Messenger RNA polyadenylation is a universal aspect of gene expression in eukaryotes. In well-established model organisms, this process is mediated by a conserved complex of 15–20 subunits. To better understand this process in apicomplexans, a group of unicellular parasites that causes serious disease in humans and livestock, a computational and high throughput sequencing study of the polyadenylation complex and poly(A) sites in several species was conducted. BLAST-based searches for orthologs of the human polyadenylation complex yielded clear matches to only two—poly(A) polymerase and CPSF73—of the 19 proteins used as queries in this analysis. As the human subunits that recognize the AAUAAA polyadenylation signal (PAS) were not immediately obvious, a computational analysis of sequences adjacent to experimentally-determined apicomplexan poly(A) sites was conducted. The results of this study showed that there exists in apicomplexans an A-rich region that corresponds in position to the AAUAAA PAS. The set of experimentally-determined sites in one species, Sarcocystis neurona, was further analyzed to evaluate the extent and significance of alternative poly(A) site choice in this organism. The results showed that almost 80% of S. neurona genes possess more than one poly(A) site, and that more than 780 sites showed differential usage in the two developmental stages–extracellular merozoites and intracellular schizonts–studied. These sites affected more than 450 genes, and included a disproportionate number of genes that encode membrane transporters and ribosomal proteins. Taken together, these results reveal that apicomplexan species seem to possess a poly(A) signal analogous to AAUAAA even though genes that may encode obvious counterparts of the AAUAAA-recognizing proteins are absent in these organisms. They also indicate that, as is the case in other eukaryotes, alternative polyadenylation is a widespread phenomenon in S. neurona that has the potential to impact growth and development.
Digital Object Identifier (DOI)
ATS was supported by a Research Experience for Undergraduates award under the auspices of National Science Foundation (www.nsf.gov) Awards MCB-1243849 and IOS-1353354 made to Dr. Hunt. Other support was from the USDA National Institute of Food and Agriculture (Hatch project accession # 227792; www.nifa.usda.gov) and funds from the Amerman Family Equine Research Fund, both to Dr. Howe.
The sequencing data generated in this study are available under Bioproject ID PRJNA436572. All other relevant data are within the paper and its Supporting Information files.
S1 Fig. Alignments of WDR33, PABN1, CFIm25 and CPSF30. https://doi.org/10.1371/journal.pone.0203317.s001 (PDF)
S2 Fig. Nucleotide composition surrounding genomic poly(A) tracts in three apicomplexan genomes. https://doi.org/10.1371/journal.pone.0203317.s002 (PDF)
S3 Fig. Number of poly(A) site clusters per gene in S. neurona. https://doi.org/10.1371/journal.pone.0203317.s003 (PDF)
S1 File. Summary of sequence libraries. https://doi.org/10.1371/journal.pone.0203317.s004 (XLSX)
S2 File. Analysis of mRNA lengthening/shortening during development in S. neurona. https://doi.org/10.1371/journal.pone.0203317.s005 (XLSX)
S3 File. Gene expression summaries. https://doi.org/10.1371/journal.pone.0203317.s006 (XLSX)
S4 File. List of poly(A) sites in S. neurona. https://doi.org/10.1371/journal.pone.0203317.s007 (XLSX)
S5 File. Analysis of differential poly(A) site usage in S. neurona. https://doi.org/10.1371/journal.pone.0203317.s008 (XLSX)
S1 Table. Oligonucleotides used in this study. https://doi.org/10.1371/journal.pone.0203317.s009 (DOCX)
Stevens, Ashley T.; Howe, Daniel K.; and Hunt, Arthur G., "Characterization of mRNA Polyadenylation in the Apicomplexa" (2018). Plant and Soil Sciences Faculty Publications. 115.
S1 Fig. Alignments of WDR33, PABN1, CFIm25 and CPSF30.
journal.pone.0203317.s002.pdf (73 kB)
S2 Fig. Nucleotide composition surrounding genomic poly(A) tracts in three apicomplexan genomes.
journal.pone.0203317.s003.pdf (29 kB)
S3 Fig. Number of poly(A) site clusters per gene in S. neurona.
journal.pone.0203317.s004.xlsx (12 kB)
S1 File. Summary of sequence libraries.
journal.pone.0203317.s005.xlsx (2987 kB)
S2 File. Analysis of mRNA lengthening/shortening during development in S. neurona.
journal.pone.0203317.s006.xlsx (2354 kB)
S3 File. Gene expression summaries.
journal.pone.0203317.s007.xlsx (1126 kB)
S4 File. List of poly(A) sites in S. neurona.
journal.pone.0203317.s008.xlsx (1298 kB)
S5 File. Analysis of differential poly(A) site usage in S. neurona.
journal.pone.0203317.s009.docx (15 kB)
S1 Table. Oligonucleotides used in this study.