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Results for “Molecular Biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2018

Deficiency of the E3 Ubiquitin Ligase RBCK1 Causes Diffuse Brain Polyglucosan Accumulation and Neurodegeneration

Glycogen synthesis is vital, malstructure resulting in precipitation and accumulation into neurotoxic polyglucosan bodies (PBs). One well-understood mechanism of PB generation is glycogen branching enzyme deficiency (GBED). Less understood is Lafora disease (LD), resulting from absence of the glycogen phosphatase laforin or the E3 ubiquitin ligase malin, and accumulation of hyperphosphorylated PBs. LD afforded first insight that glycogen sphericity depends on more than adequate branching activity. Unexpectedly, deficiencies of the Linear Ubiquitin Chain Assembly Complex (LUBAC) components RBCK1 and HOIP result in PBs in muscle tissues. Here we analyzed nervous system phenotypes of mice lacking RBCK1 and find profuse PB accumulations in brain and spinal cord with extensive neurodegeneration and neurobehavioral deficits. Brain glycogen in these mice is characterized by long chains and hyperphosphorylation, similar to LD. Like in LD, glycogen synthase and branching enzyme are unaltered. Regional PB distribution mirrors LD and not GBED. Perisynaptic PB localization is unlike LD or GBED. The results indicate that RBCK1 is part of a system supplementing laforin-malin in regulating glycogen architecture including in unique neuronal locales.

molecular biology↗

bioRxiv2018

Higher-Order Organization Principles of Pre-translational mRNPs

Compared to noncoding RNAs (ncRNAs) such as rRNAs and ribozymes, for which high resolution structures abound, little is known about the tertiary structures of mRNAs. In eukaryotic cells, newly made mRNAs are packaged with proteins in highly compacted mRNPs, but the manner of this mRNA compaction is unknown. Here we developed and implemented RIPPLiT (RNA ImmunoPrecipitation and Proximity Ligation in Tandem), a transcriptome-wide method for probing the 3D conformations of RNAs stably-associated with defined proteins, in this case exon junction complex (EJC) core factors. EJCs multimerize with other mRNP components to form megadalton sized complexes that protect large swaths of newly synthesized mRNAs from endonuclease digestion. Unlike ncRNAs, mRNAs behave more like flexible polymers without strong locus-specific interactions. Polymer analysis of proximity ligation data for hundreds of mRNA species demonstrates that pre-translational mammalian mRNPs fold as linear rod-like structures with no strong propensity for 5 and 3 end interaction.

molecular biology↗

bioRxiv2018

A Network of Noncoding Regulatory RNAs Acts in the Mammalian Brain

Noncoding RNAs (ncRNAs) play increasingly appreciated gene-regulatory roles. Here, we describe a regulatory network centered on four ncRNAs--a long ncRNA, a circular RNA, and two microRNAs--using gene editing in mice to probe the molecular consequences of disrupting key components of this network. The long ncRNA Cyrano uses an extensively paired site to miR-7 to trigger destruction of this microRNA. Cyrano-directed miR-7 degradation is much more efficient than previously described examples of target-directed microRNA degradation, which come from studies of artificial and viral RNAs. By reducing miR-7 levels, Cyrano prevents repression of miR-7-targeted mRNAs and enables the accumulation of Cdr1as, a circular RNA known to regulate neuronal activity. Without Cyrano, excess miR-7 causes cytoplasmic destruction of Cdr1as, in part through enhanced slicing of Cdr1as by a second miRNA, miR-671. Thus, several types of ncRNAs can collaborate to establish a sophisticated regulatory network.\n\nHIGHLIGHTSA long noncoding RNA, a circular RNA, and two microRNAs form a regulatory network\n\nThe Cyrano long noncoding RNA directs potent, multiple-turnover destruction of miR-7\n\nUnchecked miR-7 prevents accumulation of Cdr1as circular RNA in cytoplasm of neurons miR-7 prevents this accumulation by enhancing the miR-671-directed slicing of Cdr1as

molecular biology↗

bioRxiv2018

Telomere-loop dynamics in chromosome end protection

We used super-resolution microscopy to investigate the role of macromolecular telomere structure in chromosome end protection. In murine and human cells with reduced TRF2, we find that ATM-activation at chromosome ends occurs with a structural change from t-loops to linearized chromosome ends through t-loop unfolding. Comparably, we find Aurora B kinase regulates telomere linearity concurrent with ATM activation at telomeres during mitotic arrest. Using a separation of function allele, we find that the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that telomere linearity and ATM activation occur separately from telomere fusion via non-homologous end-joining (NHEJ). Further, we show that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1-arrest when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that regulate ATM activation at chromosome ends independent of mechanisms to suppress chromosome end fusion.

