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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Analysis of a structured intronic region of the LMP2 pre-mRNA from EBV reveals associations with human regulatory proteins.

Objective: The pre-mRNA of the Epstein-Barr virus LMP2 (latent membrane protein 2) has a region of unusual RNA structure that partially spans two consecutive exons and the entire intervening intron; suggesting RNA folding might affect splicing--particularly via interactions with human regulatory proteins. To better understand the roles of protein associations with this structured intronic region, we undertook a combined bioinformatics (motif searching) and experimental analysis (biotin pulldowns and RNA immunoprecipitations) of protein binding. Result: Characterization of the ribonucleoprotein composition of this region revealed several human proteins as interactors: regulatory proteins hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1), hnRNP U, HuR (human antigen R), and PSF (polypyrimidine tract-binding protein-associated splicing factor), as well as, unexpectedly, the cytoskeletal protein actin. Treatment of EBV-infected cells with drugs that alter actin polymerization specifically showed marked effects on splicing in this region. This suggests a potentially novel role for nuclear actin in regulation of viral RNA splicing.

molecular biology↗

bioRxiv2018

Gelbox - An Interactive Simulation Tool for Gel Electrophoresis

Gel electrophoresis enables separation and visualization of biomolecules such as DNA, RNA, or proteins. Like many powerful tools, mastering the use of gels and making the information gained from them productive, can be dificult. Gelbox is a simulation tool that helps users understand how changing experimental input parameters can affect the data output from gel electrophoresis. Our simulation model handles a wide range of settings, including many \"suboptimal\" values that may be useful for troubleshooting common mistakes made by novices.

molecular biology↗

bioRxiv2018

The Arabidopsis autophagy cargo receptors ATI1 and ATI2 bind to ATG8 via intrinsically disordered regions and are post-translationally modified upon ATG8 interaction

Selective autophagy has emerged as an important mechanism by which eukaryotic cells control the abundance of specific proteins. This mechanism relies on cargo recruitment to autophagosomes by receptors that bind to both the ubiquitin-like AUTOPHAGY8 (ATG8) protein through ATG8 interacting motifs (AIMs) and to the cargo to be degraded. In plants, two autophagy cargo receptors, ATG8 Interacting Protein 1 (ATI1) and 2 (ATI2), were identified early on, but their molecular properties remain poorly understood. Here, we show that ATI1 and ATI2 are transmembrane proteins with long N-terminal intrinsically disordered regions (IDRs). The N-terminal IDRs contain the functional AIMs, and we use nuclear magnetic resonance spectroscopy to directly observe the disorder-order transition of the AIM upon ATG8 binding. Our analyses also show that the IDRs of ATI1 and ATI2 are not equivalent, because ATI2 has properties of a fully disordered polypeptide, while ATI1 has properties consistent with a collapsed pre-molten globule-like conformation Interestingly, wild type ATI1 and ATI2 exist as distinct post-translationally modified forms. Specifically, different forms are detectable upon mutation of the AIM, suggesting that interaction of ATI1 and ATI2 to ATG8 is coupled to a change in their post-translational modification.

molecular biology↗

bioRxiv2018

Structural Adaptation of Darunavir Analogs Against Primary Resistance Mutations in HIV-1 Protease

HIV-1 protease is one of the prime targets of agents used in antiretroviral therapy against HIV. However, under selective pressure of protease inhibitors, primary mutations at the active site weaken inhibitor binding to confer resistance. Darunavir (DRV) is the most potent HIV-1 protease inhibitor in clinic; resistance is limited, as DRV fits well within the substrate envelope. Nevertheless, resistance is observed due to hydrophobic changes at residues including I50, V82 and I84 that line the S1/S1 pocket within the active site. Through enzyme inhibition assays and a series of 12 crystal structures, we interrogated susceptibility of DRV and two potent analogs to primary S1 mutations. The analogs had modifications at the hydrophobic P1 moiety to better occupy the unexploited space in the S1 pocket where the primary mutations were located. Considerable losses of potency were observed against protease variants with I84V and I50V mutations for all three inhibitors. The crystal structures revealed an unexpected conformational change in the flap region of I50V protease bound to the analog with the largest P1 moiety, indicating interdependency between the S1 subsite and the flap region. Collective analysis of protease-inhibitor van der Waals (vdW) interactions in the crystal structures using principle component analysis indicated I84V mutation underlying the largest variation in the vdW contacts. Interestingly, the principle components were able to distinguish inhibitor identity and relative potency solely based on vdW interactions of active site residues in the crystal structures. Our results reveal the interplay between inhibitor P1 moiety and primary S1 mutations, as well as suggesting a novel method for distinguishing the interdependence of resistance through principle component analyses.

