sensitivity, attenuating hypercholesterolemia and hyperinsulinemia, and blocking hepatosteatosis and liver damage. Mechanistically, HDAC11 deficiency boosts energy expenditure through promoting thermogenic capacity, which attributes to the elevation of uncoupling protein 1 (UCP1) expression and activity in brown adipose tissue. Moreover, loss of HDAC11 stimulates mitochondrial oxidation, elevates plasma adiponectin, and activates the adiponectin-AdipoR-AMPK pathway in the liver, which may contribute to a reversal in hepatosteatosis. These findings establish HDAC11 as a key regulator of metabolism and indicate that HDAC11 inhibitors may hold promise for treating overweight and obesity-related diseases.

molecular biology↗

bioRxiv2018

U1 snRNP regulates chromatin retention of noncoding RNAs

Thousands of noncoding transcripts exist in mammalian genomes, and they preferentially localize to chromatin. Here, to identify cis-regulatory elements that control RNA-chromatin association, we developed a high-throughput method named RNA element for subcellular localization by sequencing (REL-seq). Coupling REL-seq with random mutagenesis (mutREL-seq), we discovered a key 7-nt U1 recognition motif in chromatin-enriched RNA elements. Reporter assays indicated a direct role for U1 snRNP recognition in regulating RNA-chromatin localization. Globally, U1 motifs and U1 binding are strongly enriched in long noncoding RNA (lncRNA) transcripts. Inhibition of U1 snRNA, and of U2 to a lesser degree, led to global reduction in chromatin association of hundreds of lncRNAs. For promoter- and enhancer-associated noncoding RNAs, U1 binds to their genomic neighborhoods, and their chromatin association depends on both U1 and U2 snRNAs. These findings reveal that U1 snRNP, perhaps together with the splicing machinery, acts widely to promote the chromatin association of noncoding transcripts.

molecular biology↗

bioRxiv2018

Cholesterol bound Plasmodium falciparum co-chaperone ‘PFA0660w’ complexes with major virulence factor ‘PfEMP1’ via chaperone ‘PfHsp70-x’

Lethality of Plasmodium falciparum (Pf) caused malaria results from cytoadherence, which is effected by exported Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. Several exported Pf proteins (exportome) including chaperones alongside cholesterol rich microdomains are crucial for PfEMP1 translocation to infected erythrocyte surface. An exported Hsp40 (heat shock protein 40) PFA0660w functions as a co-chaperone of PfHsp70-x, and these co-localize to specialized intracellular mobile structures termed J-dots. Our studies attempt to understand the function of PFA0660w-PfHsp70-x chaperone pair using recombinant proteins. Biochemical assays reveal that N and C-terminal domains of PFA0660w and PfHsp70-x respectively are critical for their activity. We show the novel direct interaction of PfHsp70-x with the cytoplasmic tail of PfEMP1, and binding of PFA0660w with cholesterol. PFA0660w operates both as a chaperone and lipid binding molecule via its separate substrate and cholesterol binding sites. PfHsp70-x binds cholesterol linked PFA0660w and PfEMP1 simultaneously in vitro to form a complex. Collectively, our results and the past literature support the hypothesis that PFA0660w-PfHsp70-x chaperone pair assists PfEMP1 transport across the host erythrocyte through cholesterol containing J-dots. Since PFA0660w seems essential for parasite survival, characterization of its interaction with PfHsp70-x and J-dots may form the basis for development of future antimalarials.

molecular biology↗

bioRxiv2018

Benchmarking cryo-EM single particle analysis workflow

Cryo electron microscopy facilities running multiple instruments and serving users with varying skill levels need a robust and reliable method for benchmarking both the hardware and software components of their single particle analysis workflow. The workflow is complex, with many bottlenecks existing at the specimen preparation, data collection and image analysis steps; the samples and grid preparation can be of unpredictable quality, there are many different protocols for microscope and camera settings, and there is a myriad of software programs for analysis that can depend on dozens of settings chosen by the user. For this reason, we believe it is important to benchmark the entire workflow, using a standard sample and standard operating procedures, on a regular basis. This provides confidence that all aspects of the pipeline are capable of producing maps to high resolution. Here we describe benchmarking procedures using a test sample, rabbit muscle aldolase.

