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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Repression of divergent noncoding transcription by a sequence-specific transcription factor

Many active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1 regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeller. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality towards productive transcription.

molecular biology↗

bioRxiv2018

A conformational sensor based on genetic code expansion reveals an autocatalytic component in EGFR activation

Epidermal growth factor receptor (EGFR) activation by growth factors (GFs) relies on dimerization and allosteric activation of its intrinsic kinase activity, resulting in trans-phosphorylation of tyrosines on its C-terminal tail. While structural and biochemical studies identified this EGF-induced allosteric activation, imaging collective EGFR activation in cells and molecular dynamics simulations pointed at additional catalytic EGFR activation mechanisms. To gain more insight in EGFR activation mechanisms in living cells, we developed a Forster Resonance Energy Transfer (FRET) based conformational EGFR indicator (CONEGI) using genetic code expansion that reports on conformational transitions in the EGFR activation loop. Comparing conformational transitions, self-association and auto-phosphorylation of CONEGI and its Y845F mutant revealed that Y845 phosphorylation induces a catalytically active conformation in EGFR monomers. This conformational transition depends on EGFR kinase activity and auto-phosphorylation on its C-terminal tail, generating a looped causality that leads to autocatalytic amplification of EGFR phosphorylation at low EGF dose.

molecular biology↗

bioRxiv2018

The suppressive cap-binding-complex factor 4EIP is required for normal differentiation

Summary/AbstractTrypanosoma brucei live in mammals as bloodstream forms and in the Tsetse midgut as procyclic forms. Differentiation from one form to the other proceeds via a growth-arrested stumpy form with low mRNA content and translation. The parasites have six eIF4Es and five eIF4Gs. EIF4E1 pairs with the mRNA-binding protein 4EIP but not with any EIF4G. EIF4E1 and 4EIP each inhibit expression when tethered to a reporter mRNA. The 4E-binding motif in 4EIP is required for the interaction with EIF4E1 both in vivo and in a 2-hybrid assay, but not for the suppressive activity of 4EIP when tethered. However, the suppressive activity of EIF4E1 when tethered requires 4EIP. Correspondingly, in growing bloodstream forms, 4EIP is preferentially associated with unstable mRNAs. Trypanosomes lacking 4EIP have a marginal growth disadvantage as cultured bloodstream or procyclic forms. Bloodstream forms without 4EIP cannot make differentiation-competent stumpy forms, but the defect can be complemented by a truncated 4EIP that does not interact with EIF4E1. Bloodstream forms lacking EIF4E1 have a growth defect but can differentiate. We suggest that 4EIP and EIF4E1 fine-tune mRNA levels in growing cells, and that 4EIP is required for mRNA suppression during differentiation to the stumpy form.

molecular biology↗

bioRxiv2018

Discovery of a human testis-specific protein complex TEX101-DPEP3 and selection of its disrupting antibodies

TEX101 is a testis-specific protein expressed exclusively in male germ cells and is a validated biomarker of male infertility. Studies in mice suggest that TEX101 is a cell-surface chaperone which regulates, through protein-protein interactions, the maturation of proteins involved in spermatozoa transit and oocyte binding. Male TEX101-null mice are sterile. Here, we identified by co-immunoprecipitation-mass spectrometry the interactome of human TEX101 in testicular tissues and spermatozoa. The testis-specific cell-surface dipeptidase 3 (DPEP3) emerged as the top hit. We further validated the TEX101-DPEP3 complex by using hybrid immunoassays. Combinations of antibodies recognizing different epitopes of TEX101 and DPEP3 facilitated development of a simple immunoassay to screen for disruptors of TEX101-DPEP3 complex. As a proof-of-a-concept, we demonstrated that anti-TEX101 antibody T4 disrupted the native TEX101-DPEP3 complex. Disrupting antibodies may be used to study the human TEX101-DPEP3 complex, and to develop modulators for male fertility.\n\nNon-standard abbreviations