molecular biology↗

bioRxiv2018

Wheat avenin-like protein and its significant Fusarium Head Blight resistant functions

Wheat Avenin-like proteins (TaALP) are atypical storage proteins belonging to the Prolamin superfamily. Previous studies on ALPs have focused on the proteins positive effects on dough strength, whilst no correlation has been made between TaALPs and the plant immune system. Here, we performed genome-wide characterization of ALP encoding genes in bread wheat. In silico analyses indicated the presence of critical peptides in TaALPs that are active in the plant immune system. Pathogenesis-related nucleotide motifs were also identified in the putative promoter regions of TaALP encoding genes. RT-PCR was performed on TaALP and previously characterised pathogenesis resistance genes in developing wheat caryopses under control and Fusarium graminearum infection conditions. The results showed that TaALP and NMT genes were upregulated upon F. graminearum inoculation. mRNA insitu hybridization showed that TaALP genes were expressed in the embryo, aleurone and sub-aleurone layer cells. Seven TaALP genes were cloned for the expression of recombinant proteins in Escherichia coli, which displayed significant inhibitory function on F. graminearum under anti-fungal tests. In addition, FHB index association analyses showed that allelic variations of two ALP genes on chromosome 7A were significantly correlated with FHB symptoms. Over-expression of an ALP gene on chromosome 7A showed an enhanced resistance to FHB. Yeast two Hybridization results revealed that ALPs have potential proteases inhibiting effect on metacaspases and beta-glucosidases. A vital infection process related pathogen protein, F. graminearum Beta-glucosidase was found to interact with ALPs. Our study is the first to report a class of wheat storage protein or gluten protein with biochemical functions. Due to its abundance in the grain and the important multi-functions, the results obtained in the current study are expected to have a significant impact on wheat research and industry.

molecular biology↗

bioRxiv2018

Transcripts from multicopy gene families localizing to mouse Y long arm encode piRNAs and proteins

Heterochromatic long arm of mouse Y chromosome harbors the multicopy species-specific sequences Ssty, Sly, Asty, and Orly that are transcribed in testis. Of these Ssty and Sly genes encode proteins - yet all the copies of these RNAs are not translated. Using bioinformatic approaches, small RNA northern blots and electrophoretic mobility shift assays, we demonstrate here that these multicopy gene families from mouse Y-long arm generate piRNAs predominantly in testis. Thus, we identified a piRNA cluster on mouse Y chromosome and also unraveled the dual role of Y-chromosome-encoded transcripts to act as primary transcripts of piRNAs in addition to their role as protein-coding RNAs.\n\nHIGHLIGHTSO_LIFirst report of a cluster of piRNAs on a mammalian Y chromosome\nC_LIO_LIReport of primary transcripts of piRNAs\nC_LIO_LIThese piRNAs putatively regulate autosomal genes expressed in mouse testis Ssty and Sly genes code for proteins as well as generate piRNAs\nC_LI

molecular biology↗

bioRxiv2018

Quality assessment of tissue samples stored in a specialized human lung biobank

Human sample, from patients or healthy donors, are a valuable link between basic research and clinic. Especially in translational research, they play an essential role in understanding development and progression of diseases as well as in developing new diagnostic and therapeutic tools. Stored in biobanks, fast access to appropriate material becomes possible. However, biobanking in a clinical context faces several challenges. In practice, collecting samples during clinical routine does not allow to strictly adhere to protocols of sample collection in all aspects. This may influence sample quality to variable degrees. Time from sample draw to asservation is a variable factor, and influences of prolonged storage at ambient temperature of tissues are not well understood. We investigated whether delays between 5 minutes and 3 hours, and the use of RNAlater RNA-preserving reagent would lead to a relevant drop in sample quality, measured by quantitative mRNA expression analysis. Our findings suggest that even under ambient conditions, delays up to 3 hours do not have a major impact on sample quality as long as the tissue remains intact.