molecular biology↗

bioRxiv2018

Small RNAs gained during epididymal transit of sperm are essential for embryonic development in mice

The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) vs. distal (cauda) epididymis, then characterized development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, and subsequently implant inefficiently and fail soon after implantation. Remarkably, microinjection of purified cauda-specific small RNAs into caput-derived embryos not only completely rescued preimplantation molecular defects, but also suppressed the postimplantation embryonic lethality phenotype. These findings reveal an essential role for small RNA remodeling during post-testicular maturation of mammalian sperm, and identify a specific preimplantation gene expression program responsive to sperm-delivered microRNAs.

molecular biology↗

bioRxiv2018

Negative Cooperativity between Gemin2 and RNA Determines RNA Selection and Release of the SMN Complex in snRNP Assembly

The assembly of snRNP cores, in which seven Sm proteins, D1/D2/F/E/G/D3/B, form a ring around the nonameric Sm site of snRNAs, is the early step of spliceosome formation and essential to eukaryotes. It is mediated by the PMRT5 and SMN complexes sequentially in vivo. SMN deficiency causes neurodegenerative disease spinal muscular atrophy (SMA). How the SMN complex assembles snRNP cores is largely unknown, especially how the SMN complex achieves high RNA assembly specificity and how it is released. Here we show, using crystallographic and biochemical approaches, that Gemin2 of the SMN complex enhances RNA specificity of SmD1/D2/F/E/G via a negative cooperativity between Gemin2 and RNA in binding SmD1/D2/F/E/G. Gemin2, independent of its N-tail, constrains the horseshoe-shaped SmD1/D2/F/E/G from outside in a physiologically relevant, narrow state, enabling high RNA specificity. Moreover, the assembly of RNAs inside widens SmD1/D2/F/E/G, causes the release of Gemin2/SMN allosterically and allows SmD3/B to join. The assembly of SmD3/B further facilitates the release of Gemin2/SMN. This is the first to show negative cooperativity in snRNP assembly, which provides insights into RNA selection and the SMN complexs release. These findings reveal a basic mechanism of snRNP core assembly and facilitate pathogenesis studies of SMA.

molecular biology↗

bioRxiv2018

Interaction between Retinoschisin and Norrin: Physical or Functional Relationship?

BackgroundRetinoschisis and Norrie disease are X-linked recessive retinal disorders caused by mutations in RS1 and NDP genes respectively. Both are likely to be monogenic and no locus heterogeneity has been reported. However, there are reports showing overlapping clinical features of Norrie disease and retinoschisis in a NDP knock-out mouse model and also the involvement of both the genes in retinoschisis patients. Yet, the exact molecular mechanism and relationship between the two disorders have still not been understood.\n\nObjectiveIn this study, we aim to investigate the association between retinoschisin (RS1) and norrin (NDP) using in vitro and in silico approaches.\n\nMethodSpecific protein-protein interaction between RS1 and NDP was determined in human retina by co-immunoprecipitation assay and immunoblotting. The immunoprecipitated complexes of RS1 and NDP were analyzed using MALDI-TOF mass spectrometry to validate the findings. STRING database was used to explore the functional relationship.\n\nResultsCo-immunoprecipitation and immunoblotting demonstrated lack of a direct interaction between RS1 and NDP. This was further substantiated by analyzing the immunoprecipitation complexes using MALDI-TOF mass spectrometry. STRING did not reveal any direct functional association between the two proteins.\n\nConclusionWhile literature suggest the existence of digenic involvement of RS1 and NDP in the pathophysiology of retinoschisis and Norrie disease, our data provides evidence for lack of a physical interaction between the two proteins. However, we cannot exclude the possibility of an indirect functional association as our analyses point to MAP kinase signaling pathway, which is presumed to be the link between them.