molecular biology↗

bioRxiv2018

RNA polymerase II clustering through CTD phase separation

The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is an intrinsically disordered low-complexity region that is critical for pre-mRNA transcription and processing. The CTD consists of hepta-amino acid repeats varying in number from 52 in humans to 26 in yeast. Here we report that human and yeast CTDs undergo cooperative liquid phase separation at increasing protein concentration, with the shorter yeast CTD forming less stable droplets. In human cells, truncation of the CTD to the length of the yeast CTD decreases Pol II clustering and chromatin association whereas CTD extension has the opposite effect. CTD droplets can incorporate intact Pol II and are dissolved by CTD phosphorylation with the transcription initiation factor IIH kinase CDK7. Together with published data, our results suggest that Pol II forms clusters/hubs at active genes through interactions between CTDs and with activators, and that CTD phosphorylation liberates Pol II enzymes from hubs for promoter escape and transcription elongation.

molecular biology↗

bioRxiv2018

Epigenetic regulation affects fertility in Drosophila: toward the production of infertility models.

We have revealed that the chorion gene clusters amplify by repeatedly initiating DNA replication from chorion gene amplification origins in the response to developmental signals, through the transcription factors in Drosophila ovarian follicle cells. Orc1, Orc2, and Cdc6 are forms of DNA replication machinery, which are conserved from yeast to humans; and Orc1 and Orc2 mutants are lethal. Overexpression of Orc1 or Orc2 (subunits of the origin recognition complex) led to female sterility, but overexpression of Cdc6 (an Orc family member) or GFP did not. We propose that DNA replication machinery contributes to development.\n\nRecently, we found that H3K4 was trimethylated at chorion gene amplification origins, but not at the Act1 locus. Overexpression of Lsd1H3K4 dimethylase and Lid H3K4 trimethylase are female sterile but not a Lid mutant. These results showed that epigenetic regulation affected fertility. Screening strategies using Drosophila flies could also lead to the development of drugs that reduce sterility and epigenetic effects related histone modification.\n\nSummary statementThere are approximately 470,000 infertile individuals in Japan. We knockowned the prereplicative complex components and demethlases during Drosophila ovary development. In these drospohila, we could be the model of infertile.

molecular biology↗

bioRxiv2018

S phase R-loop formation is restricted by PrimPol-mediated repriming

During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here we show in vivo how R-loop formation drives a short purine-rich repeat, (GAA)10, to become a replication impediment that requires the repriming activity of the primase-polymerase PrimPol for its processive replication. Further, we show that loss of PrimPol results in a significant increase in R-loop formation around the repeat during S phase. We extend this observation by showing that PrimPol suppresses R-loop formation in genes harbouring secondary structure-forming sequences, exemplified by G quadruplex and H-DNA motifs, across the genome in both avian and human cells. Thus, R-loops promote the creation of replication blocks at susceptible sequences, while PrimPol-dependent repriming limits the extent of unscheduled R-loop formation at these sequences, mitigating their impact on replication.

molecular biology↗

bioRxiv2018

Silencing cuticular pigmentation genes enables RNA FISH in intact chemosensory appendages

Optical imaging of gene expression by RNA-fluorescent in situ hybridisation (FISH) in whole-mount sensory appendages of insects is often impeded by their highly pigmented cuticle. Since most chemical bleaching agents are incompatible with imaging fluorescent-labelled nucleotides, we developed a RNA interference-based method for clearing cuticular pigmentation that allows imaging of fluorescent mRNA in whole-mount appendages of insects. Silencing key genes of the tyrosine-derived pigmentation pathway by injecting dsRNA of laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi, Phaedon cochleariae) resulted in clearance of the highly pigmented cuticle and in significant decreased light absorbance. Intact chemosensory appendages (palps, antennae and legs) from RNAi-cleared individuals were used to image expression and spatial distribution of antisense mRNA of two chemosensory genes (gustatory receptor, odorant-binding protein) via RNA FISH and confocal laser scanning microscopy. Imaging of these genes did neither work for RNAi-controls (dsGfp) due to retained pigmentation, nor for FISH-controls using sense mRNA. Furthermore, we show that several chemical bleaching agents are not feasible with FISH, either due to significant degradation of polynucleotides, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents allowing fluorescence imaging in whole-mount appendages and organs.

molecular biology↗

bioRxiv2018

Co-transcriptional loading of RNA export factors shapes the human transcriptome.