molecular biology↗

bioRxiv2018

De novo assembly, characterization, functional annotation and expression patterns of the black tiger shrimp (Penaeus monodon) transcriptome

The black tiger shrimp (Penaeus monodon) remains the second most widely cultured shrimp species globally. However, issues with disease and domestication have seen production levels stagnate over the past two decades. To help identify innovative solutions needed to resolve bottlenecks hampering the culture of this species, it is important to generate genetic and genomic resources. Towards this aim, we have produced the most complete publicly available P. monodon transcriptome database to date. The assembly was carried out in multiple assemblers using 2x125 bp HiSeq data from PolyA selected, ribo-depleted RNA extracted from nine adult tissues and eight early life-history stages. In total, approximately 700 million high-quality sequence reads were obtained and assembled into 236,388 clusters. These were then further segregated into 99,203 adult tissue specific clusters, and 58,678 early life-history stage specific clusters. The final transcriptome had a high TransRate score of 0.37, with 88% of all reads successfully mapping back to the transcriptome. BUSCO statistics showed the assembly to be highly complete with low fragmentation, few genes missing, but higher redundancy or transcript duplication (Complete: 98.2% (Duplicated: 51.3%), Fragmented: 0.8%, Missing: 1.0%), and to greatly exceed the completeness of existing P. monodon transcriptomes. While annotation rates were low (approximately 30%), as is typical for a non-model organisms, annotated transcript clusters were successfully mapped to several hundred functional KEGG pathways. To help address the lack of annotation, transcripts were clustered into groups within tissues and early life-history stages, providing initial evidence for their roles in specific tissue functions, or developmental transitions. Additionally, transcripts of shrimp viruses previously not known to occur in Australia were also discovered. We expect the transcriptome to provide an essential resource to investigate the molecular basis of commercially relevant-significant traits in P. monodon and other shrimp species.

molecular biology↗

bioRxiv2018

Pseudomonas aeruginosa partitioning protein ParB acts as a nucleoid-associated protein binding to multiple copies of a parS-related motif.

ParA and ParB homologs are involved in accurate chromosome segregation in bacteria. ParBs participate in separation of ori domains by binding to specific parS sites, mainly localized close to oriC. In Pseudomonas aeruginosa neither a lack of parB gene nor modification of ten parSs is lethal. Remarkably, such mutants show not only defects in chromosome segregation but also growth retardation and motility dysfunctions. Moreover, a lack of parB alters expression of over one thousand genes, suggesting that ParB could interact with the chromosome outside its canonical parS targets.\n\nIndeed, DNA immunoprecipitation with anti-ParB antibodies followed by deep sequencing (ChIP-seq) revealed 420 enriched regions in WT PAO1161 strain and around 1000 in a ParB-overproducing strain and in various parS mutants. Vast majority of the ParB-enriched loci contained a heptanucleotide motif corresponding to one arm of the parS palindrome. All previously postulated parS sites with the exception of parS5 interacted with ParB in vivo. Whereas the ParB binding to the four parS sites closest to oriC, parS1-4, is involved in chromosome segregation, its genome-wide interactions with hundreds of parS half-sites could affect chromosome topology, compaction and gene expression classifying P. aeruginosa ParB as a Nucleoid Associated Protein (NAP).

