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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2016

Nol12 is a multifunctional endonuclease at the nexus of RNA and DNA metabolism

Endo- and exonucleases are major contributors to RNA metabolism through their diverse roles in maturation and turnover of different species of RNA as well as transcription. Recent data suggests RNA nucleases also affect genome stability programs and act along DNA repair pathways. Here, we describe Nol12 as a multifunctional RNA/DNA endonuclease found in different subcellular compartments - the nucleoplasm, where it co-localizes with the RNA/DNA helicase Dhx9 and paraspeckles, nucleoli as well as GW/P-bodies. We show that Nol12 is required for a key step in ribosomal RNA processing, separating large and small subunit precursors at site 2, rerouting ribosome biogenesis via an alternative pathway in its absence to ensure ribosome production. Furthermore, loss of Nol12 results in increased oxidized DNA levels followed by a rapid p53-independent ATR-Chk1-mediated apoptotic response, suggesting a role for Nol12 in the prevention or resolution of oxidative DNA damage. Identification of a complex Nol12 interactome, which includes NONO, Dhx9 and DNA-PK, further supports its diverse functions in RNA metabolism and DNA maintenance, establishing Nol12 as a multifunctional endonuclease.

Molecular Biology↗

bioRxiv2016

Gene Transfer to the Outflow Tract

Elevated intraocular pressure is the primary cause of open angle glaucoma. Outflow resistance exists within the trabecular meshwork but also at the level of Schlemms canal and further downstream within the outflow system. Viral vectors allow to take advantage of naturally evolved, highly efficient mechanisms of gene transfer, a process that is termed transduction. They can be produced at biosafety level 2 in the lab using protocols that have evolved considerably over the last 15 to 20 years. Applied by an intracameral bolus, vectors follow conventional as well as uveoscleral outflow pathways. They may affect other structures in the anterior chamber depending on their transduction kinetics which can vary among species when using the same vector. Not all vectors can express long-term, a desirable feature to address the chronicity of glaucoma. Vectors that integrate into the genome of the target cell can achieve transgene function for the life of the transduced cell but are mutagenic by definition. The most prominent long-term expressing vector systems are based on lentiviruses that are derived from HIV, FIV, or EIAV. Safety considerations make non-primate lentiviral vector systems easier to work with as they are not derived from human pathogens. Non-integrating vectors are subject to degradation and attritional dilution during cell division. Lentiviral vectors have to integrate in order to express while adeno-associated viral vectors (AAV) often persist as intracellular concatemers but may also integrate. Adeno-and herpes viral vectors do not integrate and earlier generation systems might be relatively immunogenic. Nonviral methods of gene transfer are termed transfection with few restrictions of transgene size and type but often a much less efficient gene transfer that is also short-lived. Traditional gene transfer delivers exons while some vectors (lentiviral, herpes and adenoviral) allow transfer of entire genes that include introns. Recent insights have highlighted the role of non-coding RNA, most prominently, siRNA, miRNA and lncRNA. SiRNA is highly specific, miRNA is less specific, while lncRNA uses highly complex mechanisms that involve secondary structures and intergenic, intronic, overlapping, antisense, and bidirectional location. Several promising preclinical studies have targeted the RhoA or the prostaglandin pathway or modified the extracellular matrix. TGF-{beta} and glaucoma myocilin mutants have been transduced to elevate the intraocular pressure in glaucoma models. Cell based therapies have started to show first promise. Past approaches have focused on the trabecular meshwork and the inner wall of Schlemms canal while new strategies are concerned with modification of outflow tract elements that are downstream of the trabecular meshwork.

Molecular Biology↗

bioRxiv2016

Human mismatch repair system corrects errors produced during lagging strand replication more effectively

Mismatch repair (MMR) is one of the main systems maintaining fidelity of replication. Different effectiveness in correction of errors produced during replication of the leading and the lagging DNA strands was reported in yeast, but this effect is poorly studied in humans. Here, we use MMR-deficient (MSI) and MMR-proficient (MSS) cancer samples to investigate properties of the human MMR. MSI, but not MSS, cancers demonstrate unequal mutation rates between the leading and the lagging strands. The direction of strand asymmetry in MSI cancers matches that observed in cancers with mutated exonuclease domain of polymerase {delta}, indicating that polymerase {delta} contributes more mutations than its leading-strand counterpart, polymerase {varepsilon}. As polymerase {delta} primarily synthesizes DNA during the lagging strand replication, this implies that mutations produced in wild type cells during lagging strand replication are repaired by the MMR ~3 times more effectively, compared to those produced on the leading strand.