During gene expression, RNA export factors are mainly known for driving nucleocytoplasmic transport. Whilst early studies suggested that the Exon Junction Complex provides a binding platform for them, subsequent work proposed that they are only recruited by the Cap-Binding Complex to the 5 end of RNAs, as part of TREX. Using iCLIP, we show that the export receptor Nxf1 and two TREX subunits, Alyref and Chtop, are actually recruited to the whole mRNA co-transcriptionally via splicing but before 3-end processing. Consequently, Alyref alters splicing decisions and Chtop regulates alternative polyadenylation. Alyref is recruited to the 5-end of RNAs by CBC and our data reveal subsequent binding to RNAs near EJCs. We demonstrate eIF4A3 stimulates Alyref deposition not only on spliced RNAs close to EJC sites, but also single exon transcripts. Our study reveals mechanistic insights into the co-transcriptional recruitment of mRNA export factors and how this shapes the human transcriptome.

molecular biology↗

bioRxiv2018

Widespread Alterations in Translation Elongation in the Brain of Juvenile Fmr1 Knock-Out Mice

FMRP is a polysome-associated RNA-binding protein encoded by Fmr1 that is lost in Fragile X syndrome. Increasing evidence suggests that FMRP regulates both translation initiation and elongation, but the gene-specificity of these effects is unclear. To elucidate the impact of Fmr1 loss on translation, we used ribosome profiling for genome-wide measurements of ribosomal occupancy and positioning in the cortex of 24 day-old Fmr1 knock-out mice. We found a remarkably coherent reduction in ribosome footprint abundance per mRNA for previously identified, high-affinity mRNA binding partners of FMRP, and an increase for terminal oligo-pyrimidine (TOP) motif-containing genes canonically controlled by mTOR-4EBP-eIF4E signaling. Amino acid motif- and gene-level analyses both showed a widespread reduction of translational pausing in Fmr1 knock-out mice. Our findings are consistent with a model of FMRP-mediated regulation of both translation initiation through eIF4E and elongation that is disrupted in Fragile X syndrome.

molecular biology↗

bioRxiv2018

MicroRNA-184 Is Induced By Store-Operated Calcium Entry And Regulates Early Keratinocyte Differentiation

Extracellular calcium (Ca2+) and store-operated Ca2+ entry (SOCE) govern homeostasis in the mammalian epidermis. Multiple microRNAs (miRNA) also regulate epidermal differentiation, and raised external Ca2+ modulates the expression of several such miRNAs in keratinocytes. However, little is known about the regulation of miR-184 in keratinocytes or the roles of miR-184 in keratinocyte differentiation. Here we report exogenous Ca2+ stimulates miR-184 expression in primary epidermal keratinocytes and that this occurs in a SOCE-dependent manner. Levels of miR-184 were raised by about 30-fold after exposure to 1.5 mM Ca2+ for 5 days. In contrast, neither phorbol ester nor 1, 25-dihydroxyvitamin D3 had any effect on miR-184 levels. Pharmacologic and genetic inhibitors of SOCE abrogated Ca2+-dependent miR-184 induction by 70% or more. Ectopic miR-184 inhibited keratinocyte proliferation and led to a 4-fold increase in the expression of involucrin, a marker of early keratinocyte differentiation. Exogenous miR-184 also triggered a 3-fold rise in levels of cyclin E and doubled the levels of {gamma}H2AX, a marker of DNA double strand breaks. The p21 cyclin-dependent kinase (CDK) inhibitor, which supports keratinocyte growth arrest, was also induced by miR-184. Together our findings point to a SOCE:miR-184 pathway that targets a cyclin E/DNA damage regulatory node to facilitate keratinocyte differentiation.

molecular biology↗

bioRxiv2018

C. albicans Zn Cluster Transcription Factors Tac1 and Znc1 are Activated by Farnesol to Up Regulate a Transcriptional Program Including the Multi-Drug Efflux Pump CDR1