molecular biology↗

bioRxiv2018

Utp14 interaction with the Small Subunit Processome

The SSU Processome (sometimes referred to as 90S) is an early stabile intermediate in the small ribosomal subunit biogenesis pathway of eukaryotes. Progression of the SSU Processome to a pre-40S particle requires a large-scale compaction of the RNA and release of many biogenesis factors. The U3 snoRNA is a primary component of the SSU Processome and hybridizes to the rRNA at multiple locations to organize the structure of the SSU Processome. Thus, release of U3 is prerequisite for the transition to pre-40S. Our lab proposed that the RNA helicase Dhr1 plays a crucial role in the transition by unwinding U3 and that this activity is controlled by the SSU Processome protein Utp14. How Utp14 times the activation of Dhr1 is an open question. Despite being highly conserved, Utp14 contains no recognizable domains, and how Utp14 interacts with the SSU Processome is not well characterized. Here, we used UV crosslinking and analysis of cDNA and yeast two-hybrid interaction to characterize how Utp14 interacts with the pre-ribosome. Moreover, proteomic analysis of SSU particles lacking Utp14 revealed that Utp14 is needed for efficient recruitment of the RNA exosome. Our analysis positions Utp14 to be uniquely poised to communicate the status of assembly of the SSU Processome to Dhr1 and possibly the exosome as well.

molecular biology↗

bioRxiv2018

Neurofilament light as a blood biomarker for neurodegeneration in Down syndrome

INTRODUCTIONDown syndrome (DS) may be considered a genetic form of Alzheimers disease (AD) due to universal development of AD neuropathology, but diagnosis and treatment trials are hampered by a lack of reliable blood biomarkers. A potential biomarker is neurofilament light (NF-L), due to its association with axonal damage in neurodegenerative conditions.\n\nMETHODSWe measured blood NF-L concentration in 100 adults with DS using Simoa NF-light(R) assays, and examined relationships with age, and cross-sectional and longitudinal dementia diagnosis.\n\nRESULTSNF-L levels increased with age (Spearmans rho = 0.789, p<0.001), with a steep increase after age 40, and were predictive of dementia status (p=0.022 adjusting for age, sex, and APOE4) but showed no relationship with longstanding epilepsy or premorbid ability. Baseline NF-L levels were associated with longitudinal dementia status.\n\nDISCUSSIONNF-L is a biomarker for neurodegeneration in DS, with potential for use in future clinical trials to prevent or delay dementia.\n\nResearch in contextO_ST_ABSSystematic reviewC_ST_ABSThe authors reviewed the literature using PubMed searches supplemented with our knowledge of pending papers in this research area. While blood NF-L has been associated with clinical features of progression in a number of neurodegenerative conditions, we have not identified any reports of NF-L associated with cognitive decline in DS, a genetic form of AD.\n\nInterpretationOur findings demonstrate the potential utility of NF-L as a blood biomarker of neurodegeneration in DS, a population that may not be able to tolerate more invasive procedures such as neuroimaging and lumbar punctures to track progression.\n\nFuture directionsThe association between NF-L and other markers of longitudinal AD progression should be explored further in future work.

molecular biology↗

bioRxiv2018

Reversible inhibition of specific transcription factor-DNA interactions using CRISPR

The control of gene expression by transcription factor binding sites frequently determines phenotype. However, it has been difficult to assay the function of single transcription factor binding sites within larger transcription networks. Here, we developed such a method by using deactivated Cas9 to disrupt binding to specific sites on the genome. Since CRISPR guide RNAs are longer than transcription factor binding sites, flanking sequence can be used to target specific sites. Targeting deactivated Cas9 to a specific Oct4 binding site in the Nanog promoter blocked Oct4 binding, reduced Nanog expression, and slowed division. Multiple guide RNAs allows simultaneous inhibition of multiple binding sites and conditionally-destabilized dCas9 allows rapid reversibility. The method is a novel high-throughput approach to systematically interrogate cis-regulatory function within complex regulatory networks.

molecular biology↗

bioRxiv2018

The identification of an anti-thrombin molecule via the screening of semi-random DNA libraries