Molecular Biology↗

bioRxiv2016

Probabilistic estimation of short sequence expression using RNA-Seq data and the positional bootstrap

When estimating expression of a transcript or part of a transcript using RNA-seq data, it is commonly assumed that reads are generated uniformly from positions within the transcript. While this assumption is acceptable for long transcript sequences where reads from many positions are averaged, it frequently leads to large errors for short sequences, e.g., less than 100 bp. Analysis of short sequences, such as when studying splice junctions and microRNAs, is increasingly important and necessitates addressing errors in short-sequence expression estimation. Indeed, when we examined RNA-seq data from diverse studies, we found that large errors are introduced by variations in RNA-seq coverage due to sequence content, experimental conditions and sample preparation.\n\nWe developed a technique that we call the positional bootstrap, which quantifies the level of uncertainty in expression induced by non-uniform coverage. Unlike methods that attempt to correct for biases in coverage, but do so by making strong assumptions about the form of those biases, the positional bootstrap can quantify the noise induced by all types of bias, including unknown ones. Results obtained using independently generated RNA-seq datasets show that the positional bootstrap increases the accuracy of estimates of alternative splicing levels, tissue-differential alternative splicing and tissue differential expression, by a factor of up to 10.\n\nA Python implementation of the algorithm to quantify splicing levels is freely available from github.com/PSI-Lab/BENTO-Seq.

Molecular Biology↗

bioRxiv2016

Synthetic miR-16-5p does not act as a reverse transcriptase co-factor to enhance detection of small RNA

Failure to detect low-abundance microRNAs (miRNAs), for example, in circulating plasma, may occur for a variety of reasons, including presence of enzyme inhibitors. Recently, we received the unusual but intriguing suggestion that miR-16-5p acts as a co-factor of reverse transcriptases, facilitating more efficient reverse transcription of miRNAs and thus enhanced detection of low-abundance miRNAs. We tested this hypothesis by incubating reverse transcriptase with several concentrations of synthetic miR-16-5p and then performing stem-loop RT-qPCR with serial dilutions of miRNA osa-MIR168a. Our results do not support a role for miR-16 as a co-factor of reverse transcriptase.

Molecular Biology↗

bioRxiv2016

Multiplex gene editing by CRISPR-Cpf1 through autonomous processing of a single crRNA array

Microbial CRISPR-Cas defense systems have been adapted as a platform for genome editing applications built around the RNA-guided effector nucleases, such as Cas9. We recently reported the characterization of Cpf1, the effector nuclease of a novel type V-A CRISPR system, and demonstrated that it can be adapted for genome editing in mammalian cells (Zetsche et al., 2015). Unlike Cas9, which utilizes a trans-activating crRNA (tracrRNA) as well as the endogenous RNaseIII for maturation of its dual crRNA:tracrRNA guides (Deltcheva et al., 2011), guide processing of the Cpf1 system proceeds in the absence of tracrRNA or other Cas (CRISPR associated) genes (Zetsche et al., 2015) (Figure 1a), suggesting that Cpf1 is sufficient for pre-crRNA maturation. This has important implications for genome editing, as it would provide a simple route to multiplex targeting. Here, we show for two Cpf1 orthologs that no other factors are required for array processing and demonstrate multiplex gene editing in mammalian cells as well as in the mouse brain by using a designed single CRISPR array.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC=\"FIGDIR/small/049122_fig1.gif\" ALT=\"Figure 1\">\nView larger version (35K):\norg.highwire.dtl.DTLVardef@1f0c15corg.highwire.dtl.DTLVardef@126a6fdorg.highwire.dtl.DTLVardef@9d6a2eorg.highwire.dtl.DTLVardef@a61f75_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1C_FLOATNO Cpf1 mediates processing of pre-crRNA. (a) Schematic of pre-crRNA processing for Cas9 and Cpf1. Cleavage sites indicated with red triangle. (b) In vitro processing of FnCpf1 pre-crRNA transcript (80 nM) with purified AsCpf1 or LbCpf1 protein ([~]320 nM). In the presence of Cpf1 nuclease the pre-crRNA was cleaved in a distinct pattern, indicating cleavage at similar sequence motifs. RNA molecules without Cpf1 DR features where not cleaved by Cpf1 (control RNA). (c) RNAseq analysis of FnCpf1 pre-crRNA cleavage products, as shown in (b). A high fraction of sequence reads smaller than 65nt are cleavage products of spacers flanked by DR sequences.\n\nC_FIG