molecular biology↗

bioRxiv2018

USP21 and OTUD3 Antagonize Regulatory Ribosomal Ubiquitylation and Ribosome-Associated Quality Control

Defects within mRNAs or nascent chains that halt ribosomal progression can trigger ribosome-associated quality control (RQC) pathways that facilitate mRNA and nascent polypeptide destruction as well as ribosome recycling. Failure to remove defective mRNAs or nascent chains can lead to the accumulation of cytotoxic protein aggregates and proteotoxic stress. We previously established that the E3 ligase ZNF598 catalyzes regulatory ribosomal ubiquitylation of specific 40S ribosomal proteins required for downstream RQC events. Utilizing an optical RQC reporter we identify OTUD3 and USP21 as deubiquitylating enzymes that antagonize ZNF598-mediated 40S ubiquitylation and facilitate ribosomal deubiquitylation following RQC activation. Overexpression of either USP21 or OTUD3 enhances readthrough of stall-inducing sequences as compared to knock-in cells lacking individual RRub sites suggesting that combinatorial ubiquitylation of RPS10 (eS10) and RPS20 (uS10) is required for optimal resolution of RQC events and that deubiquitylating enzymes can limit RQC activation.

molecular biology↗

bioRxiv2018

Secondary structure of pre-mRNA introns for genes of 15q11-12 locus. Mapping of functionally significant motives of RNA binding proteins and f nucleosome positioning signals

In this study, we identified reproducible substructures in the folded structures of long intron RNAs for recursive spliced variants and annotated pre-mRNA for GABRB3 and GABRA5. We mapped the RNA motives recognized by RNA-binding proteins for the specified locus and characterized the area of preferred localization. A comparison of pre-mRNA variants revealed the dominant type of protein potential effects. We determined the structural specifics of RNA in the dense Alu cluster and clarified the analogy of apical substructure to the A-Xist fragment of transcriptional variant. Mapping of the nucleosome potential reveals alternation of strong and weak signals at the 3-end portion of GABRB3 and clusters of nucleosome positioning signal in the vicinity of the Alu cluster. Distribution of simple oligonucleotides among reproducible substructures revealed an enrichment in Py-tracts; for some of them, this may be considered as a complementary supplement to the Pu-tract enrichment of ncRNA Malat1 as a component of nuclear speckles. The secondary structure elements of bidirectional transcripts are predisposed for somatic homolog pairing in this locus, as was previously shown experimentally.\n\nA model of potential intron RNA influence on splicing has been suggested based on its interaction with Py-tract-binding RNP, serine-arginine SRSF proteins, ncRNA Malat1, as well as the action of Alu cluster.

molecular biology↗

bioRxiv2018

FUS controls the processing of snoRNAs into smaller RNA fragments that can regulate gene expression

FUS is a multifunctional protein involved in many steps of RNA metabolism, including transcription, splicing, miRNA processing and replication-dependent histone gene expression. In this paper, we show for the first time that FUS binds and negatively regulates the levels of a subset of snoRNAs in cells. Scanning of available human small RNA databases revealed the existence of smaller RNA fragments that can be processed from FUS-dependent snoRNAs. Therefore, we suggest that FUS mediates the biogenesis of snoRNA-derived small RNAs, called sdRNAs. Further in silico approaches enabled us to predict putative targets of selected FUS-dependent sdRNAs. Our results indicate that sdRNAs may bind to different regions of target mRNAs as well as to noncoding transcripts and influence the posttranscriptional level or translation of these targets. SIGNIFICANCE STATEMENTRNA metabolism is orchestrated by a complex network of RNA-protein interactions and involves various classes of RNA molecules. Small nucleolar RNAs (snoRNAs) are commonly considered essential components of the ribosome biogenesis pathway. However, recent studies have revealed that snoRNAs can also be fragmented into small entities called snoRNA-derived RNAs (sdRNAs), which have been linked to multiple cancer types and thus may serve as next-generation prognostic or diagnostic biomarkers. In this paper, a multifunctional protein, FUS, was shown to be involved in the biogenesis of snoRNA-derived fragments. Furthermore, we combined bioinformatic analyses with complementary experimental approaches to elucidate the role of FUS-dependent sdRNAs in gene expression regulation. Our findings reveal the considerable regulatory potential of this new class of small noncoding RNAs.