Farnesol, a quorum-sensing molecule, inhibits C. albicans hyphal formation, affects its biofilm formation and dispersal, and impacts its stress response. Several aspects of farnesols mechanism of action remain incompletely uncharacterized. Among these are a thorough accounting of the cellular receptors and transporters for farnesol. This work suggests these themes are linked through the Zn cluster transcription factors Tac1 and Znc1, and their induction of the multi-drug efflux pump Cdr1. Specifically, we have demonstrated that Tac1 and Znc1 are functionally activated by farnesol through a mechanism that mimics other means of hyperactivation of Zn cluster transcription factors. This is consistent with our observation that many genes acutely induced by farnesol are dependent on TAC1, ZNC1, or both. A related molecule, 1-dodecanol, invokes a similar TAC1/ZNC1 response, while several other proposed C. albicans quorum sensing molecules do not. TAC1 and ZNC1 both bind to and up-regulate the CDR1 promoter in response to farnesol. Differences in inducer and DNA binding specificity lead to Tac1 and Znc1 having overlapping, but non-identical, regulons. TAC1 and ZNC1 dependent farnesol induction of their target genes was inversely related to the level of CDR1 present in the cell, suggesting a model in which induction of CDR1 by Tac1 and Znc1 leads to an increase in farnesol efflux. Consistent with this premise, our results show that CDR1 expression, and its regulation by TAC1 and ZNC1, facilitates growth in the presence of high farnesol concentrations in C. albicans, and certain strains of its close relative C. dubliniensis.

molecular biology↗

bioRxiv2018

Transcription apparatus of the yeast killer DNA plasmids: Architecture, function, and evolutionary origin

Transcription of extrachromosomal elements such as organelles, viruses, and plasmids is dependent on cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast Kluyveromyces lactis contains killer DNA plasmids that bear putative non-canonical RNAP genes. Here, we describe the architecture and evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two plasmid-encoded proteins - the mRNA capping enzyme, and a putative helicase which interacts with plasmid-specific DNA. Further, we identify a promoter element that causes 5 polyadenylation of plasmid-specific transcripts via RNAP slippage during transcription initiation, and structural elements that precede the termination sites. As a result, we present a first model of the yeast killer plasmid transcription initiation and intrinsic termination. Finally, we demonstrate that plasmid RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively.

molecular biology↗

bioRxiv2018

The interplay between small RNA pathways shapes chromatin landscape in C. elegans

The nematode C. elegans contains several types of endogenous small interfering RNAs (endo-siRNAs) produced by RNA-dependent RNA polymerase (RdRP) complexes. Both \"silencing\" siRNAs bound by Worm-specific Argonautes (WAGO) and \"activating\" siRNAs bound by the CSR-1 Argonaute require the DRH-3 helicase, an RdRP component. Here we show that, in the drh-3(ne4253) mutant deficient in RdRP-produced secondary endo-siRNAs, the silencing histone mark H3K9me3 is largely depleted, whereas in the csr-1 partial loss-of-function mutant this mark is ectopically deposited on CSR-1 target genes. Moreover, we observe ectopic H3K9me3 at enhancer elements in both drh-3 and csr-1 partial loss-of-function mutants and describe small RNAs matching enhancers. Finally, we detect accumulation of H3K27me3 at highly expressed genes in the drh-3(ne4253) mutant, which correlates with their reduced transcription. Our study shows that when abundant RdRP-produced siRNAs are depleted, there is ectopic elevation of noncoding RNAs linked to increase in silencing chromatin marks. Moreover, our results suggest that enhancer small RNAs may guide local H3K9 methylation.

molecular biology↗

bioRxiv2018

Condensin-dependent chromatin condensation represses transcription globally during quiescence

