Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites

ABSTRACT Alternative splicing is a widespread phenomenon in metazoans by which single genes are able to produce multiple isoforms of the gene product. However, this has been poorly characterized in apicomplexans, a major phylum of some of the most important global parasites. Efforts have been hamper...

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Autores principales:	V. Vern Lee, Louise M. Judd, Aaron R. Jex, Kathryn E. Holt, Christopher J. Tonkin, Stuart A. Ralph
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2021
Materias:	Plasmodium RNA splicing RNA-seq Toxoplasma nanopore transcriptional regulation Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/632f680011c440afafa95b35d849df06
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spelling	oai:doaj.org-article:632f680011c440afafa95b35d849df062021-12-02T19:22:27ZDirect Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites10.1128/mSystems.01081-202379-5077https://doaj.org/article/632f680011c440afafa95b35d849df062021-04-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mSystems.01081-20https://doaj.org/toc/2379-5077ABSTRACT Alternative splicing is a widespread phenomenon in metazoans by which single genes are able to produce multiple isoforms of the gene product. However, this has been poorly characterized in apicomplexans, a major phylum of some of the most important global parasites. Efforts have been hampered by atypical transcriptomic features, such as the high AU content of Plasmodium RNA, but also the limitations of short-read sequencing in deciphering complex splicing events. In this study, we utilized the long read direct RNA sequencing platform developed by Oxford Nanopore Technologies to survey the alternative splicing landscape of Toxoplasma gondii and Plasmodium falciparum. We find that while native RNA sequencing has a reduced throughput, it allows us to obtain full-length or nearly full-length transcripts with comparable quantification to Illumina sequencing. By comparing these data with available gene models, we find widespread alternative splicing, particularly intron retention, in these parasites. Most of these transcripts contain premature stop codons, suggesting that in these parasites, alternative splicing represents a pathway to transcriptomic diversity, rather than expanding proteomic diversity. Moreover, alternative splicing rates are comparable between parasites, suggesting a shared splicing machinery, despite notable transcriptomic differences between the parasites. This study highlights a strategy in using long-read sequencing to understand splicing events at the whole-transcript level and has implications in the future interpretation of transcriptome sequencing studies. IMPORTANCE We have used a novel nanopore sequencing technology to directly analyze parasite transcriptomes. The very long reads of this technology reveal the full-length genes of the parasites that cause malaria and toxoplasmosis. Gene transcripts must be processed in a process called splicing before they can be translated to protein. Our analysis reveals that these parasites very frequently only partially process their gene products, in a manner that departs dramatically from their human hosts.V. Vern LeeLouise M. JuddAaron R. JexKathryn E. HoltChristopher J. TonkinStuart A. RalphAmerican Society for MicrobiologyarticlePlasmodiumRNA splicingRNA-seqToxoplasmananoporetranscriptional regulationMicrobiologyQR1-502ENmSystems, Vol 6, Iss 2 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Plasmodium RNA splicing RNA-seq Toxoplasma nanopore transcriptional regulation Microbiology QR1-502
spellingShingle	Plasmodium RNA splicing RNA-seq Toxoplasma nanopore transcriptional regulation Microbiology QR1-502 V. Vern Lee Louise M. Judd Aaron R. Jex Kathryn E. Holt Christopher J. Tonkin Stuart A. Ralph Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites
description	ABSTRACT Alternative splicing is a widespread phenomenon in metazoans by which single genes are able to produce multiple isoforms of the gene product. However, this has been poorly characterized in apicomplexans, a major phylum of some of the most important global parasites. Efforts have been hampered by atypical transcriptomic features, such as the high AU content of Plasmodium RNA, but also the limitations of short-read sequencing in deciphering complex splicing events. In this study, we utilized the long read direct RNA sequencing platform developed by Oxford Nanopore Technologies to survey the alternative splicing landscape of Toxoplasma gondii and Plasmodium falciparum. We find that while native RNA sequencing has a reduced throughput, it allows us to obtain full-length or nearly full-length transcripts with comparable quantification to Illumina sequencing. By comparing these data with available gene models, we find widespread alternative splicing, particularly intron retention, in these parasites. Most of these transcripts contain premature stop codons, suggesting that in these parasites, alternative splicing represents a pathway to transcriptomic diversity, rather than expanding proteomic diversity. Moreover, alternative splicing rates are comparable between parasites, suggesting a shared splicing machinery, despite notable transcriptomic differences between the parasites. This study highlights a strategy in using long-read sequencing to understand splicing events at the whole-transcript level and has implications in the future interpretation of transcriptome sequencing studies. IMPORTANCE We have used a novel nanopore sequencing technology to directly analyze parasite transcriptomes. The very long reads of this technology reveal the full-length genes of the parasites that cause malaria and toxoplasmosis. Gene transcripts must be processed in a process called splicing before they can be translated to protein. Our analysis reveals that these parasites very frequently only partially process their gene products, in a manner that departs dramatically from their human hosts.
format	article
author	V. Vern Lee Louise M. Judd Aaron R. Jex Kathryn E. Holt Christopher J. Tonkin Stuart A. Ralph
author_facet	V. Vern Lee Louise M. Judd Aaron R. Jex Kathryn E. Holt Christopher J. Tonkin Stuart A. Ralph
author_sort	V. Vern Lee
title	Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites
title_short	Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites
title_full	Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites
title_fullStr	Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites
title_full_unstemmed	Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites
title_sort	direct nanopore sequencing of mrna reveals landscape of transcript isoforms in apicomplexan parasites
publisher	American Society for Microbiology
publishDate	2021
url	https://doaj.org/article/632f680011c440afafa95b35d849df06
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Direct Nanopore Sequencing of mRNA Reveals Landscape of Transcript Isoforms in Apicomplexan Parasites

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