MicroExonator enables systematic discovery and quantification of microexons across mouse embryonic development

AbstractMicroexons, exons that are ≤30 nucleotides, were shown to play key roles in neuronal development, but are difficult to detect and quantify using standard RNA-Seq alignment tools. Here, we present MicroExonator, a novel pipeline for reproducible de novo discovery and quantification of microexons. We processed 289 RNA-seq datasets from eighteen mouse tissues corresponding to nine embryonic and postnatal stages, providing the most comprehensive survey of microexons available for mouse. We detected 2,984 microexons, 332 of which are differentially spliced throughout mouse embryonic brain development, including 29 that are not present in mouse transcript annotation databases. Unsupervised clustering of microexons alone segregates brain tissues by developmental time and further analysis suggest a key function for microexon inclusion in axon growth and synapse formation. Finally, we analysed single-cell RNA-seq data from the mouse visual cortex and we report differential inclusion between neuronal subpopulations, suggesting that some microexons could be cell-type specific.

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MicroExonator enables systematic discovery and quantification of microexons across mouse embryonic development

Genome Biology ◽

10.1186/s13059-020-02246-2 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Guillermo E. Parada ◽

Roberto Munita ◽

Ilias Georgakopoulos-Soares ◽

Hugo J. R. Fernandes ◽

Veronika R. Kedlian ◽

...

Keyword(s):

Single Cell ◽

Synapse Formation ◽

De Novo ◽

System Development ◽

Axon Growth ◽

Nervous System Development ◽

Rna Seq ◽

Mouse Development ◽

Mouse Tissues ◽

The Impact

Abstract Background Microexons, exons that are ≤ 30 nucleotides, are a highly conserved and dynamically regulated set of cassette exons. They have key roles in nervous system development and function, as evidenced by recent results demonstrating the impact of microexons on behaviour and cognition. However, microexons are often overlooked due to the difficulty of detecting them using standard RNA-seq aligners. Results Here, we present MicroExonator, a novel pipeline for reproducible de novo discovery and quantification of microexons. We process 289 RNA-seq datasets from eighteen mouse tissues corresponding to nine embryonic and postnatal stages, providing the most comprehensive survey of microexons available for mice. We detect 2984 microexons, 332 of which are differentially spliced throughout mouse embryonic brain development, including 29 that are not present in mouse transcript annotation databases. Unsupervised clustering of microexons based on their inclusion patterns segregates brain tissues by developmental time, and further analysis suggests a key function for microexons in axon growth and synapse formation. Finally, we analyse single-cell RNA-seq data from the mouse visual cortex, and for the first time, we report differential inclusion between neuronal subpopulations, suggesting that some microexons could be cell type-specific. Conclusions MicroExonator facilitates the investigation of microexons in transcriptome studies, particularly when analysing large volumes of data. As a proof of principle, we use MicroExonator to analyse a large collection of both mouse bulk and single-cell RNA-seq datasets. The analyses enabled the discovery of previously uncharacterized microexons, and our study provides a comprehensive microexon inclusion catalogue during mouse development.

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Functional characterization of Neurofilament Light b splicing and misbalance in zebrafish

10.1101/2020.04.27.064063 ◽

2020 ◽

Author(s):

DL Demy ◽

ML Campanari ◽

R Munoz-Ruiz ◽

HD Durham ◽

BJ Gentil ◽

...

Keyword(s):

Motor Neurons ◽

Neuronal Development ◽

Rna Binding ◽

De Novo ◽

Functional Characterization ◽

Axon Growth ◽

Motor Deficits ◽

Neurofilament Light ◽

Cytoplasmic Inclusions

AbstractNeurofilaments (NFs), a major cytoskeletal component of motor neurons, play a key role in their differentiation, establishment and maintenance of their morphology and mechanical strength. The de novo assembly of these neuronal intermediate filaments requires the presence of the neurofilament light subunit, NEFL, which expression is reduced in motor neurons in Amyotrophic Lateral Sclerosis (ALS). This study used zebrafish as a model to characterize the NEFL homologue neflb, which encodes two different isoforms via splicing of the primary transcript (neflbE4 and neflbE3). In vivo imaging showed that neflb is crucial for proper neuronal development, and that disrupting the balance between its two isoforms specifically affects NF assembly and motor axon growth, with resulting motor deficits. This equilibrium is also disrupted upon partial depletion of TDP-43, a RNA binding protein that is mislocalized into cytoplasmic inclusions in ALS. The study supports interaction of NEFL expression and splicing with TDP-43 in a common pathway, both biologically and pathogenetically.

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Single-particle fluorescence tracking combined with TrackMate assay reveals highly heterogeneous and discontinuous lysosomal transport in freely orientated axons

10.22541/au.164219078.86486130/v1 ◽

2022 ◽

Author(s):

Yongyang Liu ◽

Yaxin Lu ◽

Zhiyong Tang ◽

Yuheng Cao ◽

Dehua Huang ◽

...