Molecular Biology↗

bioRxiv2016

Direct binding to RPA-coated ssDNA allows recruitment of the ATR activator TopBP1 to sites of DNA damage

A critical event for the ability of cells to tolerate DNA damage and replication stress is activation of the ATR kinase. ATR activation is dependent on the BRCT repeat-containing protein TopBP1. Previous work has shown that recruitment of TopBP1 to sites of DNA damage and stalled replication forks is necessary for downstream events in ATR activation, however the mechanism for this recruitment was not known. Here, we use protein binding assays and functional studies in Xenopus egg extracts to show that TopBP1 makes a direct interaction, via its BRCT2 domain, with RPA-coated ssDNA. We identify a point mutant that abrogates this interaction, and show that this mutant fails to accumulate at sites of DNA damage, and that the mutant cannot activate ATR. These data thus supply a mechanism for how the critical ATR activator, TopBP1, senses DNA damage and stalled replication forks to initiate assembly of checkpoint signaling complexes.

Molecular Biology↗

bioRxiv2016

Shh and ZRS enhancer co-localisation is specific to the zone of polarizing activity

Limb-specific Shh expression is regulated by the (~1 Mb distant) ZRS enhancer. In the mouse, limb bud restricted spatiotemporal expression of Shh occurs from ~E10-E11.5 at the distal posterior margin and is essential for correct autopod formation. Here, we have analysed the higher-order chromatin conformation of Shh in expressing and non-expressing tissues, both by fluorescence in situ hybridisation (FISH) and by chromosome conformation capture (5C). Conventional and super-resolution light microscopy identified significantly elevated frequencies of Shh/ZRS co-localisation only in the Shh expressing regions of the limb bud. However, Shh-ZRS spatial distances were consistently shorter than intervening distances to a neural enhancer in all tissues and developmental stages analysed. 5C identified a topologically associating domain (TAD) over the Shh/ZRS genomic region and enriched interactions between Shh and ZRS throughout E11.5 embryos. Shh/ZRS co-localisation, therefore, correlates with the spatiotemporal domain of limb bud-specific Shh expression, but close Shh/ZRS proximity in the nucleus occurs regardless of whether the gene or enhancer is active. We suggest that this constrained chromatin configuration optimises the opportunity for the active enhancer to locate and instigate Shh expression.

Molecular Biology↗

bioRxiv2016

Neighborhood regulation by lncRNA promoters, transcription, and splicing

Mammalian genomes are pervasively transcribed to produce thousands of spliced long noncoding RNAs (lncRNAs), whose functions remain poorly understood. Because recent evidence has implicated several specific lncRNA loci in the local regulation of gene expression, we sought to determine whether such local regulation is a property of many lncRNA loci. We used genetic manipulations to dissect 12 genomic loci that produce lncRNAs and found that 5 of these loci influence the expression of a neighboring gene in cis. Surprisingly, however, none of these effects required the specific lncRNA transcripts themselves and instead involved general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Interestingly, such effects are not limited to lncRNA loci: we found similar effects on local gene expression at 4 of 6 protein-coding loci. These results demonstrate that crosstalk among neighboring genes is a prevalent phenomenon that can involve multiple mechanisms and cis regulatory signals, including a novel role for RNA splicing. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs.

Molecular Biology↗

bioRxiv2016

Characterization of a putative NsrR homologue in Streptomyces venezuelae reveals a new member of the Rrf2 superfamily

Members of the Rrf2 superfamily of transcription factors are widespread in bacteria but their functions are largely unexplored. The few that have been characterized in detail sense nitric oxide (NsrR), iron limitation (RirA), cysteine availability (CymR) and the iron sulfur (Fe-S) cluster status of the cell (IscR). In this study we combined ChIP- and dRNA-seq with in vitro biochemistry to characterize a putative NsrR homologue in Streptomyces venezuelae. ChIP-seq analysis revealed that rather than regulating the nitrosative stress response like Streptomyces coelicolor NsrR, Sven6563 binds to a conserved motif at a different, much larger set of genes with a diverse range of functions, including a number of regulators, genes required for glutamine synthesis, NADH/NAD(P)H metabolism, as well as general DNA/RNA and amino acid/protein turn over. Our biochemical experiments further show that Sven6563 has a [2Fe-2S] cluster and that the switch between oxidized and reduced cluster controls its DNA binding activity in vitro. To our knowledge, both the sensing domain and the putative target genes are novel for an Rrf2 protein, suggesting Sven6563 represents a new member of the Rrf2 superfamily. Given the redox sensitivity of its Fe-S cluster we have tentatively named the protein RsrR for Redox sensitive response Regulator.