Thrombosis remains one of the leading causes of mortality and morbidity in the world. Thrombin is a key enzyme involved in the blood clotting processes, which can be intervened by low concentrations of Hirudin. The C-terminal dodecapeptide of Hirudin was capable of inhibiting thrombosis. This peptide has been partially randomized in this report, and the coding sequences have been expressed in yeast as chimerical peptides for secretion into the culture media. Two other semi-random modules have been processed likewise. The supernatant was subsequently tested for anti-thrombin activities. DNA sequencing indicated that the putative positive clone encoded a single serine residue followed by a stop codon. The Ninhydrin assay of the culture supernatant of the positive clone indicated a high content of amino acid. Electrospray Mass Spectrometry showed a distinct peak at 430.5 when the expression products from Pichia pastoris were examined, suggesting that the compound may be a dimannosylated serine, as yeast possesses glycosylation at serine residues. The observed effects of -Mannosidase treatments on the function of yeast induction products are consistent with this assumption. Partial randomization of peptides and proteins may accelerate directed evolution, yielding unprecedented number of variants for functional interrogation and drug development.

molecular biology↗

bioRxiv2018

RAD51 paralogs regulate double strand break repair pathway choice by limiting Ku complex retention

RAD51 paralogs are a group of conserved proteins in eukaryotes that are involved in the repair of DNA breaks at several levels. On one hand, they help the strand invasion step catalyzed by RAD51. Also, they play late roles in Holliday Junction metabolism. Here we uncover a new role of the RAD51 paralogs at an earlier event in the repair of broken chromosomes. All five RAD51 paralogs affect the balance between double strand break repair pathways. Specifically, they favor homology-mediated repair over non-homologous end-joining. Such role is independent of RAD51 or the checkpoint activity of these proteins. Moreover, it defines a novel control point of double strand break repair independent and subsequent to DNA-end resection initiation.\n\nMechanistically, RAD51 paralogs limit the retention of Ku80 at the sites of DNA breaks. Thus, our data extend the role of this family of proteins to the earliest event of double strand break repair.

molecular biology↗

bioRxiv2018

RppH can faithfully replace TAP to allow cloning of 5’-triphosphate carrying small RNAs

RNA interference was first described in the nematode Caenorhabditis elegans. Ever since, several new endogenous small RNA pathways have been described and characterized to different degrees. Much like plants, but unlike Drosophila and mammals, worms have RNA-dependent RNA Polymerases (RdRPs) that directly synthesize small RNAs using other transcripts as a template. The very prominent secondary small interfering RNAs, also called 22G-RNAs, produced by the RdRPs RRF-1 and EGO-1 in C. elegans, maintain the 5 triphosphate group, stemming from RdRP activity, also after loading into an Argonaute protein. This creates a technical issue, since 5PPP groups decrease cloning efficiency for small RNA sequencing. To increase cloning efficiency of these small RNA species, a common practice in the field is the treatment of RNA samples, prior to library preparation, with Tobacco Acid pyrophosphatase (TAP). Recently, TAP production and supply was discontinued, so an alternative must be devised. We turned to RNA 5 pyrophosphohydrolase (RppH), a commercially available pyrophosphatase isolated from E. coli. Here we directly compare TAP and RppH in their use for small RNA library preparation. We show that RppH-treated samples faithfully recapitulate TAP-treated samples. Specifically, there is enrichment for 22G-RNAs and mapped small RNA reads show no small RNA transcriptome-wide differences between RppH and TAP treatment. We propose that RppH can be used as a small RNA pyrophosphatase to enrich for triphosphorylated small RNA species and show that RppH- and TAP-derived datasets can be used in direct comparison.

molecular biology↗

bioRxiv2018

E93 expression and links to the juvenile hormone in hemipteran mealybugs with insights on female neoteny