Keyword(s):

Axonal Transport ◽

Single Particle ◽

Neuronal Development ◽

Synapse Formation ◽

Transport Model ◽

Gene Transfection ◽

Axon Growth ◽

Physical Factors ◽

Size Of Particles ◽

Number Of Bends

Axonal transport plays a significant role in the establishment of neuronal polarity, axon growth, and synapse formation during neuronal development. The axon of a naturally growing neuron is a highly complex and multifurcated structure with a large number of bends and branches. Nowadays, the study of dynamic axonal transport in morphologically complex neurons is greatly limited by the technological barrier. Here, a sparse gene transfection strategy was developed to locate fluorescent mCherry in the lysosome of primary neurons, thus enabling us to track the lysosome-based axonal transport with a single-particle resolution. Thereby, several axonal transport models were observed, including forward or backward transport model, stop-and-go model, repeated back-and-forth transport model, and cross-branch transport model. Then, the accurate single-particle velocity quantification by TrackMate revealed a highly heterogeneous and discontinuous transportation process of lysosome-based axonal transport in freely orientated axons. And, multiple physical factors, such as the axonal structure and the size of particles, were disclosed to affect the velocity of particle transporting in freely orientated axons. The combined single-particle fluorescence tracking and TrackMate assay can be served as a facile tool for evaluating axonal transport in neuronal development and axonal transport-related diseases.

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nanotatoR: a tool for enhanced annotation of genomic structural variants

BMC Genomics ◽

10.1186/s12864-020-07182-w ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Surajit Bhattacharya ◽

Hayk Barseghyan ◽

Emmanuèle C. Délot ◽

Eric Vilain

Keyword(s):

De Novo ◽

Genome Mapping ◽

Gene List ◽

Sufficient Information ◽

Rna Seq ◽

Structural Variants ◽

De Novo Genome Assembly ◽

Pathogenic Variants ◽

Increased Sensitivity

Abstract Background Whole genome sequencing is effective at identification of small variants, but because it is based on short reads, assessment of structural variants (SVs) is limited. The advent of Optical Genome Mapping (OGM), which utilizes long fluorescently labeled DNA molecules for de novo genome assembly and SV calling, has allowed for increased sensitivity and specificity in SV detection. However, compared to small variant annotation tools, OGM-based SV annotation software has seen little development, and currently available SV annotation tools do not provide sufficient information for determination of variant pathogenicity. Results We developed an R-based package, nanotatoR, which provides comprehensive annotation as a tool for SV classification. nanotatoR uses both external (DGV; DECIPHER; Bionano Genomics BNDB) and internal (user-defined) databases to estimate SV frequency. Human genome reference GRCh37/38-based BED files are used to annotate SVs with overlapping, upstream, and downstream genes. Overlap percentages and distances for nearest genes are calculated and can be used for filtration. A primary gene list is extracted from public databases based on the patient’s phenotype and used to filter genes overlapping SVs, providing the analyst with an easy way to prioritize variants. If available, expression of overlapping or nearby genes of interest is extracted (e.g. from an RNA-Seq dataset, allowing the user to assess the effects of SVs on the transcriptome). Most quality-control filtration parameters are customizable by the user. The output is given in an Excel file format, subdivided into multiple sheets based on SV type and inheritance pattern (INDELs, inversions, translocations, de novo, etc.). nanotatoR passed all quality and run time criteria of Bioconductor, where it was accepted in the April 2019 release. We evaluated nanotatoR’s annotation capabilities using publicly available reference datasets: the singleton sample NA12878, mapped with two types of enzyme labeling, and the NA24143 trio. nanotatoR was also able to accurately filter the known pathogenic variants in a cohort of patients with Duchenne Muscular Dystrophy for which we had previously demonstrated the diagnostic ability of OGM. Conclusions The extensive annotation enables users to rapidly identify potential pathogenic SVs, a critical step toward use of OGM in the clinical setting.

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Identification and Expression Analysis of the Genes Involved in the Raffinose Family Oligosaccharides Pathway of Phaseolus vulgaris and Glycine max

Plants ◽

10.3390/plants10071465 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1465

Author(s):

Ramon de Koning ◽

Raphaël Kiekens ◽

Mary Esther Muyoka Toili ◽

Geert Angenon

Keyword(s):

