Splicing in immune cells—mechanistic insights and emerging topics

Schaub, Annalisa; Glasmacher, Elke

doi:10.1093/intimm/dxx026

Cited by 61 publications

(58 citation statements)

References 100 publications

(140 reference statements)

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“…Of the DU peaks, on average 20% were tagged as near an A-rich region, and potentially due to internal priming, while an average of 32% flanked the canonical polyA motif. Among the DTU genes, there were known examples of alternatively spliced genes in immune cells, including IKZF1 (Ikaros) and PTPRC (CD45) [29]. Although PTPRC gene expression is ubiquitous among immune cell types, we found we could distinguish clear patterns of alternative peak usage in monocytes compared to other cell types ( Figure 2E) according to t-SNE visualisations of relative peak expression, demonstrating that we are able to detect cell type gene expression activity masked when only considering an aggregate of the reads across a gene.…”

Section: Differential Transcript Usage Among Human Pbmcsmentioning

confidence: 99%

Sierra: discovery of differential transcript usage from polyA-captured single-cell RNA-seq data

Patrick

Humphreys

Oshlack

et al. 2019

Preprint

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Background: High-throughput single-cell RNA-seq (scRNA-seq) is a powerful technology for studying gene expression variability in single cells; however, standard analysis approaches only consider the overall expression of each gene, masking additional heterogeneity that could exist through cell type-specific expression of alternative mRNA transcripts.Results: Here we show that differential transcript usage (DTU) can be readily detected in data-sets generated from commonly used polyA-captured nanodroplet scRNA-seq technology.Our computational pipeline, Sierra, detects and quantifies polyadenylation sites in scRNA-seq data-sets, which are utilised to evaluate DTU between single-cell populations. We validate our approach by comparing cardiac cell populations derived from scRNA-seq to bulk RNAseq of matched populations obtained through fluorescent activated cell sorting (FACS), demonstrating that we detect a significant overlap in cell-type DTU between the congruous data-sets. We further illustrate the utility of our method by detecting alternative transcript usage in human peripheral blood mononuclear cells (PBMCs), 3'UTR shortening in activated and proliferating cardiac fibroblasts from injured mouse hearts and finally by building an initial atlas of cell type-specific transcript usage across 12 mouse tissues. Conclusions:We anticipate that Sierra will enable new avenues of transcriptional complexity and regulation to be explored in single-cell transcriptomic experiments. Sierra is available at https://github.com/VCCRI/Sierra.

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Section: Differential Transcript Usage Among Human Pbmcsmentioning

confidence: 99%

Sierra: discovery of differential transcript usage from polyA-captured single-cell RNA-seq data

Patrick

Humphreys

Oshlack

et al. 2019

Preprint

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“…29 The spliceosome involves multicomponent complexes including five snRNPs that coordinate mRNA splicing. 30 Our finding that components of the spliceosome are both up-regulated and found interacting with UBR5 in MCL cell lines identifies a new pathway of UBR5 involvement. The striking phenotype of IgD in HECT domain mutants supports defects in splicing since IgM and IgD are generated from alternative splicing of the heavy chain gene.…”

Section: Discussionmentioning

confidence: 77%

E3 Ligase UBR5 HECT domain mutations in lymphoma control maturation of B cells via alternative splicing

Swenson

Gilbreath

Vahle

et al. 2019

Preprint

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194, Text: 3,968, Figures: 7, References: 38 27 Scientific category: Immunobiology and Lymphoid Neoplasms 28 29 KEY POINTS 30 • Utilizing a novel mouse model mimicking MCL patient mutations, the loss of UBR5's HECT 31 domain causes alterations in B cell development. 32 33 • UBR5 mutations lead to stabilization of UBR5 and aberrant splicing. 34 35 2 ABSTRACT 36 Coordination of a number of molecular mechanisms including transcription, alternative splicing, 37 and class switch recombination are required to facilitate development, activation, and survival of B 38 cells. Disruption of these pathways can result in malignant transformation. Recently, next generation 39 sequencing has identified a number of novel mutations in mantle cell lymphoma (MCL) patients 40 including the ubiquitin E3 ligase UBR5. Approximately 18% of MCL patients were found to have 41 mutations in UBR5 with the majority of mutations within the HECT domain of the protein which can 42 accept and transfer ubiquitin molecules to the substrate. Determining if UBR5 controls the maturation 43 of B cells is important to fully understand malignant transformation to MCL. To elucidate the role of 44UBR5 in B cell maturation and activation we generated a conditional mutant disrupting UBR5's C-45 terminal HECT domain. Loss of the UBR5 HECT domain leads to a block in maturation of B cells in 46 the spleen and up-regulation of proteins associated with mRNA splicing via the spliceosome. Our 47 55 Mantle Cell Lymphoma (MCL) is a rare, aggressive form of Non-Hodgkin's Lymphoma 56 (NHL). 1 Although MCL represents only ~6% of NHL lymphoma cases, it has one of the highest 57 mortality rates of all lymphomas with only a 50% five year survival. 2 Given the high mortality 58 rate and propensity for recurrence, having better comprehension of mutations found in MCL 59 and how disease develops in B cells will open avenues for identifying new therapies. Recently, 60 the ubiquitin protein ligase E3 component n-recognin 5 (UBR5) was found mutated in ~18% of 61 patients with MCL. 3 The majority of mutations identified in UBR5 were frame shift mutations 62 found within its HECT domain, which can accept and transfer ubiquitin molecules to the 63 substrate, leading to a premature stop codon prior to the cysteine residue associated with 64 ubiquitin transfer. 65 66 UBR5 is a large ~300kDa protein HECT E3 ligase with a conserved carboxyl-terminal 67 HECT domain. In HECT E3 ligases, the N-terminal portion (N-lobe) of the enzyme interacts 68 with E2 ubiquitin-conjugating enzymes and determines substrate specificity while the C-69 terminal HECT domain (C-lobe) contains a catalytic cysteine residue that binds ubiquitin. 4 The 70 two lobes are connected by a flexible linker that allows for shifting orientation between N-and 71 C-lobes during ubiquitin transfer to allow for efficient movement of ubiquitin from the E3 ligase 72 to the substrate protein. UBR5 regulates a number of cellular processes including metabolism, 73 apoptosis, angiogenesis, gene expression, and genome integrity. 5-1...

