Direct nanopore sequencing of mRNA reveals landscape of transcript isoforms in apicomplexan parasites

Lee, V Vern; Judd, Louise M.; Jex, Aaron R.; Holt, Kathryn E.; Tonkin, Christopher J.; Ralph, Stuart A.

doi:10.1101/2020.02.16.946699

Cited by 9 publications

(19 citation statements)

References 108 publications

(130 reference statements)

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“…Generating similar datasets from other life-cycle stages of Toxoplasma will be important in assessing stage-specifically–expressed genes that could not be captured in the present study. In addition, integrating our stage-specific 5′-end data with emerging long-read RNA-seq data from published ( Lee et al., 2020 ) and future studies will likely improve the association of (alternative) TSSs to annotated genes.…”

Section: Discussionmentioning

confidence: 99%

High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii

Markus

Waldman

Lorenzi

et al. 2021

Front. Cell. Infect. Microbiol.

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Toxoplasma gondii is a common parasite of humans and animals, causing life-threatening disease in the immunocompromized, fetal abnormalities when contracted during gestation, and recurrent ocular lesions in some patients. Central to the prevalence and pathogenicity of this protozoan is its ability to adapt to a broad range of environments, and to differentiate between acute and chronic stages. These processes are underpinned by a major rewiring of gene expression, yet the mechanisms that regulate transcription in this parasite are only partially characterized. Deciphering these mechanisms requires a precise and comprehensive map of transcription start sites (TSSs); however, Toxoplasma TSSs have remained incompletely defined. To address this challenge, we used 5′-end RNA sequencing to genomically assess transcription initiation in both acute and chronic stages of Toxoplasma. Here, we report an in-depth analysis of transcription initiation at promoters, and provide empirically-defined TSSs for 7603 (91%) protein-coding genes, of which only 1840 concur with existing gene models. Comparing data from acute and chronic stages, we identified instances of stage-specific alternative TSSs that putatively generate mRNA isoforms with distinct 5′ termini. Analysis of the nucleotide content and nucleosome occupancy around TSSs allowed us to examine the determinants of TSS choice, and outline features of Toxoplasma promoter architecture. We also found pervasive divergent transcription at Toxoplasma promoters, clustered within the nucleosomes of highly-symmetrical phased arrays, underscoring chromatin contributions to transcription initiation. Corroborating previous observations, we asserted that Toxoplasma 5′ leaders are among the longest of any eukaryote studied thus far, displaying a median length of approximately 800 nucleotides. Further highlighting the utility of a precise TSS map, we pinpointed motifs associated with transcription initiation, including the binding sites of the master regulator of chronic-stage differentiation, BFD1, and a novel motif with a similar positional arrangement present at 44% of Toxoplasma promoters. This work provides a critical resource for functional genomics in Toxoplasma, and lays down a foundation to study the interactions between genomic sequences and the regulatory factors that control transcription in this parasite.

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Section: Discussionmentioning

confidence: 99%

High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii

Markus

Waldman

Lorenzi

et al. 2021

Front. Cell. Infect. Microbiol.

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“…falciparum genome ( Chappell et al, 2020 ). To our knowledge, the largest available dataset of complete transcript structures was obtained by Nanopore long-read direct RNA sequencing ( Lee et al, 2021 ). In direct RNA sequencing, the transcript is sequenced from the 3′ end.…”

Section: Resultsmentioning

confidence: 99%

“…Direct RNA sequencing data from P . falciparum obtained using the Oxford Nanopore MinION platform reported in Lee et al (2021) under SRA accession number SRR11094274 were uploaded to the public server at usegalaxy.org and aligned to the P . falciparum 3D7 v3.2 reference genome ( Böhme et al, 2019 ) using Minimap2 ( Li, 2018 ) with settings long-read spliced alignment, maximum intron 5 kb, and search GT-AG on the transcript strand only.…”

