Characterization of mRNA polyadenylation in the apicomplexa

Stevens, Ashley T.; Howe, Daniel K.; Hunt, Arthur G.

doi:10.1371/journal.pone.0203317

Cited by 24 publications

(23 citation statements)

References 58 publications

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“…As recently reported for apicomplexan genomes (Stevens et al. ), high matches in all intestinal parasite genomes were to CPSF73 and PAP; in C. parvum and Entamoeba spp. there were also significant matches to CFIm25.…”

Section: Discussionsupporting

confidence: 80%

mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites

Ospina-Villa¹,

Tovar-Ayona

López‐Camarillo

et al. 2020

J Eukaryotic Microbiology

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In humans, mRNA polyadenylation involves the participation of about 20 factors in four main complexes that recognize specific RNA sequences. Notably, CFIm25, CPSF73, and PAP have essential roles for poly(A) site selection, mRNA cleavage, and adenosine residues polymerization. Besides the relevance of polyadenylation for gene expression, information is scarce in intestinal protozoan parasites that threaten human health. To better understand polyadenylation in Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum, which represent leading causes of diarrhea worldwide, genomes were screened for orthologs of human factors. Results showed that Entamoeba histolytica and C. parvum have 16 and 12 proteins out of the 19 human proteins used as queries, respectively, while G. lamblia seems to have the smallest polyadenylation machinery with only six factors. Remarkably, CPSF30, CPSF73, CstF77, PABP2, and PAP, which were found in all parasites, could represent the core polyadenylation machinery. Multiple genes were detected for several proteins in Entamoeba, while gene redundancy is lower in Giardia and Cryptosporidium. Congruently with their relevance in the polyadenylation process, CPSF73 and PAP are present in all parasites, and CFIm25 is only missing in Giardia. They conserve the functional domains and predicted folding of human proteins, suggesting they may have the same roles in polyadenylation.

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Section: Discussionsupporting

confidence: 80%

mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites

Ospina-Villa¹,

Tovar-Ayona

López‐Camarillo

et al. 2020

J Eukaryotic Microbiology

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“…In mammals, recognition of the canonical poly(A) signal AAUAAA involves direct binding by CPSF30 41, 42 . Notably, alternative polyadenylation controls the expression of an Arabidopsis CPSF30 isoform that encodes a YT521-B homology (YTH) domain with the potential to bind and read m 6 A 43 . A recent study indicated that this YTH domain-containing isoform is required to supress chimera formation 38 .…”