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“…As a proof of the concept, we investigated PTPRC , one of the best-studied examples, to examine the isotypes in T cells. PTPRC is essential during lymphocyte differentiation, using exons 4, 5, and 6 variably(13, 30). Two transcript isoforms are highlighted according to ENSEMBL(24): PTPRC-209, which retains exons 4,5,6 and translates into protein isoform PTPRC-RABC, and PTPRC-201, which splices out all three exons and translates into PTPRC-RO ( Supplementary Figure 1b ).…”

Section: Resultsmentioning

confidence: 99%

“…The ImmGen Consortium integrated high-throughput data and revealed pervasive events of alternative splicing (AS) in T cells, with ~60% of genes showing different AS events linking to the differentiation of different T cell lineages(8). Multiple studies identified specific transcript isoforms involved in T cell-related biology(9), including T cell activation (10–12), T cell differentiation(13, 14), and apoptosis(15). For instance, PTPRC , which encodes the transmembrane tyrosine phosphatase CD45, is critical for T cell receptor (TCR) signal transduction(16).…”

Section: Introductionmentioning

confidence: 99%

Landscape of transcript isoforms in single T cells infiltrating in non-small cell lung cancer

Zhang

Ren

2020

Preprint

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11Single cell RNA-seq has enabled high-resolution characterization of molecular signatures of tumor-12 infiltrating lymphocytes. However, analyses at the transcript isoform level are rarely reported. As 13 alternative splicing is critical to T cell differentiation and activation, here we proposed a computational 14 method named as IDEA to comprehensively detect and annotate differentially used isoforms across 15 cell subtypes. We applied IDEA on a scRNA-seq dataset of 12,346 T cells from non-small cell lung 16 cancer. We found most genes tend to dominantly express one isoform in single T cells, enabling 17 typing T cells according to the isotypes given a gene. Isotype analysis suggested that tumor-infiltrating 18 T cells significantly preferred specific isotypes for 245 genes in CD8+ T cells and 456 genes in CD4+ 19 T cells. Functional annotation suggests that the preferred isoforms involved in coding/non-coding 20 switches, transcription start site changes, gains/losses of domains and subcellular translocation. 21 Clonal analysis revealed that isoform switching occurred during T cell activation/differentiation. Our 22 analysis provides precise characterization of the molecular events in tumor-infiltrating T cells and 23 sheds new lights into the regulatory mechanisms of tumor-infiltrating T cells. 24 25 65 Altogether, our work provides a comprehensive analysis of the transcript isoform landscape of tumor-66 infiltrating T cells in non-small cell lung cancer at the single-cell resolution. The abundant isoform 67 switching events identified among tumor-infiltrating T cell subtypes may serve as a rich data resource 68for precisely analyzing the mechanisms underlying T cell-based antitumor immunity. Our analysis also 69 highlights the necessity of splicing analysis at the single-cell resolution, which can be applied to a 70 wide range of biological questions.

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Splicing in immune cells—mechanistic insights and emerging topics

Abstract: Splicing mechanisms—relevance for immune cells

Cited by 61 publications

References 100 publications

Sierra: discovery of differential transcript usage from polyA-captured single-cell RNA-seq data

Sierra: discovery of differential transcript usage from polyA-captured single-cell RNA-seq data

E3 Ligase UBR5 HECT domain mutations in lymphoma control maturation of B cells via alternative splicing

Landscape of transcript isoforms in single T cells infiltrating in non-small cell lung cancer

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