molecular biology↗

bioRxiv2018

Membrane insertion of α-xenorhabdolysin in near-atomic detail

-Xenorhabdolysins (Xax) are -pore-forming toxins (-PFT) from pathogenic bacteria that form 1-1.3 MDa large pore complexes to perforate the host cell membrane. PFTs are used by a variety of bacterial pathogens as an offensive or defensive mechanism to attack host cells. Due to the lack of structural information, the molecular mechanism of action of Xax toxins is poorly understood. Here, we report the cryo-EM structure of the XaxAB pore complex from Xenorhabdus nematophila at an average resolution of 4.0 [A] and the crystal structures of the soluble monomers of XaxA and XaxB at 2.5 [A] and 3.4 [A], respectively. The structures reveal that XaxA and XaxB are built similarly and appear as heterodimers in the 12-15 subunits containing pore. The structure of the XaxAB pore represents therefore the first structure of a bi-component -PFT. Major conformational changes in XaxB, including the swinging out of an amphipathic helix are responsible for membrane insertion. XaxA acts as an activator and stabilizer for XaxB that forms the actual transmembrane pore. Based on our results, we propose a novel structural model for the mechanism of action of Xax toxins.

molecular biology↗

bioRxiv2018

Proteostasis perturbation destabilizes respiratory complex assembly-intermediates via aggregation of subunits

Proteostasis is maintained by optimum expression, folding, transport, and clearance of proteins. Deregulation of any of these processes triggers widespread protein aggregation and loss of function. Here, we perturbed proteostasis by blocking proteasome-mediated protein degradation and investigated proteome partitioning from soluble to insoluble fraction. Aggregation of Respiratory Chain Complex (RCC) subunits highlights the early destabilization event as revealed by proteome redistribution. Sequence analyses followed by microscopy suggest that low complexity regions at the N-terminus are capable to facilitate aggregation of RCC subunits. As a result, respiratory complex assembly process is impaired due to destabilization of sub-complexes marking the onset of mitochondrial dysfunction and ROS accumulation. Redistribution of Histone proteins and their modifications indicated reprogramming of transcription as adaptive response. Together, we demonstrate susceptibility of RCC subunits to aggregation under multiple proteotoxic stresses providing an explanation for the simultaneous deregulation of proteostasis and bioenergetics in age-related degenerative conditions.

molecular biology↗

bioRxiv2018

DNA Gap Repair-Mediated Site-Directed Mutagenesis is Different from Mandecki and Recombineering Approaches

Site-directed mutagenesis allows the generation of mutant DNA sequences for downstream functional analysis of genetic variants involved in human health and disease. Understanding the mechanisms of different mutagenesis methods can help select the best approach for specific needs. We compared three different approaches for in vivo site-directed DNA mutagenesis that utilize a mutant single-stranded DNA oligonucleotide (ssODN) to target a wild type DNA sequence in the host Escherichia coli (E. coli). The first method, Mandecki, uses restriction nucleases to introduce a double stranded break (DSB) into a DNA sequence which needs to be denatured prior to co-transformation. The second method, recombineering (recombination-mediated genetic engineering), requires lambda red gene products and a mutant ssODN with homology arms of at least 20 nucleotides. In a third method described here for the first time, DNA gap repair, a mutant ssODN targets a DNA sequence containing a gap introduced by PCR. Unlike recombineering, both DNA gap repair and Mandecki can utilize homology arms as short as 10 nucleotides. DNA gap repair requires neither red gene products as recombineering nor DNA denaturation or nucleases as Mandecki, and unlike other methods is background-free. We conclude that Mandecki, recombineering, and DNA gap repair have at least partly different mechanisms, and that DNA gap repair provides a new, straightforward approach for effective site-directed mutagenesis.