molecular biology↗

bioRxiv2018

A SKI subcomplex specifically required for the degradation of ribosome-free RNA regions

The Ski2-Ski3-Ski8 (SKI) complex assists the RNA exosome during the 3-5 degradation of cytoplasmic transcripts. Previous reports showed that the SKI complex is involved in the 3-5 degradation of mRNA, including 3 untranslated regions (UTRs), devoid of ribosomes. Paradoxically, we recently showed that the SKI complex directly interacts with ribosomes during the co-translational mRNA decay and that this interaction is necessary for its RNA degradation promoting activity. Here, we characterized a new SKI-associated factor, Ska1, which antagonizes the SKI-ribosome interaction. We showed that the SKI-Ska1-subcomplex is specifically involved in the degradation of ribosome-free RNA regions such as long mRNA 3UTRs and cytoplasmic lncRNAs. We propose a model in which the SKI-exosome complex first targets ribosome-free RNA 3ends in its Ska1-associated form. When the complex reaches the mRNA coding sequence, the Ska1-SKI-exosome complex is exchanged for the SKI-exosome, which interacts directly with ribosomes in order to resume the degradation process.

molecular biology↗

bioRxiv2018

Comparative analysis of miR-155 tissue expression profiles of different breeds of chicken

miR-155 is an important microRNA which has multiple functions in many physiological and pathological processes. In this study, partial pri-miR-155 sequences were cloned from AA+ broiler, Sanhuang broiler and Hi-Line Brown layer, respectively. Stem-loop RT-qPCR was performed to detect the miR-155 spatiotemporal expression profiles of each chicken breed. The results showed that the partial pri-miR-155 sequences of different breeds of chicken were high conserved. The expression patterns of miR-155 between broiler and layer were basically similar, and miR-155 is expressed highly in immune related tissues. Interestingly, miR-155 expression activity had higher level in fat tissue of the three chicken breeds (14-day-old), but it decreased significantly in fat of the Hi-Line Brown layer (10-month-old and 24-month-old). In addition, the expression activities of miR-155 in 14-day-old broilers (AA+ broiler and Sanhuang broiler) were significantly lower than that of Hi-Line Brown layer (14-day-old) (P<0.05). Moreover, miR-155 expression activities in skeletal muscle of 14-day-old and 10-month-old Hi-Line Brown layer were also significantly lower than that of 24-month-old layer (P<0.05). The results indicated that miR-155 might be one of the important factors affecting the differences in skeletal muscle development and adipogenesis between different chicken breeds. These data can serve as a foundation for further study the functions and mechanisms of miR-155 in the physiological and pathological contexts.

molecular biology↗

bioRxiv2018

Identification of miRNAs and associated pathways regulated by Leukemia Inhibitory Factor in trophoblastic cell lines

