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

Parada, Guillermo E.; Munita, Roberto; Georgakopoulos-Soares, Ilias; Fernandez, Hugo F.; Metzakopian, Emmanouil; Andrés, Marı́a Estela; Miska, Eric A.; Hemberg, Martin

doi:10.1101/2020.02.12.945683

Cited by 4 publications

(7 citation statements)

References 80 publications

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“…5a). To quantify AS patterns, we used Whippet, a computationally lightweight and accurate quantification method, previously integrated in computational workflows dedicated to process scRNA-seq data 35,36 . Because splicing node coverage is limited at the single-cell level (Extended Data Fig.…”

Section: Comprehensive Profiling Of As Across Mouse Gastrulation and ...mentioning

confidence: 99%

High-throughput total RNA sequencing in single cells using VASA-seq

et al. 2022

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Most methods for single-cell transcriptome sequencing amplify the termini of polyadenylated transcripts, capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts and hinders alternative splicing analysis. We, therefore, developed VASA-seq to detect the total transcriptome in single cells, which is enabled by fragmenting and tailing all RNA molecules subsequent to cell lysis. The method is compatible with both plate-based formats and droplet microfluidics. We applied VASA-seq to more than 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. Analyzing the dynamics of the total single-cell transcriptome, we discovered cell type markers, many based on non-coding RNA, and performed in vivo cell cycle analysis via detection of non-polyadenylated histone genes. RNA velocity characterization was improved, accurately retracing blood maturation trajectories. Moreover, our VASA-seq data provide a comprehensive analysis of alternative splicing during mammalian development, which highlighted substantial rearrangements during blood development and heart morphogenesis.

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Section: Comprehensive Profiling Of As Across Mouse Gastrulation and ...mentioning

confidence: 99%

High-throughput total RNA sequencing in single cells using VASA-seq

et al. 2022

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“…Every splicing node is associated with different types of AS, alternative transcriptional start sites or alternative polyadenylation events and their inclusion rates are calculated as percent-spliced-in (ψ) values, which is quantified by taking the ratio of reads that support the inclusion of a given splicing node (Figure 6A). To quantify AS patterns, we used Whippet, a computationally light-weight and accurate quantification method, previously integrated in computational workflows dedicated to process scRNA-seq data (Parada et al, 2021;Sterne-Weiler et al, 2018). Since splicing node coverage is limited at the single-cell level (Figure S4A), we implemented a pseudo-bulk pooling approach, developed as part of MicroExonator (Parada et al, 2021), where reads from the same cell type are pooled in silico before splicing node quantification.…”

Section: Comprehensive Profiling Of Alternative Splicing Across Mouse Gastrulation and Early Organogenesismentioning

confidence: 99%

“…To quantify AS patterns, we used Whippet, a computationally light-weight and accurate quantification method, previously integrated in computational workflows dedicated to process scRNA-seq data (Parada et al, 2021;Sterne-Weiler et al, 2018). Since splicing node coverage is limited at the single-cell level (Figure S4A), we implemented a pseudo-bulk pooling approach, developed as part of MicroExonator (Parada et al, 2021), where reads from the same cell type are pooled in silico before splicing node quantification.…”

Section: Comprehensive Profiling Of Alternative Splicing Across Mouse Gastrulation and Early Organogenesismentioning

confidence: 99%

“…This filter is particularly important for transcripts assembled from reads that are mapped to repetitive sequences or exons that are ≤30 nt, which can arise from HISAT2 misalignments. To annotate potentially novel microexons, we used MicroExonator, a specialized computational workflow for discovering and quantifying microexons (Parada et al, 2021). After running the MicroExonator's discovery module we obtained a transcriptome annotation, which was later processed with custom scripts to limit the number of alternative transcription start and end sites.…”

Section: Expanding the Transcriptome Annotationmentioning

confidence: 99%

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Droplet-based Single-cell Total RNA-seq Reveals Differential Non-Coding Expression and Splicing Patterns during Mouse Development

Salmén

Jonghe

Kamiński

et al. 2021

Preprint

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In recent years, single-cell transcriptome sequencing has revolutionized biology, allowing for the unbiased characterization of cellular subpopulations. However, most methods amplify the termini of polyadenylated transcripts capturing only a small fraction of the total cellular transcriptome. This precludes the detection of many long non-coding, short non-coding and non-polyadenylated protein-coding transcripts. Additionally, most workflows do not sequence the full transcript hindering the analysis of alternative splicing. We therefore developed VASA- seq to detect the total transcriptome in single cells. VASA-seq is compatible with both plate- based formats and droplet microfluidics. We applied VASA-seq to over 30,000 single cells in the developing mouse embryo during gastrulation and early organogenesis. The dynamics of the total single-cell transcriptome result in the discovery of novel cell type markers many based on non-coding RNA, an in vivo cell cycle analysis and an improved RNA velocity characterization. Moreover, it provides the first comprehensive analysis of alternative splicing during mammalian development.

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“…Advances in our understanding of pre‐mRNA splicing, coupled with high‐throughput sequencing technology, have led to the identification of many AS events, and the RBPs and their binding RNA elements influencing AS selection (Pan et al, 2008). With these achievements, small sized exons (≤51 bp) termed “microexons” were discovered to be present in human tissues, and their alternative splicing to have a role in neuronal differentiation, embryonic differentiation, cellular physiology, and disease progression (Head et al, 2021; Irimia et al, 2014; Lee et al, 2020; Parada et al, 2021; Vecellio Reane et al, 2016). In the human genome, approximately 13,095 microexons are highly conserved across species and are differentially expressed between tissue types (Y. I. Li et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Microexon alternative splicing of small GTPase regulators: Implication in central nervous system diseases

2021

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Microexons are small sized (≤51 bp) exons which undergo extensive alternative splicing in neurons, microglia, embryonic stem cells, and cancer cells, giving rise to cell type specific protein isoforms. Due to their small sizes, microexons provide a unique challenge for the splicing machinery. They frequently lack exon splicer enhancers/repressors and require specialized neighboring trans-regulatory and cis-regulatory elements bound by RNA binding proteins (RBPs) for their inclusion. The functional consequences of including microexons within mRNAs have been extensively documented in the central nervous system (CNS) and aberrations in their inclusion have been observed to lead to abnormal processes. Despite the increasing evidence for microexons impacting cellular physiology within CNS, mechanistic details illustrating their functional importance in diseases of the CNS is still limited. In this review, we discuss the unique characteristics of microexons, and how RBPs participate in regulating their inclusion and exclusion during splicing. We consider recent findings of microexon alternative splicing and their implication for regulating the function of small GTPases in the context of the microglia, and we extrapolate these findings to what is known in neurons. We further discuss the emerging evidence for dysregulation of the Rho GTPase pathway in CNS diseases and the consequences contributed by the mis-splicing of microexons.

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

Cited by 4 publications

References 80 publications

High-throughput total RNA sequencing in single cells using VASA-seq

High-throughput total RNA sequencing in single cells using VASA-seq

Droplet-based Single-cell Total RNA-seq Reveals Differential Non-Coding Expression and Splicing Patterns during Mouse Development

Microexon alternative splicing of small GTPase regulators: Implication in central nervous system diseases

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