molecular biology↗

bioRxiv2018

Environmental factors shape methionine metabolism in p16/MTAP deleted cells

The co-deletion of a common tumor suppressor locus and neighboring metabolic gene is an attractive possible synthetic dependency of tumor suppression on metabolism. However, the general impact that these co-deletions have on metabolism, which also dependent on nutrient availability and the tissue of origin, is unknown. As a model to investigate this question, we considered a set of tissue-matched cancer cells with homozygous co-deletions in CDKN2a and MTAP, genes respectively encoding the most commonly deleted tumor suppressor p16 and an enzyme involved in methionine metabolism. A comparative metabolomics analysis revealed that while there exists a definite pan-cancer metabolic signature of MTAP-deletion, this signature was not preserved when cells were subjected to changes in the availability of methionine, serine, or cysteine, nutrients related to methionine metabolism. Notably, the heterogeneity exhibited by these cells in their responsiveness to nutrient availability dominated both MTAP status and tissue-of-origin. Furthermore, re-expression of MTAP exerted a modest effect on metabolism. Together these findings demonstrate that environmental factors, particularly nutrition and tissue identity, may overwhelm the genetic effects of collateral deletions of metabolic genes.

molecular biology↗

bioRxiv2018

A Rev-CBP80-eIF4AI complex drives Gag synthesis from the HIV-1 unspliced mRNA

Gag synthesis from the full-length unspliced mRNA is critical for the production of the viral progeny during human immunodeficiency virus type-1 (HIV-1) replication. While most spliced mRNAs follow the canonical gene expression pathway in which the recruitment of the nuclear cap-binding complex (CBC) and the exon junction complex (EJC) largely stimulates the rates of nuclear export and translation, the unspliced mRNA relies on the viral protein Rev to reach the cytoplasm and recruit the host translational machinery. Here, we confirm that Rev ensures high levels of Gag synthesis by driving nuclear export and translation of the unspliced mRNA. These functions of Rev are supported by the CBC subunit CBP80, which binds Rev and the unspliced mRNA in the nucleus and the cytoplasm. We also demonstrate that Rev interacts with the DEAD-box RNA helicase eIF4AI, which translocates to the nucleus and cooperates with Rev to promote Gag synthesis. Interestingly, molecular docking analyses revealed the assembly of a Rev-CBP80-eIF4AI complex that is organized around the Rev response element (RRE). Together, our results provide further evidence towards the understanding of the molecular mechanisms by which Rev drives Gag synthesis from the unspliced mRNA during HIV-1 replication.

molecular biology↗

bioRxiv2018

A single N1-methyladenosine on the large ribosomal subunit rRNA impacts locally its structure and the translation of key metabolic enzymes

The entire chemical modification repertoire of yeast ribosomal RNAs and the enzymes responsible for it have recently been identified. Nonetheless, in most cases the precise roles played by these chemical modifications in ribosome structure, function and regulation remain totally unclear. Previously, we demonstrated that yeast Rrp8 methylates m1A645 of 25S rRNA in yeast. Here, using mung bean nuclease protection assays in combination with quantitative RP-HPLC and primer extension, we report that 25S/28S rRNA of S. pombe, C. albicans and humans also contain a single m1A methylation in the helix 25.1. We characterized nucleomethylin (NML) as a human homolog of yeast Rrp8 and demonstrate that NML catalyzes the m1A1322 methylation of 28S rRNA in humans. Our in vivo structural probing of 25S rRNA, using both DMS and SHAPE, revealed that the loss of the Rrp8-catalyzed m1A modification alters the conformation of domain I of yeast 25S rRNA causing translation initiation defects detectable as halfmers formation, likely because of incompetent loading of 60S on the 43S-preinitiation complex. Quantitative proteomic analysis of the yeast{Delta} rrp8 mutant strain using 2D-DIGE, revealed that loss of m1A645 impacts production of specific set of proteins involved in carbohydrate metabolism, translation and ribosome synthesis. In mouse, NML has been characterized as a metabolic disease-associated gene linked to obesity. Our findings in yeast also point to a role of Rrp8 in primary metabolism. In conclusion, the m1A modification is crucial for maintaining an optimal 60S conformation, which in turn is important for regulating the production of key metabolic enzymes.

molecular biology↗