IntroductionLeukemia Inhibitory Factor (LIF) regulates behavior of trophoblast cells and their interaction with immune and endothelial cells. In vitro, trophoblast cell response to LIF may vary depending on the cell model. Reported differences in the miRNA profile of trophoblastic cells may be responsible for these observations. Therefore, miRNA expression was investigated in four trophoblastic cell lines under LIF stimulation followed by in silico analysis of altered miRNAs and their associated pathways.\n\nMethodsLow density TaqMan miRNA assays were used to quantify levels of 762 mature miRNAs under LIF stimulation in three choriocarcinoma-derived (JEG-3, ACH-3P and AC1-M59) and a trophoblast immortalized (HTR-8/SVneo) cell lines. Expression of selected miRNAs was confirmed in primary trophoblast cells and cell lines by qPCR. Targets and associated pathways of the differentially expressed miRNAs were inferred from the miRTarBase followed by a KEGG Pathway Enrichment Analysis. HTR-8/SVneo and JEG-3 cells were transfected with miR-21-mimics and expression of miR-21 targets was assessed by qPCR.\n\nResultsA similar number of miRNAs changed in each tested cell line upon LIF stimulation, however, low coincidence of individual miRNA species was observed and occurred more often among choriocarcinoma-derived cells (complete data set at http://www.ncbi.nlm.nih.gov/geo/ under GEO accession number GSE130489). Altered miRNAs were categorized into pathways involved in human diseases, cellular processes and signal transduction. Six cascades were identified as significantly enriched, including JAK/STAT and TGFB-SMAD. Upregulation of miR-21-3p was validated in all cell lines and primary cells and STAT3 was confirmed as its target.\n\nDiscussionDissimilar miRNA responses may be involved in differences of LIF effects on trophoblastic cell lines.

molecular biology↗

bioRxiv2018

Dimerisation of APOBEC1 is dispensable for its RNA editing activity

The AID/APOBECs are DNA/RNA deaminases whose mutagenic activity has been linked to cancer. Among them, APOBEC1 physiologically partakes into a complex that edits a CAA codon into UAA Stop codon in the transcript of Apolipoprotein B (APOB), a protein crucial in the transport of lipids in the blood. Catalytically inactive mutants of APOBEC1 have a dominant negative effect on its activity, as they compete for the targeting of the APOB mRNA. Here we titrate APOBEC1-mediated editing in presence of catalytically inactive chimeras and mutants of APOBEC1, and we show that APOBEC1 inability to dimerise is the main determinant for its activity. This property is especially evident in an APOBEC1 mutant (L173A G227A) with increased activity on RNA despite decreased self-interaction. Moreover, dimerisation protects APOBEC1 from degradation and regulates its availability. Considering APOBEC1 capability to target DNA, we demonstrate that increased availability of the protein due to dimerisation leads to increase in the DNA damage induced by APOBEC1. These findings demonstrate that dimerisation, a property common to other APOBECs targeting DNA, might represent another layer in the regulation of this editing enzyme. BULLET POINTSO_LIAPOBEC1 inability to dimerise is the main determinant for its activity. C_LIO_LIDimerisation protects APOBEC1 from degradation and regulates its availability. C_LIO_LIAlterations in the balance between monomeric and dimeric APOBEC1 increase DNA damage. C_LI

molecular biology↗

bioRxiv2018

Molecular characterization of effector protein SAP54 in Bellis Virescence Phytoplasma (16SrIII-J)

Phytoplasmas are wall-less bacteria, with a parasitic life style responsible for numerous plant diseases worldwide. The genomic landscape of phytoplasmas has been scarcely studied. Nevertheless, since the introduction of next generation sequencing technologies, genome wide studies of these pathogens are flourishing and a handful of phytoplasmas genomes are available in public databases. In South America, phytoplasmas from 16SrIII group (X-disease) are the most widely distributed, and only a draft genome from a phytoplasmas 16SrIII-J infected periwinkle from Chile has been generated (Phytoplasma Vc33). Here, in grafting experiments we characterized the phenotypic signatures of an Argentinian daisy derived isolate of a 16SrIII-J phytoplasma (Bellis Virescence Phytoplasma) infecting periwinkle. Moreover, we applied a pipeline for genome wide annotation of the Vc33 genome and identified the effector protein SAP54. We then employed the obtained data to amplify, clone, sequence and characterize a SAP54 orthologue protein of Bellis Virescence Phytoplasma. Structural and phylogenetic analyses suggested that the identified SAP54 is highly conserved, and that its co-divergence among phytoplasma is not directly consistent with the evolutionary trajectories derived from rRNA analyses. The results gathered here could provide the basis for reverse genetics experiments using 16SrIII-J SAP54 proteins to assess their eventual influence in pathogenesis.