Quiescence is a stress-resistant state in which cells reversibly exit the mitotic cell cycle and suspend most cellular processes. Quiescence is essential for stem cell maintenance and its misregulation is implicated in tumor formation. One of the conserved hallmarks of quiescent cells, from Saccharomyces cerevisiae to humans, is highly condensed chromatin. Here, we use Micro-C XL to map chromatin contacts at single-nucleosome resolution genome-wide to elucidate mechanisms and functions of condensed chromatin in quiescent S. cerevisiae cells. We describe previously uncharacterized chromatin domains on the order of 10-60 kilobases that in quiescent cells are formed by condensin-mediated chromatin loops. Conditional depletion of condensin prevents chromatin condensation during quiescence entry and leads to widespread transcriptional de-repression. We further demonstrate that condensin-dependent chromatin compaction is conserved in quiescent human fibroblasts. We propose that condensin-dependent condensation of chromatin represses transcription throughout the quiescent cell genome.

molecular biology↗

bioRxiv2018

Versatile in vivo genome editing with the prototypical Streptococcus thermophilus CRISPR1-Cas9 system

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

molecular biology↗

bioRxiv2018

Kaposi’s sarcoma-associated herpesvirus ORF68 is a DNA binding protein required for viral genome cleavage and packaging

Herpesviral DNA packaging into nascent capsids requires multiple conserved viral proteins that coordinate genome encapsidation. Here, we investigated the role of the ORF68 protein of Kaposis sarcoma-associated herpesvirus (KSHV), a protein required for viral DNA encapsidation whose function remains largely unresolved across the herpesviridae. We found that KSHV ORF68 is expressed with early kinetics and localizes predominantly to viral replication compartments, although it is dispensable for viral DNA replication and gene expression. However, in agreement with its proposed role in viral DNA packaging, KSHV-infected cells lacking ORF68 failed to cleave viral DNA concatemers, accumulated exclusively immature B-capsids, and released no infectious progeny virions. ORF68 has no predicted domains aside from a series of putative zinc finger motifs. However, in vitro biochemical analyses of purified ORF68 protein revealed that it robustly binds DNA and is associated with nuclease activity. These activities provide new insights into the role of KSHV ORF68 in viral genome encapsidation.\n\nImportanceKaposis sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposis sarcoma and several B-cell cancers, causing significant morbidity and mortality in immunocompromised individuals. A critical step in the production of infectious viral progeny is the packaging of the newly replicated viral DNA genome into the capsid, which involves coordination between at least seven herpesviral proteins. While the majority of these packaging factors have been well studied in related herpesviruses, the role of the KSHV ORF68 protein and its homologs remains unresolved. Here, using a KSHV mutant lacking ORF68, we confirm its requirement for viral DNA processing and packaging in infected cells. Furthermore, we show that the purified ORF68 protein directly binds DNA and is associated with a metal-dependent cleavage activity on double stranded DNA in vitro. These activities suggest a novel role for ORF68 in herpesviral genome processing and encapsidation.

molecular biology↗

bioRxiv2018

ATPase and protease domain movements in the bacterial AAA+ protease FtsH are driven by thermal fluctuations

AAA+ proteases are essential players in cellular pathways of protein degradation. Elucidating their conformational behavior is key for understanding their reaction mechanism and, importantly, for elaborating our understanding of mutation-induced protease deficiencies. Here, we study the structural dynamics of the Thermotoga maritima AAA+ hexameric ring metalloprotease FtsH (TmFtsH). Using a single-molecule Forster resonance energy transfer approach to monitor ATPase and protease inter-domain conformational changes in real time, we show that TmFtsH--even in the absence of nucleotide--is a highly dynamic protease undergoing conformational transitions between five states on the second timescale in a sequential manner. Addition of ATP does not influence the number of states nor change the timescale of domain motions, but affects the state occupancy distribution leading to an inter-domain compaction. These findings suggest that thermal energy, but not chemical energy, provides the major driving force for conformational switching, while ATP, through a state reequilibration, introduces directionality into this process. The TmFtsH A359V mutation, a homolog of the human pathogenic A510V mutation of paraplegin (SPG7) causing hereditary spastic paraplegia (HSP), does not affect the dynamic behavior of the protease but impairs the ATP-coupled domain compaction and, thus, may account for protease malfunctioning and pathogenesis in HSP.

molecular biology↗