Molecular Biology↗

bioRxiv2016

Genomic positional conservation identifies topological anchor point (tap)RNAs linked to developmental loci

The mammalian genome is transcribed into large numbers of long noncoding RNAs (lncRNAs), but the definition of functional lncRNA groups has proven difficult, partly due to their low sequence conservation and lack of identified shared properties. Here we consider positional conservation across mammalian genomes as an indicator of functional commonality. We identify 665 conserved lncRNA promoters in mouse and human genomes that are preserved in genomic position relative to orthologous coding genes. The identified positionally conserved lncRNA genes are primarily associated with developmental transcription factor loci with which they are co-expressed in a tissue-specific manner. Strikingly, over half of all positionally conserved RNAs in this set are linked to distinct chromatin organization structures, overlapping the binding sites for the CTCF chromatin organizer and located at chromatin loop anchor points and borders of topologically associating domains (TADs). These topological anchor point (tap)RNAs possess conserved sequence domains that are enriched in potential recognition motifs for Zinc Finger proteins. Characterization of these noncoding RNAs and their associated coding genes shows that they are functionally connected: they regulate each others expression and influence the metastatic phenotype of cancer cells in vitro in a similar fashion. Thus, interrogation of positionally conserved lncRNAs identifies a new subset of tapRNAs with shared functional properties. These results provide a large dataset of lncRNAs that conform to the \"extended gene\" model, in which conserved developmental genes are genomically and functionally linked to regulatory lncRNA loci across mammalian evolution.

Molecular Biology↗

bioRxiv2016

Evolutionary dynamics of abundant stop codon readthrough in Anopheles and Drosophila

Translational stop codon readthrough was virtually unknown in eukaryotic genomes until recent developments in comparative genomics and new experimental techniques revealed evidence of readthrough in hundreds of fly genes and several human, worm, and yeast genes. Here, we use the genomes of 21 species of Anopheles mosquitoes and improved comparative techniques to identify evolutionary signatures of conserved, functional readthrough of 353 stop codons in the malaria vector, Anopheles gambiae, and 51 additional Drosophila melanogaster stop codons, with several cases of double and triple readthrough including readthrough of two adjacent stop codons, supporting our earlier prediction of abundant readthrough in pancrustacea genomes. Comparisons between Anopheles and Drosophila allow us to transcend the static picture provided by single-clade analysis to explore the evolutionary dynamics of abundant readthrough. We find that most differences between the readthrough repertoires of the two species are due to readthrough gain or loss in existing genes, rather than to birth of new genes or to gene death; that RNA structures are sometimes gained or lost while readthrough persists; and that readthrough is more likely to be lost at TAA and TAG stop codons. We also determine which characteristic properties of readthrough predate readthrough and which are clade-specific. We estimate that there are more than 600 functional readthrough stop codons in A. gambiae and 900 in D. melanogaster. We find evidence that readthrough is used to regulate peroxisomal targeting in two genes. Finally, we use the sequenced centipede genome to refine the phylogenetic extent of abundant readthrough.

Molecular Biology↗

bioRxiv2016

A hyperactive transcriptional state marks genome reactivation at the mitosis-G1 transition

During mitosis, RNA polymerase II (Pol II) and many transcription factors dissociate from chromatin, and transcription ceases globally. Transcription is known to restart in bulk by telophase, but whether de novo transcription at the mitosis-G1 transition is in any way distinct from later in interphase remains unknown. We tracked Pol II occupancy genome-wide in mammalian cells progressing from mitosis through late G1. Unexpectedly, during the earliest rounds of transcription at the mitosis-G1 transition, ~50% of active genes and distal enhancers exhibit a spike in transcription, exceeding levels observed later in G1 phase. Enhancer-promoter chromatin contacts are depleted during mitosis and restored rapidly upon G1 entry, but do not spike. Of the chromatin-associated features examined, histone H3 lysine 27 acetylation levels at individual loci in mitosis best predict the mitosis-G1 transcriptional spike. Single-molecule RNA imaging supports that the mitosis-G1 transcriptional spike can constitute the maximum transcriptional activity per DNA copy throughout the cell division cycle. The transcriptional spike occurs heterogeneously and propagates to cell-to-cell differences in mature mRNA expression. Our results raise the possibility that passage through the mitosis-G1 transition might predispose cells to diverge in gene expression states.

