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Co-Transcriptional Splicing in Murin...

Reimer, Kirsten Audrey.

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Co-Transcriptional Splicing in Murine Erythroblasts.

Record Type:	Electronic resources : Monograph/item
Title/Author:	Co-Transcriptional Splicing in Murine Erythroblasts./
Author:	Reimer, Kirsten Audrey.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2021,
Description:	148 p.
Notes:	Source: Dissertations Abstracts International, Volume: 83-02, Section: B.
Contained By:	Dissertations Abstracts International83-02B.
Subject:	Biochemistry. -
Online resource:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=28321805
ISBN:	9798522998721

Co-Transcriptional Splicing in Murine Erythroblasts.
Reimer, Kirsten Audrey.

Co-Transcriptional Splicing in Murine Erythroblasts. - Ann Arbor : ProQuest Dissertations & Theses, 2021 - 148 p.

Source: Dissertations Abstracts International, Volume: 83-02, Section: B.

Thesis (Ph.D.)--Yale University, 2021.

This item must not be sold to any third party vendors.

Eukaryotic genes contain non-coding sequences called introns. The removal of introns from pre-mRNAs, termed splicing, is carried out by the spliceosome, a multi-megadalton molecular complex of proteins and RNAs. Splicing occurs co-transcriptionally across multiple cell types and species. The Neugebauer lab has developed single molecule nascent RNA sequencing methods-including single molecule intron tracking (SMIT) and long-read sequencing (LRS) of nascent RNA-to visualize the precursors, intermediates, and products of transcription and splicing in budding and fission yeasts. Using these methods, the lab was able to estimate the kinetics of single intron removal in both yeasts by relating the 30 end of nascent RNA (the position of RNA Polymerase II) to progress of the splicing reaction. In both species of yeast, splicing proceeded rapidly and co-transcriptionally.In comparison to yeast, mammalian genes are much more complex-on average they contain eight long introns surrounded by short exons. It was unclear how the presence of many more long introns, often with more poorly conserved splice site sequences, would affect how splicing and transcription are coordinated. Thus, I have optimized new methods to isolate nascent RNA and analyze co-transcriptional splicing in mammalian cells.To determine how splicing is integrated with transcription elongation and 30end formation in mammalian cells, I performed long-read sequencing of individual nascent RNAs and PRO-seq during murine erythropoiesis. I chose murine erythroidleukemia (MEL) cells as a model system, as they can be easily differentiated in vitro, and they express a subset of erythroid-specific genes at high levels. Many studies of gene expression have historically been carried out in erythroblasts, and the biogenesis of β-globin mRNA - the most highly expressed transcription erythroblasts - was the focus of many seminal studies on the mechanisms of pre-mRNA splicing.I isolated nascent, chromatin-associated RNAs from MEL cells before and after induction of terminal erythroid differentiation and performed long-read sequencing on the Pacific Biosciences Sequel platform. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (PolII) was within 75 - 300 nucleotides of 30 splice sites, 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 of splicing. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 30 end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 30 splice site improves both splicing and 30 end cleavage of individual-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.Thus, I conclude that highly expressed pre-mRNAs in MEL cells are largely spliced co-transcriptionally, and that the mammalian spliceosome can assemble and act rapidly on this set of pre-mRNAs. A previously unappreciated level of cross-talk between splicing and 30 end cleavage efficiencies is involved in erythroid development. Together, this work provides a high-resolution description of mammalian gene expression and shows that short-read RNA sequencing of bulk RNA can conceal coordinated behaviours that can only be observed at the level of individual nascent transcripts.

ISBN: 9798522998721Subjects--Topical Terms:

518028
Biochemistry.
Subjects--Index Terms:

Co-transcriptional splicing

Co-Transcriptional Splicing in Murine Erythroblasts.
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245 1 0 $a Co-Transcriptional Splicing in Murine Erythroblasts.
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300 $a 148 p.
500 $a Source: Dissertations Abstracts International, Volume: 83-02, Section: B.
500 $a Advisor: Neugebauer, Karla M.
502 $a Thesis (Ph.D.)--Yale University, 2021.
506 $a This item must not be sold to any third party vendors.
520 $a Eukaryotic genes contain non-coding sequences called introns. The removal of introns from pre-mRNAs, termed splicing, is carried out by the spliceosome, a multi-megadalton molecular complex of proteins and RNAs. Splicing occurs co-transcriptionally across multiple cell types and species. The Neugebauer lab has developed single molecule nascent RNA sequencing methods-including single molecule intron tracking (SMIT) and long-read sequencing (LRS) of nascent RNA-to visualize the precursors, intermediates, and products of transcription and splicing in budding and fission yeasts. Using these methods, the lab was able to estimate the kinetics of single intron removal in both yeasts by relating the 30 end of nascent RNA (the position of RNA Polymerase II) to progress of the splicing reaction. In both species of yeast, splicing proceeded rapidly and co-transcriptionally.In comparison to yeast, mammalian genes are much more complex-on average they contain eight long introns surrounded by short exons. It was unclear how the presence of many more long introns, often with more poorly conserved splice site sequences, would affect how splicing and transcription are coordinated. Thus, I have optimized new methods to isolate nascent RNA and analyze co-transcriptional splicing in mammalian cells.To determine how splicing is integrated with transcription elongation and 30end formation in mammalian cells, I performed long-read sequencing of individual nascent RNAs and PRO-seq during murine erythropoiesis. I chose murine erythroidleukemia (MEL) cells as a model system, as they can be easily differentiated in vitro, and they express a subset of erythroid-specific genes at high levels. Many studies of gene expression have historically been carried out in erythroblasts, and the biogenesis of β-globin mRNA - the most highly expressed transcription erythroblasts - was the focus of many seminal studies on the mechanisms of pre-mRNA splicing.I isolated nascent, chromatin-associated RNAs from MEL cells before and after induction of terminal erythroid differentiation and performed long-read sequencing on the Pacific Biosciences Sequel platform. Splicing was not accompanied by transcriptional pausing and was detected when RNA polymerase II (PolII) was within 75 - 300 nucleotides of 30 splice sites, 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 of splicing. Overall, splicing efficiencies were correlated among introns within the same transcript, and intron retention was associated with inefficient 30 end cleavage. Remarkably, a thalassemia patient-derived mutation introducing a cryptic 30 splice site improves both splicing and 30 end cleavage of individual-globin transcripts, demonstrating functional coupling between the two co-transcriptional processes as a determinant of productive gene output.Thus, I conclude that highly expressed pre-mRNAs in MEL cells are largely spliced co-transcriptionally, and that the mammalian spliceosome can assemble and act rapidly on this set of pre-mRNAs. A previously unappreciated level of cross-talk between splicing and 30 end cleavage efficiencies is involved in erythroid development. Together, this work provides a high-resolution description of mammalian gene expression and shows that short-read RNA sequencing of bulk RNA can conceal coordinated behaviours that can only be observed at the level of individual nascent transcripts.
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653 $a Co-transcriptional splicing
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