Section: Resultsmentioning

confidence: 99%

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Parker

Knop

Sherwood

et al. 2019

Preprint

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21Understanding genome organization and gene regulation requires insight into RNA transcription, 22 processing and modification. We adapted nanopore direct RNA sequencing to examine RNA from a 23 wild-type accession of the model plant Arabidopsis thaliana and a mutant defective in mRNA 24 methylation (m 6 A). Here we show that m 6 A can be mapped in full-length mRNAs transcriptome-wide 25 and reveal the combinatorial diversity of cap-associated transcription start sites, splicing events, 26 poly(A) site choice and poly(A) tail length. Loss of m 6 A from 3' untranslated regions is associated 27 with decreased relative transcript abundance and defective RNA 3′ end formation. A functional 28 consequence of disrupted m 6 A is a lengthening of the circadian period. We conclude that nanopore 29 direct RNA sequencing can reveal the complexity of mRNA processing and modification in full-length 30 single molecule reads. These findings can refine Arabidopsis genome annotation. Further, applying 31 this approach to less well-studied species could transform our understanding of what their genomes 32 encode. 33 34 misidentification of 3′ ends through internal priming 3 , spurious antisense and splicing events 46 produced by RT template switching 4,5 , and the inability to detect all base modifications in the 47 copying process 6 . The fragmentation of RNA prior to short-read sequencing makes it difficult to 48 interpret the combination of authentic RNA processing events and remains an unsolved problem 7 . 49We investigated whether long-read direct RNA sequencing (DRS) with nanopores 8 could 50 reveal the complexity of Arabidopsis mRNA processing and modifications. In nanopore DRS, the 51 protein pore (nanopore) sits in a membrane through which an electrical current is passed, and intact 52 RNA is fed through the nanopore by a motor protein 8 . Each RNA sequence within the nanopore 53 (5 bases) can be identified by the magnitude of signal it produces. Arabidopsis is a pathfinder model 54 in plant biology, and its genome annotation strongly influences the annotation and our 55 understanding of what other plant genomes encode. We applied nanopore DRS and Illumina RNAseq 56 to wild-type Arabidopsis (Col-0) and mutants defective in m 6 A 9 and exosome-mediated RNA decay 10 . 57We reveal m 6 A and combinations of RNA processing events (alternative patterns of 5′ capped 58 transcription start sites, splicing, 3′ polyadenylation and poly(A) tail length) in full-length Arabidopsis 59 mRNAs transcriptome-wide. 60 61 Results 62Nanopore DRS detects long, complex mRNAs and short, structured non-coding RNAs 63We purified poly (A)+ RNA from four biological replicates of 14-day-old Arabidopsis Col-0 seedlings. 64We incorporated synthetic External RNA Controls Consortium (ERCC) RNA Spike-In mixes into all 65 replicates 11,12 and carried out nanopore DRS. Illumina RNAseq was performed in parallel on similar 66 material. Using Guppy base-calling (Oxford Nanopore Technologies) and minimap2 alignment 67 software 13 , we identified around 1 mi...

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“…In several eukaryotes, proteins containing a YTH domain have been identified as specific m 6 A 'reader' proteins, mediating both mRNA degradation and translational activation in an m 6 A-dependent manner 24 . The P. falciparum genome encodes two distinct proteins containing a YTH domain: PfYTH.1 (PF3D7_1419900, previously annotated as 'cleavage and polyadenylation specificity factor subunit 4, CPSF4) 36 and PfYTH.2 (PF3D7_0309800, Fig. 6c).…”

Section: P Falciparum Encodes An Evolutionarily Conserved Diverse Smentioning

confidence: 99%

Transcriptome-wide dynamics of extensive m6A mRNA methylation during Plasmodium falciparum blood-stage development

et al. 2019

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Malaria pathogenesis results from the asexual replication of Plasmodium falciparum within human red blood cells, which relies on a precisely timed cascade of gene expression over a 48-h life cycle. Although substantial post-transcriptional regulation of this hardwired program has been observed, it remains unclear how these processes are mediated on a transcriptome-wide level. To this end, we identified mRNA modifications in the P. falciparum transcriptome and performed a comprehensive characterization of N 6-methyladenosine (m 6 A) over the course of blood-stage development. Using mass spectrometry and m 6 A RNA sequencing, we demonstrate that m 6 A is highly developmentally regulated, exceeding m 6 A levels known in any other eukaryote. We characterize a distinct m 6 A writer complex and show that knockdown of the putative m 6 A methyltransferase, PfMT-A70, by CRISPR interference leads to increased levels of transcripts that normally contain m 6 A. In accordance, we find an inverse correlation between m 6 A methylation and mRNA stability or translational efficiency. We further identify two putative m 6 Abinding YTH proteins that are likely to be involved in the regulation of these processes across the parasite's life cycle. Our data demonstrate unique features of an extensive m 6 A mRNA methylation programme in malaria parasites and reveal its crucial role in dynamically fine-tuning the transcriptional cascade of a unicellular eukaryote.

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Characterization of mRNA polyadenylation in the apicomplexa

Cited by 24 publications

References 58 publications

mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites

mRNA Polyadenylation Machineries in Intestinal Protozoan Parasites

Nanopore direct RNA sequencing maps an Arabidopsis N6 methyladenosine epitranscriptome

Transcriptome-wide dynamics of extensive m6A mRNA methylation during Plasmodium falciparum blood-stage development

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