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 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.

The Elusive Mitochondrial Genomes of Apicomplexa: Where Are We Now?

“…With this in mind, we explored the recently published direct ONT mRNA data from T. gondii (Lee et al, 2021). We searched for clues of the underlying transcription mechanism of gene fragments in the T. gondii mitogenome.…”

“…We searched for clues of the underlying transcription mechanism of gene fragments in the T. gondii mitogenome. Although this sequencing was performed to identify possible isoforms in the nuclear genes (Lee et al, 2021), a total of 355 ONT reads (from 112 to 2,962 bp long) mapped in the mitogenome of T. gondii. The longest mRNAs correspond almost exactly to the series of discrete sequence blocks described by Namasivayam et al (2021).…”

Identifying transcript 5′ capped ends in Plasmodium falciparum

“…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. The .bam alignment file was used as input to the Full-Length Alternative Isoform analysis of RNA (FLAIR) program (Tang et al, 2020a) with the-nvrna option using reference genome sequence and annotation (PlasmoDB release 44) for correction of isoform sequences.…”

“…In order to annotate transcripts, data of complete (end-toend) transcript structures are required, which can be difficult to obtain from short-read cDNA data owing to breaks in coverage at highly AT-rich or unmappable regions of the P. 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.…”

“…Figure6Annotation of 5 capped transcripts in Plasmodium falciparum. P. falciparum transcripts were annotated from FLAIR analysis(Tang et al, 2020a) of direct RNA sequencing data(Lee et al, 2021). 5 capped transcripts were identified by the overlap of 5 end nucleotide position with 5 capped nucleotide enrichment data.…”