title: Strand-specific RNA sequencing in Plasmodium falciparum malaria identifies developmentally regulated long non-coding RNA and circular RNA.
abstract: BACKGROUND
  The human malaria parasite Plasmodium falciparum has a complex and multi-stage life cycle that requires extensive and precise gene regulation to allow invasion and hijacking of host cells, transmission, and immune escape. To date, the regulatory elements orchestrating these critical parasite processes remain largely unknown. Yet it is becoming increasingly clear that long non-coding RNAs (lncRNAs) could represent a missing regulatory layer across a broad range of organisms.
  
  
  RESULTS
  To investigate the regulatory capacity of lncRNA in P. falciparum, we harvested fifteen samples from two time-courses. Our sample set profiled 56 h of P. falciparum blood stage development. We then developed and validated strand-specific, non-polyA-selected RNA sequencing methods, and pursued the first assembly of P. falciparum strand-specific transcript structures from RNA sequencing data. This approach enabled the annotation of over one thousand lncRNA transcript models and their comprehensive global analysis: coding prediction, periodicity, stage-specificity, correlation, GC content, length, location relative to annotated transcripts, and splicing. We validated the complete splicing structure of three lncRNAs with compelling properties. Non-polyA-selected deep sequencing also enabled the prediction of hundreds of intriguing P. falciparum circular RNAs, six of which we validated experimentally.
  
  
  CONCLUSIONS
  We found that a subset of lncRNAs, including all subtelomeric lncRNAs, strongly peaked in expression during invasion. By contrast, antisense transcript levels significantly dropped during invasion. As compared to neighboring mRNAs, the expression of antisense-sense pairs was significantly anti-correlated during blood stage development, indicating transcriptional interference. We also validated that P. falciparum produces circRNAs, which is notable given the lack of RNA interference in the organism, and discovered that a highly expressed, five-exon antisense RNA is poised to regulate P. falciparum gametocyte development 1 (PfGDV1), a gene required for early sexual commitment events.
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. k8broadbent@post.harvard.edu.|Broad Institute, Cambridge, Massachusetts, USA. k8broadbent@post.harvard.edu.
authors.forename: Kate M
authors.initials: KM
authors.lastname: Broadbent
authors.affiliation: FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA. jbroadbent@vetmed.wsu.edu.|Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USA. jbroadbent@vetmed.wsu.edu.
authors.forename: Jill C
authors.initials: JC
authors.lastname: Broadbent
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA. uribacke@hsph.harvard.edu.|Department of Cell and Molecular Biology, BMC, Uppsala University, Uppsala, Sweden. uribacke@hsph.harvard.edu.
authors.forename: Ulf
authors.initials: U
authors.lastname: Ribacke
authors.affiliation: Broad Institute, Cambridge, Massachusetts, USA. dfwirth@hsph.harvard.edu.|Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, USA. dfwirth@hsph.harvard.edu.
authors.forename: Dyann
authors.initials: D
authors.lastname: Wirth
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. john_rinn@harvard.edu.|Broad Institute, Cambridge, Massachusetts, USA. john_rinn@harvard.edu.|Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA. john_rinn@harvard.edu.
authors.forename: John L
authors.initials: JL
authors.lastname: Rinn
authors.affiliation: Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA. psabeti@oeb.harvard.edu.|Broad Institute, Cambridge, Massachusetts, USA. psabeti@oeb.harvard.edu.|FAS Center for Systems Biology, Harvard University, Cambridge, Massachusetts, USA. psabeti@oeb.harvard.edu.|Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts, USA. psabeti@oeb.harvard.edu.
authors.forename: Pardis C
authors.initials: PC
authors.lastname: Sabeti
mesh_terms: D001769:Blood / Q000469:parasitology; D018507:Gene Expression Regulation, Developmental; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006801:Humans; D058977:Molecular Sequence Annotation; D010963:Plasmodium falciparum / Q000235:genetics / Q000254:growth & development*; D012326:RNA Splicing; D062085:RNA, Long Noncoding / Q000235:genetics*; D016053:RNA, Protozoan / Q000097:blood* / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D062085:RNA, Long Noncoding; D016053:RNA, Protozoan