Author
Kirsten A. Reimer
Bio: Kirsten A. Reimer is an academic researcher from Yale University. The author has contributed to research in topics: RNA splicing & Intron. The author has an hindex of 4, co-authored 5 publications receiving 46 citations.
Topics: RNA splicing, Intron, Gene, Transcription (biology), RNA polymerase II
Papers
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TL;DR: In this paper, the authors performed long-read sequencing of individual nascent RNAs and precision run-on sequencing (PRO-seq) during mouse erythropoiesis, and found that splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II was within 75-300 nucleotides of 3' splice sites (3'SSs), often during transcription of the downstream exon.
77 citations
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TL;DR: A thalassemia patient-derived mutation introducing a cryptic 3’SS improves both splicing and3’ end cleavage of individual β-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.
Abstract: Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3’ end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and PRO-seq during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75 – 300 nucleotides of 3’ splice sites (3’SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3’ end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3’SS improves both splicing and 3’ end cleavage of individual β-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.
58 citations
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TL;DR: A patient-derived mutation in β-globin that causes thalassemia improves splicing efficiency and proper termination, revealing co-transcriptionalsplicing efficiency is a determinant of productive gene output.
Abstract: Pre-mRNA splicing is tightly coordinated with transcription in yeasts, and introns can be removed soon after they emerge from RNA polymerase II (Pol II). To determine if splicing is similarly rapid and efficient in mammalian cells, we performed long read sequencing of nascent RNA during mouse erythropoiesis. Remarkably, 50% of splicing occurred while Pol II was within 150 nucleotides of 3′ splice sites. PRO-seq revealed that Pol II does not pause around splice sites, confirming that mammalian and yeast spliceosomes can act equally rapidly. Two exceptions were observed. First, several hundred introns displayed abundant splicing intermediates, suggesting that the spliceosome can stall after the first catalytic step. Second, some genes – notably globins – displayed poor splicing coupled to readthrough transcription. Remarkably, a patient-derived mutation in β-globin that causes thalassemia improves splicing efficiency and proper termination, revealing co-transcriptional splicing efficiency is a determinant of productive gene output.
12 citations
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TL;DR: Changes in the developing erythroblast transcriptome caused by alternative splicing, the molecular basis of splicing-related blood diseases, and therapeutic advances in disease treatment using CRISPR/Cas9 gene editing are focused on.
Abstract: During erythropoiesis, hematopoietic stem and progenitor cells transition to erythroblasts en route to terminal differentiation into enucleated red blood cells. Transcriptome-wide changes underlie distinct morphological and functional characteristics at each cell division during this process. Many studies of gene expression have historically been carried out in erythroblasts, and the biogenesis of β-globin mRNA—the most highly expressed transcript in erythroblasts—was the focus of many seminal studies on the mechanisms of pre-mRNA splicing. We now understand that pre-mRNA splicing plays an important role in shaping the transcriptome of developing erythroblasts. Recent advances have provided insight into the role of alternative splicing and intron retention as important regulatory mechanisms of erythropoiesis. However, dysregulation of splicing during erythropoiesis is also a cause of several hematological diseases, including β-thalassemia and myelodysplastic syndromes. With a growing understanding of the role that splicing plays in these diseases, we are well poised to develop gene-editing treatments. In this review, we focus on changes in the developing erythroblast transcriptome caused by alternative splicing, the molecular basis of splicing-related blood diseases, and therapeutic advances in disease treatment using CRISPR/Cas9 gene editing.
7 citations
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01 Dec 2020TL;DR: How to isolate nascent RNA from mammalian cells through subcellular fractionation of chromatin‐associated RNA, as well as how to deplete poly(A)+ RNA and rRNA, and how to generate a full‐length cDNA library for use on long read sequencing platforms is described.
Abstract: Long read sequencing technologies now allow high-quality sequencing of RNAs (or their cDNAs) that are hundreds to thousands of nucleotides long. Long read sequences of nascent RNA provide single-nucleotide-resolution information about co-transcriptional RNA processing events-e.g., splicing, folding, and base modifications. Here, we describe how to isolate nascent RNA from mammalian cells through subcellular fractionation of chromatin-associated RNA, as well as how to deplete poly(A)+ RNA and rRNA, and, finally, how to generate a full-length cDNA library for use on long read sequencing platforms. This approach allows for an understanding of coordinated splicing status across multi-intron transcripts by revealing patterns of splicing or other RNA processing events that cannot be gained from traditional short read RNA sequencing. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Subcellular fractionation Basic Protocol 2: Nascent RNA isolation and adapter ligation Basic Protocol 3: cDNA amplicon preparation.
4 citations
Cited by
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TL;DR: This Review provides insight into the regulation of lncRNA transcription and processing gleaned from the application of recently devised nascent transcriptomics technology, and compares and contrast different methodologies for studying nascent transcription.
90 citations
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TL;DR: In this paper , the authors perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells.
79 citations
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TL;DR: In this paper, the authors performed long-read sequencing of individual nascent RNAs and precision run-on sequencing (PRO-seq) during mouse erythropoiesis, and found that splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II was within 75-300 nucleotides of 3' splice sites (3'SSs), often during transcription of the downstream exon.
77 citations
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Queen Mary University of London1, University of Edinburgh2, University of Cambridge3, University of British Columbia4, Semmelweis University5, St James's University Hospital6, University of Paris7, Boston Children's Hospital8, Norwegian University of Science and Technology9, Royal Bournemouth Hospital10, Children's of Alabama11, Imperial College London12, Great Ormond Street Hospital13, University Hospitals Bristol NHS Foundation Trust14, University Medical Center Utrecht15, Autonomous University of Barcelona16, Foothills Medical Centre17, University College London18, French Institute of Health and Medical Research19, Broad Institute20, Nottingham University Hospitals NHS Trust21, St George's Hospital22, The Royal Marsden NHS Foundation Trust23, Katholieke Universiteit Leuven24, Hochschule Hannover25, Qatar Airways26, St Bartholomew's Hospital27, Barts Health NHS Trust28
TL;DR: A cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci are reported, offering novel insights into the etiology of myeloids malignancies and a framework to prioritize variants for inclusion into routine diagnostics and patient management.
Abstract: The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management. Familial myeloid malignancies have recently been classified as separate disease entities. Here, using whole-exome sequencing of affected pedigrees - the authors highlight genetic variants associated with these conditions.
61 citations
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TL;DR: A thalassemia patient-derived mutation introducing a cryptic 3’SS improves both splicing and3’ end cleavage of individual β-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.
Abstract: Pre-mRNA processing steps are tightly coordinated with transcription in many organisms. To determine how co-transcriptional splicing is integrated with transcription elongation and 3’ end formation in mammalian cells, we performed long-read sequencing of individual nascent RNAs and PRO-seq during mouse erythropoiesis. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (Pol II) was within 75 – 300 nucleotides of 3’ splice sites (3’SSs), often during transcription of the downstream exon. Interestingly, several hundred introns displayed abundant splicing intermediates, suggesting that splicing delays can take place between the two catalytic steps. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 3’ end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 3’SS improves both splicing and 3’ end cleavage of individual β-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.
58 citations