Insect metamorphosis generates reproductive adults and is commonly accompanied by the direct or indirect development of wings. In some winged insects, the imago is altered by life history changes. For instance, in scale insects and mealybugs, reproductive females retain juvenile features and are wingless. The transcription factor E93 triggers metamorphosis and plays in concert with the juvenile hormone pathway to guarantee the successful transition from juvenile to adult. We previously provided evidence of an atypical down-regulation of the juvenile hormone pathway during female adult development in the Japanese mealybug. Here, we further investigate how E93 is involved in the production of neotenic wingless females, by identifying its isoforms, assessing their expression patterns and evaluating the effect of exogenous juvenile hormone mimic treatment on E93. This study identifies three E93 isoforms on the 5 end based on Japanese mealybug cDNA and shows that female development occurs with the near absence of E93 transcripts, as opposed to male metamorphosis. Additionally, while male development is typically affected by exogenous juvenile hormone mimic treatments, females seem to remain insensitive to the treatment, and up-regulation of the juvenile hormone signaling is not observed. Furthermore, juvenile hormone mimic treatment on female nymphs did not have obvious effect on E93 transcription, while treatment on male prepupae resulted in decreased E93 transcripts. In this study, we emphasize the importance of examining cases of atypical metamorphosis as complementary systems to provide a better understanding on the molecular mechanisms underlying insect metamorphosis. For instance, the factors regulating the expression of E93 are largely unclear. Investigating the regulatory mechanism of E93 transcription could provide clues towards identifying the factors that induce or suppress E93 transcription, in turn triggering male adult development or female neoteny.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC=\"FIGDIR/small/283556v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (21K):\norg.highwire.dtl.DTLVardef@1dca37corg.highwire.dtl.DTLVardef@7142a5org.highwire.dtl.DTLVardef@1ce7675org.highwire.dtl.DTLVardef@1c30267_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Neotenic female Planococcus kraunhiae (Japanese mealybug) develops with low E93 expression.\n- E93 expression pattern during male development is typical to other insects.\n- Juvenile hormone mimic treatment on male prepupae results in decreased E93 transcripts.\n- Juvenile hormone mimic treatment on female nymphs does not have obvious effects on E93 transcription.\n- Female mealybugs have low sensitivity to juvenile hormone mimic treatments compared to males and other insects.

molecular biology↗

bioRxiv2018

Compound signaling activates endogenous retroviruses by inducing enhancer and gene-neighborhood transcription

SummaryMultiple sclerosis (MS) is a neuroinflammatory and autoimmune disease, in which various immune cell types and autoreactive T cells exert a pathogenic activity. This disease is also associated with increased transcription of several endogenous retroviruses (HERVs) normally kept in check by heterochromatin. Here, we have uncovered an organic pollutant dieldrin that activates several HERVs associated with MS and allowing us to examine the mechanism of their activation. Dieldrin singles out by its ability to simultaneously activate the MAP kinase and the PI3K pathways, while also triggering calcium dependent peptidylarginine deiminase activity. It was this association of pathways that caused HERV activation, a phenomenon that was only part of more generally increased transcription of heterochromatic regions. The HERV transcripts were generally not polyadenylated. Some arose as a consequence of activation of HERV-based enhancers, while others were the result of unusually strong activation at some mostly transcription factor genes causing transcription to leak out of the HERV-free region that surrounds them. Altogether, our data emphasized the hazard associated with simultaneous activation of multiples signaling pathways by xenobiotics, while also providing a very general toolbox for the interpretation of HERV transcription.

molecular biology↗

bioRxiv2018

Identification of cis elements for spatio-temporal control of DNA replication

The temporal order of DNA replication (replication timing, RT) is highly coupled with genome architecture, but cis-elements regulating spatio-temporal control of replication have remained elusive. We performed an extensive series of CRISPR mediated deletions and inversions and high-resolution capture Hi-C of a pluripotency associated domain (DppA2/4) in mouse embryonic stem cells. Whereas CTCF mediated loops and chromatin domain boundaries were dispensable, deletion of three intra-domain prominent CTCF-independent 3D contact sites caused a domain-wide delay in RT, shift in sub-nuclear chromatin compartment and loss of transcriptional activity, These \"early replication control elements\" (ERCEs) display prominent chromatin features resembling enhancers/promoters and individual and pair-wise deletions of the ERCEs confirmed their partial redundancy and interdependency in controlling domain-wide RT and transcription. Our results demonstrate that discrete cis-regulatory elements mediate domain-wide RT, chromatin compartmentalization, and transcription, representing a major advance in dissecting the relationship between genome structure and function.\n\nHighlightsO_LIcis-elements (ERCEs) regulate large scale chromosome structure and function\nC_LIO_LIMultiple ERCEs cooperatively control domain-wide replication\nC_LIO_LIERCEs harbor prominent active chromatin features and form CTCF-independent loops\nC_LIO_LIERCEs enable genetic dissection of large-scale chromosome structure-function.\nC_LI

molecular biology↗

bioRxiv2018

Unmodified, autologous adipose-derived regenerative cells improve cardiac function, structure and revascularization in a porcine model of chronic myocardial infarction

