| 1. |
- Holmqvist, Isak, et al.
(författare)
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FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
- 2021
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Ingår i: Rna. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 27:10, s. 1127-1139
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Tidskriftsartikel (refereegranskat)abstract
- Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this work-flow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.
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| 2. |
- Kristenson, Linnea, 1991, et al.
(författare)
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Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity
- 2024
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Ingår i: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. - 0027-8424 .- 1091-6490. ; 121:15
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Tidskriftsartikel (refereegranskat)abstract
- Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-gamma signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7 - H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta- subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock - out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM - 3, restored the NK cell ability to eliminate TMEM30A- mutated cells. The key role of the TIM - 3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM - 3 in the realm of cancer immunotherapy.
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| 3. |
- Xie, Guojiang
(författare)
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Characterization of Epstein-Barr virus non-coding RNAs in infected cells
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Epstein-Barr virus (EBV) causes multiple types of lymphoid and epithelial malignancies. Unraveling the viral gene expression patterns in tumors and identifying interaction partners between EBV and host elements is crucial for understanding EBV tumorigenesis. We characterized the EBV RNA in primary tumors by using publicly available bulk and single-cell RNA sequencing data. Our findings show that the dominant polyadenylated RNA transcript in all EBV-associated malignancies originated from the viral long non-coding RNA RPMS1. Additionally, we identified and characterized three novel RNA elements by using full-length single-molecule sequencing together with our own bioinformatics tool, FLAME. These transcripts were co-expressed at high levels with RPMS1 in tumors. Furthermore, the viral immunoevasin BNLF2 was the highest-expressed protein-coding gene in all tumor types. These findings contradict previously proposed EBV latency models. Single-cell sequencing of primary nasopharyngeal carcinoma tissues and B cells from a splenectomized patient confirmed the EBV expression pattern observed in bulk sequencing, with a dominant RPMS1 expression. Comparative analysis between EBV-positive malignant cells and adjacent healthy epithelium in nasopharyngeal carcinoma revealed a RPMS1 microRNA-mediated downregulation of immune regulatory pathways and tumor suppressor pathways, and induction of oncogene pathways. In a similar manner, EBV-infected B cells displayed a higher propensity for cell proliferation compared with uninfected B cells. EBV-infected blood cells from immunosuppressed patients expressed RPMS1 as well as the short non-polyadenylated non-coding RNAs EBER1 and EBER2. To investigate the role of these EBV-encoded non-coding RNAs, we utilized chromatin isolation by RNA purification, ChIRP, to identify host interaction partners. RPMS1 was mainly bound by proteins associated with the splicing machinery. Despite the fact that EBERs are neither spliced nor translated, proteins involved in the spliceosome, ribosome, and DNA repair pathway were identified to interact with EBERs. Functionally, EBERs reduced polyribosome formation and inhibited protein translation. Furthermore, EBER2 was found to interact with DNA in a sequence-dependent manner and selectively suppress the cell cycle and cell division. Our studies provide a comprehensive landscape of EBV expression in latently infected cells, including malignancies, and unveil interactions between the highly expressed viral non-coding RNAs and host elements. The results show that RPMS1 functions as a driver of cancer hallmarks while EBERs induce a cellular state corresponding with cell quiescence observed during latent infection. The findings enhance our comprehension of EBV biology and may guide future research toward identifying drugs for treating EBV-related disease.
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