Molecular Biology↗

bioRxiv2016

APOBEC3A/B-induced mutagenesis is responsible for 20% of heritable mutations in the TpCpW context

APOBEC3A/B cytidine deaminase is responsible for the majority of cancerous mutations in a large fraction of cancer samples. However, its role in heritable mutagenesis remains very poorly understood. Recent studies have demonstrated that both in yeast and in human cancerous cells, most of APOBEC3A/B-induced mutations occur on the lagging strand during replication. Here, we use data on rare human polymorphisms, interspecies divergence, and de novo mutations to study germline mutagenesis, and analyze mutations at nucleotide contexts prone to attack by APOBEC3A/B. We show that such mutations occur preferentially on the lagging strand. Moreover, we demonstrate that APOBEC3A/B-like mutations tend to produce strand-coordinated clusters, which are also biased towards the lagging strand. Finally, we show that the mutation rate is increased 3 of C[->]G mutations to a greater extent than 3 of C[->]T mutations, suggesting pervasive translesion bypass of the APOBEC3A/B-induced damage. Our study demonstrates that 20% of C[->]T and C[->]G mutations segregating as polymorphisms in human population are attributable to APOBEC3A/B activity.

Molecular Biology↗

bioRxiv2016

Cryptic diversity and population structure at small scales: The freshwater snail Ancylus (Planorbidae, Pulmonata) in the Montseny mountain range

Anthropogenic impacts like intensified land use and climate change are severe threats to freshwater biodiversity and effective biodiversity monitoring is therefore one of the most urgent tasks. This is however often hampered by the lack of knowledge regarding the number and ecology of species. Molecular tools have shown many freshwater taxa to comprise morphologically cryptic species, which often occur in sympatry on a small geographic scale. Here, we studied the freshwater snail Ancylus fluviatilis (MUELLER, 1774) species complex in the Iberian Montseny Mountains. We hypothesised 1) that several species of A. fluviatilis sensu lato occur in the Montseny, 2) that different Ancylus species seldom co-occur in syntopy due to different ecological demands or interspecific competition, and 3) that species show a pattern of strong population structure within streams or catchments due to ecological preferences or local adaptation. We barcoded 180 specimens from 36 sites in the Montseny for the cytochrome c oxidase subunit I (COI) barcoding gene and molecularly identified two Ancylus species. These species seldom occurred in syntopy and a species distribution modelling approach showed differing bioclimatic preferences of the species. One species mainly occurs in cooler, higher altitude streams while the second species occurs in lower-altitude areas with higher temperatures. Tests of population structure showed that both species possibly do not disperse well in the study area and that populations within species are likely adapted to certain bioclimatic conditions in different regions of the Montseny. Our results highlight the need to incorporate molecular techniques into routine monitoring programmes.

Molecular Biology↗

bioRxiv2016

C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Caseffector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage.In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.

Molecular Biology↗

bioRxiv2016

Evolution of local mutation rate and its determinants

Mutation rate varies along the human genome, and part of this variation is explainable by measurable local properties of the DNA molecule. Moreover, mutation rates differ between orthologous genomic regions of different species, but the drivers of this change are unclear. Here, we compare the local mutation rates of several species. We show that these rates are very similar between human and apes, implying that their variation has a strong underlying cryptic component not explainable by the known genomic features. Mutation rates become progressively less similar in more distant species, and these changes are partially explainable by changes in the local genomic features of orthologous regions, most importantly, in the recombination rate. However, they are much more rapid, implying that the cryptic component underlying the mutation rate is more ephemeral than the known genomic features. These findings shed light on the determinants of mutation rate evolution.

Molecular Biology↗

bioRxiv2016

Prediction of allosteric sites and mediating interactions through bond-to-bond propensities

Allosteric regulation is central to many biochemical processes. Allosteric sites provide a target to fine-tune protein activity, yet we lack computational methods to predict them. Here, we present an efficient graph-theoretical approach for identifying allosteric sites and the mediating interactions that connect them to the active site. Using an atomistic graph with edges weighted by covalent and non-covalent bond energies, we obtain a bond-to-bond propensity that quantifies the effect of instantaneous bond fluctuations propagating through the protein. We use this propensity to detect the sites and communication pathways most strongly linked to the active site, assessing their significance through quantile regression and comparison against a reference set of 100 generic proteins. We exemplify our method in detail with three well-studied allosteric proteins: caspase-1, CheY, and h-Ras, correctly predicting the location of the allosteric site and identifying key allosteric interactions. Consistent prediction of allosteric sites is then attained in a further set of 17 proteins known to exhibit allostery. Because our propensity measure runs in almost linear time, it offers a scalable approach to high-throughput searches for candidate allosteric sites.

Molecular Biology↗