Numerous studies have investigated cell-based therapies for myocardial infarction (MI), with mixed results. In the present study the left anterior descending (LAD) artery of pigs was occluded for 180 min. Four weeks later, the mean left ventricular ejection fraction (LVEF) was shown to have been reduced to approximately 35%. At that time, 18x106 unmodified, autologous adipose-derived regenerative cells (UA-ADRCs) were delivered into the LAD vein (control: delivery of saline). Six weeks following UA-ADRCs/saline delivery, the mean LVEF had increased by 18% (p<0.01) after delivery of UA-ADRCs, but was unchanged after delivery of saline. This is among the best outcome ever reported in studies on porcine animal models of cell-based therapies for MI in which functional outcome was assessed with cardiac magnetic resonance imaging. The unique combination of the procedure used for isolating UA-ADRCs, the late cell delivery time and the uncommon cell delivery route applied in the present study may open new horizons for cell-based therapies for MI.

molecular biology↗

bioRxiv2018

Transcription-dependent regulation of replication dynamics modulates genome stability

Replication stress is a primary threat to genome stability and has been implicated in tumorigenesis1, 2. Common fragile sites (CFSs) are loci hypersensitive to replication stress3 and are hotspots for chromosomal rearrangements in cancers4. CFSs replicate late in S-phase3, are cell-type dependent4-6 and nest within very large genes4, 7-9. The mechanisms responsible for CFS instability are still discussed, notably the relative impact of transcription-replication conflicts7, 8, 10 versus their low density in replication initiation events5, 6. Here we address the relationships between transcription, replication, gene size and instability by manipulating the transcription of three endogenous large genes, two in chicken and one in human cells. Remarkably, moderate transcription destabilises large genes whereas high transcription levels alleviate their instability. Replication dynamics analyses showed that transcription quantitatively shapes the replication program of large genes, setting both their initiation profile and their replication timing as well as regulating internal fork velocity. Noticeably, high transcription levels advance the replication time of large genes from late to mid S-phase, which most likely gives cells more time to complete replication before mitotic entry. Transcription can therefore contribute to maintaining the integrity of some difficult-to-replicate loci, challenging the dominant view that it is exclusively a threat to genome stability.

molecular biology↗

bioRxiv2018

A naturally-occurring dominant-negative competitor of Keap1 against its inhibition of Nrf2

Transcription factor Nrf2 is a master regulator of antioxidant and/or electrophile response elements (AREs/EpREs)driven genes involved in homeostasis, detoxification and adaptation to various stresses. The cytoprotective activity of Nrf2, though being oppositely involved in both cancer prevention and progression, is critically controlled by Keap1 (Kelch-like ECH-associated protein 1) as an adaptor subunit of Cullin 3-based E3 ubiquitin ligase, that is a key sensor for oxidative and electrophilic stresses. Now, we first report a novel naturally-occurring mutant of Keap1, designated Keap1{Delta}C, which lacks most of its C-terminal Nrf2-interacting domain essential for inhibition of the CNC-bZIP factor. This mutant Keap1{Delta}C is yielded by translation from an alternatively mRNA-spliced variant lacking the fourth and fifth exons, but their coding sequences are retained in the wild-type Keap1 locus (with no genomic deletions). Although this variant was found primarily in the human highly-metastatic hepatoma (MHCC97H) cells, it was widely expressed at very lower levels in all other cell lines examined. No matter whether Keap1{Delta}C retains less or no ability to inhibit Nrf2, it functions as a dominant-negative competitor of Keap1 against its inhibition of Nrf2-target genes. This is due to its antagonist effect on Keap1-mediated turnover of Nrf2 protein.

molecular biology↗