Quantifying alternative splicing from paired-end RNA-sequencing data

RNA-sequencing has revolutionized biomedical research and, in particular, our ability to study gene alternative splicing. The problem has important implications for human health, as alternative splicing may be involved in malfunctions at the cellular level and multiple diseases. However, the high-dimensional nature of the data and the existence of experimental biases pose serious data analysis challenges. We find that the standard data summaries used to study alternative splicing are severely limited, as they ignore a substantial amount of valuable information. Current data analysis methods are based on such summaries and are hence suboptimal. Further, they have limited flexibility in accounting for technical biases. We propose novel data summaries and a Bayesian modeling framework that overcome these limitations and determine biases in a nonparametric, highly flexible manner. These summaries adapt naturally to the rapid improvements in sequencing technology. We provide efficient point estimates and uncertainty assessments. The approach allows to study alternative splicing patterns for individual samples and can also be the basis for downstream analyses. We found a severalfold improvement in estimation mean square error compared popular approaches in simulations, and substantially higher consistency between replicates in experimental data. Our findings indicate the need for adjusting the routine summarization and analysis of alternative splicing RNA-seq studies. We provide a software implementation in the R package casper.Comment: Published in at http://dx.doi.org/10.1214/13-AOAS687 the Annals of Applied Statistics (http://www.imstat.org/aoas/) by the Institute of Mathematical Statistics (http://www.imstat.org). With correction

Evolving Towards the Hypercycle: A Spatial Model of Molecular Evolution

We extend earlier cellular automata models of spatially extended hypercycles by including an explicit genetic component into the model. This allows us to study the sequence evolution of hypercyclically coupled molecular replicators in addition to considering their population dynamics and spatial organization. In line with previous models, that considered either spatial organization or sequence evolution alone, we find both temporal oscillations of the relative concentration of the species forming the hypercycles as well as the formation of spatial organisations including spiral waves. We also confirm the greatly increased robustness of the spatially extended hypercycle against various classes of parasites. We find the sequence evolution of each of the hypercyclically coupled populations proceeds (after an inital selection-dominated phase) in a drift-like manner that can be described by a diffusion process in sequence space. Kimura's theory of neutral evolution is therefore applicable on long time-scales despite the fact that the hypercycle exhibits extreme periodic changes in population sizes and that are governed solely by frequency-dependent selection

Quantifying alternative splicing from paired-end RNA-sequencing data

RNA-sequencing has revolutionized biomedical research and, in particular, our ability to study gene alternative splicing. The problem has important implications for human health, as alternative splicing is involved in malfunctions at the cellular level and multiple diseases. However, the high-dimensional nature of the data and the existence of experimental biases pose serious data analysis challenges. We find that the standard data summaries used to study alternative splicing are severely limited, as they ignore a substantial amount of valuable information. Current data analysis methods are based on such summaries and are hence sub-optimal. Further, they have limited flexibility in accounting for technical biases. We propose novel data summaries and a Bayesian modeling framework that overcome these limitations and determine biases in a non-parametric, data-dependent manner. These summaries adapt naturally to the rapid improvements in sequencing technology. We provide efficient point estimates and uncertainty assessments. The approach allows to study alternative splicing patterns for individual samples and can also be the basis for downstream differential expression analysis. We found an over 5 fold improvement in estimation mean square error compared to a popular approach in simulations, and substantially higher correlations between replicates in experimental data. Our findings indicate the need for modifying the routine summarization and analysis of alternative splicing RNA-seq studies. We provide a software implementation in the R package casper

Neutral Networks of Interacting RNA Secondary Structures

RNA molecules interact by forming inter-molecular base pairs that compete with the intra-molecular base pairs of their secondary structures. Here we investigate the patterns of neutral mutations in RNAs whose function is the interaction with other RNAs, i.e., the co-folding with one or more other RNA molecules. We find that (1) the degree of neutrality is much smaller in interacting RNAs compared to RNAs that just have to coform to a single externally prescribed target structure, and (2) strengthening this contraint to the conservation of the co-folded structure with two or more partners essentially eliminates neutrality. It follows that RNAs whose function depends on the formation of a specific interaction complex with a target RNA molecule will evolve much more slowly than RNAs with a function depending only on their own structure