Common Bean ◽

Seed Development ◽

Expression Analysis ◽

De Novo ◽

Expression Patterns ◽

Gene Families ◽

Rna Seq ◽

Raffinose Family Oligosaccharides ◽

Specific Expression ◽

Raffinose Synthase

Raffinose family oligosaccharides (RFO) play an important role in plants but are also considered to be antinutritional factors. A profound understanding of the galactinol and RFO biosynthetic gene families and the expression patterns of the individual genes is a prerequisite for the sustainable reduction of the RFO content in the seeds, without compromising normal plant development and functioning. In this paper, an overview of the annotation and genetic structure of all galactinol- and RFO biosynthesis genes is given for soybean and common bean. In common bean, three galactinol synthase genes, two raffinose synthase genes and one stachyose synthase gene were identified for the first time. To discover the expression patterns of these genes in different tissues, two expression atlases have been created through re-analysis of publicly available RNA-seq data. De novo expression analysis through an RNA-seq study during seed development of three varieties of common bean gave more insight into the expression patterns of these genes during the seed development. The results of the expression analysis suggest that different classes of galactinol- and RFO synthase genes have tissue-specific expression patterns in soybean and common bean. With the obtained knowledge, important galactinol- and RFO synthase genes that specifically play a key role in the accumulation of RFOs in the seeds are identified. These candidate genes may play a pivotal role in reducing the RFO content in the seeds of important legumes which could improve the nutritional quality of these beans and would solve the discomforts associated with their consumption.

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RNA-Seq reveals divergent gene expression between larvae with contrasting trophic modes in the poecilogonous polychaete Boccardia wellingtonensis

Scientific Reports ◽

10.1038/s41598-021-94646-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Álvaro Figueroa ◽

Antonio Brante ◽

Leyla Cárdenas

Keyword(s):

De Novo ◽

Juvenile Stage ◽

Type I ◽

Rna Seq ◽

Mrna Synthesis ◽

De Novo Transcriptome ◽

Larval Stages ◽

Divergent Gene ◽

Genetic Mechanisms ◽

Planktotrophic Larvae

AbstractThe polychaete Boccardia wellingtonensis is a poecilogonous species that produces different larval types. Females may lay Type I capsules, in which only planktotrophic larvae are present, or Type III capsules that contain planktotrophic and adelphophagic larvae as well as nurse eggs. While planktotrophic larvae do not feed during encapsulation, adelphophagic larvae develop by feeding on nurse eggs and on other larvae inside the capsules and hatch at the juvenile stage. Previous works have not found differences in the morphology between the two larval types; thus, the factors explaining contrasting feeding abilities in larvae of this species are still unknown. In this paper, we use a transcriptomic approach to study the cellular and genetic mechanisms underlying the different larval trophic modes of B. wellingtonensis. By using approximately 624 million high-quality reads, we assemble the de novo transcriptome with 133,314 contigs, coding 32,390 putative proteins. We identify 5221 genes that are up-regulated in larval stages compared to their expression in adult individuals. The genetic expression profile differed between larval trophic modes, with genes involved in lipid metabolism and chaetogenesis over expressed in planktotrophic larvae. In contrast, up-regulated genes in adelphophagic larvae were associated with DNA replication and mRNA synthesis.

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Multiple Alu exonization in 3’UTR of a primate specific isoform of CYP20A1 creates a potential miRNA sponge

Genome Biology and Evolution ◽

10.1093/gbe/evaa233 ◽

2020 ◽

Author(s):

Aniket Bhattacharya ◽

Vineet Jha ◽

Khushboo Singhal ◽

Mahar Fatima ◽

Dayanidhi Singh ◽

...

Keyword(s):

Heat Shock ◽

Cortical Neurons ◽

Regulatory Networks ◽

Large Scale ◽

Neuronal Development ◽

Random Sets ◽

Rna Seq ◽

Orphan Gene ◽

Mirna Sponge ◽

Human Neurons

Abstract Alu repeats contribute to phylogenetic novelties in conserved regulatory networks in primates. Our study highlights how exonized Alus could nucleate large-scale mRNA-miRNA interactions. Using a functional genomics approach, we characterize a transcript isoform of an orphan gene, CYP20A1 (CYP20A1_Alu-LT) that has exonization of 23 Alus in its 3’UTR. CYP20A1_Alu-LT, confirmed by 3’RACE, is an outlier in length (9 kb 3’UTR) and widely expressed. Using publically available datasets, we demonstrate its expression in higher primates and presence in single nucleus RNA-seq of 15928 human cortical neurons. miRanda predicts ∼4700 miRNA recognition elements (MREs) for ∼1000 miRNAs, primarily originated within these 3’UTR-Alus. CYP20A1_Alu-LT could be a potential multi-miRNA sponge as it harbors ≥10 MREs for 140 miRNAs and has cytosolic localization. We further tested whether expression of CYP20A1_Alu-LT correlates with mRNAs harboring similar MRE targets. RNA-seq with conjoint miRNA-seq analysis was done in primary human neurons where we observed CYP20A1_Alu-LT to be downregulated during heat shock response and upregulated in HIV1-Tat treatment. 380 genes were positively correlated with its expression (significantly downregulated in heat shock and upregulated in Tat) and they harbored MREs for nine expressed miRNAs which were also enriched in CYP20A1_Alu-LT. MREs were significantly enriched in these 380 genes compared to random sets of differentially expressed genes (p = 8.134e-12). Gene ontology suggested involvement of these genes in neuronal development and hemostasis pathways thus proposing a novel component of Alu-miRNA mediated transcriptional modulation that could govern specific physiological outcomes in higher primates.

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