Section: Methodsmentioning

confidence: 99%

Identifying transcript 5′ capped ends in Plasmodium falciparum

Shaw

Piriyapongsa

Kaewprommal

et al. 2021

PeerJ

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Background The genome of the human malaria parasite Plasmodium falciparum is poorly annotated, in particular, the 5′ capped ends of its mRNA transcripts. New approaches are needed to fully catalog P. falciparum transcripts for understanding gene function and regulation in this organism. Methods We developed a transcriptomic method based on next-generation sequencing of complementary DNA (cDNA) enriched for full-length fragments using eIF4E, a 5′ cap-binding protein, and an unenriched control. DNA sequencing adapter was added after enrichment of full-length cDNA using two different ligation protocols. From the mapped sequence reads, enrichment scores were calculated for all transcribed nucleotides and used to calculate P-values of 5′ capped nucleotide enrichment. Sensitivity and accuracy were increased by combining P-values from replicate experiments. Data were obtained for P. falciparum ring, trophozoite and schizont stages of intra-erythrocytic development. Results 5′ capped nucleotide signals were mapped to 17,961 non-overlapping P. falciparum genomic intervals. Analysis of the dominant 5′ capped nucleotide in these genomic intervals revealed the presence of two groups with distinctive epigenetic features and sequence patterns. A total of 4,512 transcripts were annotated as 5′ capped based on the correspondence of 5′ end with 5′ capped nucleotide annotated from full-length cDNA data. Discussion The presence of two groups of 5′ capped nucleotides suggests that alternative mechanisms may exist for producing 5′ capped transcript ends in P. falciparum. The 5′ capped transcripts that are antisense, outside of, or partially overlapping coding regions may be important regulators of gene function in P. falciparum.

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“…rely on post-transcriptional regulation for parasite development, including processes such as mRNA sequestration (17) and alternative splicing (18). Some studies have speculated that NMD coupled to intron retention -the predominant form of alternative splicing in apicomplexans -coupled to NMD could contribute to widespread regulation of mRNA abundance (10,19) and hence we are interested in characterising NMD in P. falciparum.…”

Section: Crispr-cas9 Ribonucleoproteinsmentioning

confidence: 99%

“…The malaria parasite P. falciparum has high levels of intron retention compared to other forms of alternative splicing, which is markedly different from the proportions of alternative splice variants in humans (10). We therefore investigated whether intron retention coupled to NMD has a role in the regulation of gene expression in P. falciparum.…”

Section: Introductionmentioning

confidence: 99%

A divergent nonsense-mediated decay machinery inPlasmodium falciparumis inefficient and non-essential

McHugh

Bulloch²,

Batinovic³

et al. 2021

Preprint

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Nonsense-mediated decay (NMD) is a conserved mRNA quality control process that eliminates transcripts bearing a premature termination codon. In addition to its role in removing erroneous transcripts, NMD is involved in post-transcriptional regulation of gene expression via programmed intron retention in metazoans. The apicomplexan parasite Plasmodium falciparum shows relatively high levels of intron retention, but it is unclear whether these variant transcripts are functional targets of NMD. In this study, we use CRISPR-Cas9 to disrupt and epitope-tag two core NMD components: PfUPF1 (PF3D7_1005500) and PfUPF2 (PF3D7_0925800). Using RNA-seq, we find that NMD in P. falciparum is highly derived and requires UPF2, but not UPF1 for transcript degradation. Furthermore, our work suggests that the majority of intron retention in P. falciparum has no functional role and that NMD is not required for parasite growth ex vivo. We localise both PfUPF1 and PfUPF2 to puncta within the parasite cytoplasm, which may represent processing bodies - ribonucleoparticles that are sites of cytoplasmic mRNA decay. Finally, we identify a number of mRNA-binding proteins that co-immunoprecipitate with the NMD core complex and propose a model for a divergent NMD that does not require PfUPF1 and incorporates novel accessory proteins to elicit mRNA decay.

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Direct nanopore sequencing of mRNA reveals landscape of transcript isoforms in apicomplexan parasites

Cited by 9 publications

References 108 publications

High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii

High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii

Identifying transcript 5′ capped ends in Plasmodium falciparum

A divergent nonsense-mediated decay machinery inPlasmodium falciparumis inefficient and non-essential

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