CelloS: a Multi-level Approach to Evolutionary Dynamics

We study the evolution of simple cells that are equipped with a genome, a rudimentary gene regulation network at transcription level and two classes of functional genes: motion effectors allow the cell to move in response to nutrient gradients while nutrient importers are required to actually feed from the environment. The model is inspired by the protist Naegleria gruberi which can switch between a feeding and dividing amoeboid state and a mobile agellate state depending on environmental conditions. Simulation results demonstrate how selection in a variable environment affects the gene number and effciency so that the cells can rapidly switch from one expression regime to the other depending on the external conditions

Genomic organization of eukaryotic tRNAs

BACKGROUND: Surprisingly little is known about the organization and distribution of tRNA genes and tRNA-related sequences on a genome-wide scale. While tRNA gene complements are usually reported in passing as part of genome annotation efforts, and peculiar features such as the tandem arrangements of tRNA gene in Entamoeba histolytica have been described in some detail, systematic comparative studies are rare and mostly restricted to bacteria. We therefore set out to survey the genomic arrangement of tRNA genes and pseudogenes in a wide range of eukaryotes to identify common patterns and taxon-specific peculiarities. RESULTS: In line with previous reports, we find that tRNA complements evolve rapidly and tRNA gene and pseudogene locations are subject to rapid turnover. At phylum level, the distributions of the number of tRNA genes and pseudogenes numbers are very broad, with standard deviations on the order of the mean. Even among closely related species we observe dramatic changes in local organization. For instance, 65% and 87% of the tRNA genes and pseudogenes are located in genomic clusters in zebrafish and stickleback, resp., while such arrangements are relatively rare in the other three sequenced teleost fish genomes. Among basal metazoa, Trichoplax adherens has hardly any duplicated tRNA gene, while the sea anemone Nematostella vectensis boasts more than 17000 tRNA genes and pseudogenes. Dramatic variations are observed even within the eutherian mammals. Higher primates, for instance, have 616 +/- 120 tRNA genes and pseudogenes of which 17% to 36% are arranged in clusters, while the genome of the bushbaby Otolemur garnetti has 45225 tRNA genes and pseudogenes of which only 5.6% appear in clusters. In contrast, the distribution is surprisingly uniform across plant genomes. Consistent with this variability, syntenic conservation of tRNA genes and pseudogenes is also poor in general, with turn-over rates comparable to those of unconstrained sequence elements. Despite this large variation in abundance in Eukarya we observe a significant correlation between the number of tRNA genes, tRNA pseudogenes, and genome size. CONCLUSIONS: The genomic organization of tRNA genes and pseudogenes shows complex lineage-specific patterns characterized by an extensive variability that is in striking contrast to the extreme levels of sequence-conservation of the tRNAs themselves. The comprehensive analysis of the genomic organization of tRNA genes and pseudogenes in Eukarya provides a basis for further studies into the interplay of tRNA gene arrangements and genome organization in general

Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture

GEMC1 and MCIDAS interactions with SWI/SNF complexes regulate the multiciliated cell-specific transcriptional program

Cell signalling; TranscriptionSeñalización celular; TranscripciónSenyalització cel·lular; TranscripcióMulticiliated cells (MCCs) project dozens to hundreds of motile cilia from their apical surface to promote the movement of fluids or gametes in the mammalian brain, airway or reproductive organs. Differentiation of MCCs requires the sequential action of the Geminin family transcriptional activators, GEMC1 and MCIDAS, that both interact with E2F4/5-DP1. How these factors activate transcription and the extent to which they play redundant functions remains poorly understood. Here, we demonstrate that the transcriptional targets and proximal proteomes of GEMC1 and MCIDAS are highly similar. However, we identified distinct interactions with SWI/SNF subcomplexes; GEMC1 interacts primarily with the ARID1A containing BAF complex while MCIDAS interacts primarily with BRD9 containing ncBAF complexes. Treatment with a BRD9 inhibitor impaired MCIDAS-mediated activation of several target genes and compromised the MCC differentiation program in multiple cell based models. Our data suggest that the differential engagement of distinct SWI/SNF subcomplexes by GEMC1 and MCIDAS is required for MCC-specific transcriptional regulation and mediated by their distinct C-terminal domains.We thank F. Guillemot, C. Lynch, M. Serrano, and S. Brody for antibodies, F. Supek for cells and reagents, A. Holland and C. Jewett for DEUP1 antibody and expansion microscopy suggestions, J. St-Germain for data analysis, J. Lüders for help with expansion microscopy, T. Dantas for sharing unpublished data and support from the IRB Functional Genomics and Biostatistics/Bioinformatics, Protein Expression and Mass Spectrometry Core Facilities. ML and BT were funded by Severo Ochoa FPI fellowships from the Ministry of Science, Innovation and Universities (MCIU), PK by an Advanced Postdoc Mobility fellowship from the Swiss National Science Foundation and the Kurt and Senta Herrmann Foundation and I.G.C by an AECC fellowship. THS was funded by the MCIU (PGC2018-095616-B-I00/GINDATA) and by the NIH Intramural Research Program, National Cancer Institute, Center for Cancer Research. X.S. was supported by MINECO (PID2019-110198RB-I00) and the European Research Council (CONCERT, contract number 648201). IRB Barcelona is the recipient of institutional funding from FEDER and the Centres of Excellence Severo Ochoa award to IRB Barcelona from MINECO (Government of Spain). MRM was funded by the National Heart, Lung and Blood Institute of the NIH (R01-HL128370). VC was funded by the Associazione Italiana per la Ricerca sul Cancro (AIRC), the European Research Council (ERC) grant 614541 and the GiovanniArmenise foundation career development award to VC. SR was funded by a Singapore National Medical Research Council (NMRC) Open Fund-Individual Research Grant (OFIRG19nov-0037). HZ was supported by National Cancer Institute (R01 CA220551)

DDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes

© 2015 Macmillan Publishers Limited. All rights reserved. dDsk2 is a conserved extraproteasomal ubiquitin receptor that targets ubiquitylated proteins for degradation. Here we report that dDsk2 plays a nonproteolytic function in transcription regulation. dDsk2 interacts with the dHP1c complex, localizes at promoters of developmental genes and is required for transcription. Through the ubiquitin-binding domain, dDsk2 interacts with H2Bub1, a modification that occurs at dHP1c complex-binding sites. H2Bub1 is not required for binding of the complex; however, dDsk2 depletion strongly reduces H2Bub1. Co-depletion of the H2Bub1 deubiquitylase dUbp8/Nonstop suppresses this reduction and rescues expression of target genes. RNA polymerase II is strongly paused at promoters of dHP1c complex target genes and dDsk2 depletion disrupts pausing. Altogether, these results suggest that dDsk2 prevents dUbp8/Nonstop-dependent H2Bub1 deubiquitylation at promoters of dHP1c complex target genes and regulates RNA polymerase II pausing. These results expand the catalogue of nonproteolytic functions of ubiquitin receptors to the epigenetic regulation of chromatin modifications.This work was supported by grants from MICINN (CSD2006-49, BFU2009-07111 and BFU2012-30724) and the Generalitat de Catalunya (SGR2009-1023 and SGR2014-204). This work was carried out within the framework of the ‘Centre de Referència en Biotecnologia’ of the ‘Generalitat de Catalunya’. R.K. acknowledges receipt of a ‘La Caixa’ PhD fellowshipPeer Reviewe

Differential distribution of the wlaN and cgtB genes, Associated with Guillain-Barré Syndrome, in Campylobacter jejuni isolates from humans, broiler chickens, and wild birds

Campylobacter jejuni causes campylobacteriosis, a bacterial gastroenteritis with high incidence worldwide. Moreover, C. jejuni infection can trigger the polyneuropathic disorder denominated Guillain-Barré syndrome (GBS). The C. jejuni strains that can elicit GBS carry either wlaN or cgtB, coding both genes for a β-1,3-galactosyltransferase enzyme that is required for the production of sialylated lipooligosaccharide (LOSSIAL). We described a differential prevalence of the genes wlaN and cgtB in C. jejuni isolates from three different ecological niches: humans, broiler chickens, and wild birds. The distribution of both genes, which is similar between broiler chicken and human isolates and distinct when compared to the wild bird isolates, suggests a host-dependent distribution. Moreover, the prevalence of the wlaN and cgtB genes seems to be restricted to some clonal complexes. Gene sequencing identified the presence of new variants of the G- homopolymeric tract within the wlaN gene. Furthermore, we detected two variants of a G rich region within the cgtB gene, suggesting that, similarly to wlaN, the G-tract in the cgtB gene mediates the phase variation control of cgtB expression. Caco-2 cell invasion assays indicate that there is no evident correlation between the production of LOSSIAL and the ability to invade eukaryotic cells.info:eu-repo/semantics/publishedVersio