molecular biology↗

bioRxiv2018

The functional circular RNA, ciRS-7 (CDR1as), is biosynthesized using back-splicing promoted by inverted mammalian-wide MIRs but not primate-specific Alus

Circular RNAs (circRNAs) are stable noncoding RNAs with a closed circular structure. One of the first and best studied circRNAs is ciRS-7 (CDR1as) that acts as a regulator of the microRNA miR-7, however, the biosynthesis pathway has remained an enigma. Here we delineate the biosynthesis pathway of ciRS-7. The back-splicing events that form circRNAs are often facilitated by flanking inverted repeats of the primate-specific Alu elements. ciRS-7 gene lacks these elements but, instead, we identified a set of flanking inverted elements belonging to the mammalian-wide interspersed repeat (MIR) family. Splicing reporter assays in HEK293 cells demonstrated that these inverted MIRs are required to generate ciRS-7 through a back-splicing and CRISPR/Cas9-mediated deletions confirmed the requirement of the endogenous MIR elements in SH-SY5Y cells. Using bioinformatics searches, we identified several other MIR-dependent circRNAs that we confirmed experimentally. We propose that MIR-mediated RNA circularization constitutes a new widespread biosynthesis principle for mammalian circRNAs.

molecular biology↗

bioRxiv2018

Enhancing the cell-free expression of native membrane proteins by in-silico optimization of the coding sequence - an experimental study of the human voltage-dependent anion channel

The investigation of membrane proteins, key constituents of cells, is hampered by the difficulty and complexity of their in vitro synthesis, of unpredictable yield. Cell-free synthesis is herein employed to unravel the impact of the expression construct on gene transcription and translation, without the complex regulatory mechanisms of cellular systems. Through the systematic design of plasmids in the immediacy of the start of the target gene, it was possible to identify translation initiation and the conformation of mRNA as the main factors governing the cell-free expression efficiency of the human voltage dependent anion channel (VDAC), a relevant membrane protein in drug-based therapy. A simple translation initiation model was developed to quantitatively assess the expression potential for the designed constructs. A scoring function is proposed that quantifies the feasibility of formation of the translation initiation complex through the ribosome-mRNA hybridization energy and the accessibility of the mRNA segment binding to the ribosome. The scoring function enables to optimize plasmid sequences and semi-quantitatively predict protein expression efficiencies.

molecular biology↗

bioRxiv2018

Translation of upstream open reading frames in a model of neuronal differentiation

Upstream open reading frames (uORFs) initiate translation within mRNA 5 leaders, and have the potential to alter main coding sequence (CDS) translation on transcripts in which they reside. Ribosome profiling (RP) studies suggest that translating ribosomes are pervasive within 5 leaders across model systems. However, the significance of this observation remains unclear. To explore a role for uORF usage in neuronal differentiation, we performed RP on undifferentiated and differentiated human neuroblastoma cells. Using a spectral coherence algorithm (SPECtre), we identify 4,954 uORFs across 31% of all neuroblastoma transcripts. These uORFs predominantly utilize non-AUG initiation codons and exhibit translational efficiencies (TE) comparable to annotated coding regions. Usage of both AUG initiated uORFs and a conserved and consistently translated subset of non-AUG initiated uORFs correlates with repressed CDS translation. Ribosomal protein transcripts are enriched in uORFs, and select uORFs on such transcripts were validated for expression. With neuronal differentiation, we observed an overall positive correlation between translational shifts in uORF/CDS pairs. However, a subset of transcripts exhibit inverse shifts in translation of uORF/CDS pairs. These uORFs are enriched in AUG initiation sites, non-overlapping, and shorter in length.\n\nCumulatively, CDSs downstream of uORFs characterized by persistent translation show smaller shifts in TE with neuronal differentiation relative to CDSs without a predicted uORF, suggesting that fluctuations in CDS translation are buffered by uORF translation. In sum, this work provides insights into the dynamic relationships and potential regulatory functions of uORF/CDS pairs in a model of neuronal differentiation.

molecular biology↗