Person:

Broadbent, Kate Mariel

Background:

Mounting evidence suggests a major role for epigenetic feedback in Plasmodium falciparum transcriptional regulation. Long non-coding RNAs (lncRNAs) have recently emerged as a new paradigm in epigenetic remodeling. We therefore set out to investigate putative roles for lncRNAs in P. falciparum transcriptional regulation.

Results:

We used a high-resolution DNA tiling microarray to survey transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. We identified 872 protein-coding genes and 60 putative P. falciparum lncRNAs under developmental regulation during the parasite's pathogenic human blood stage. Further characterization of lncRNA candidates led to the discovery of an intriguing family of lncRNA telomere-associated repetitive element transcripts, termed lncRNA-TARE. We have quantified lncRNA-TARE expression at 15 distinct chromosome ends and mapped putative transcriptional start and termination sites of lncRNA-TARE loci. Remarkably, we observed coordinated and stage-specific expression of lncRNA-TARE on all chromosome ends tested, and two dominant transcripts of approximately 1.5 kb and 3.1 kb transcribed towards the telomere.

Conclusions:

We have characterized a family of 22 telomere-associated lncRNAs in P. falciparum. Homologous lncRNA-TARE loci are coordinately expressed after parasite DNA replication, and are poised to play an important role in P. falciparum telomere maintenance, virulence gene regulation, and potentially other processes of parasite chromosome end biology. Further study of lncRNA-TARE and other promising lncRNA candidates may provide mechanistic insight into P. falciparum transcriptional regulation.

The mechanisms underpinning gene regulation in P. falciparum malaria remain largely elusive, though mounting evidence suggests a major role for epigenetic feedback. Interestingly, long non-(protein)-coding RNAs (lncRNAs) have been found to play a dominant role in initiating and guiding the transcriptional, epigenetic, and post-transcriptional status of specific loci across a broad range of organisms. LncRNAs are uniquely poised to act co-transcriptionally on neighboring loci, and/or to remain physically tethered at their site of origin, and through sequence-specific binding activities can impart temporal and spatial specificity to ubiquitously expressed nuclear protein complexes. Proteins, on the other hand, must be translated in the cytoplasm, and hence lose memory of their transcriptional origins. Encouraged by these features of lncRNAs, we set out to investigate the regulatory capacity of P. falciparum lncRNAs on a genome-wide scale. First, we surveyed transcriptional activity across approximately one quarter of the P. falciparum genome using a custom high-density DNA tiling array. We predicted a set of 60 developmentally regulated intergenic lncRNAs, and found that many of these novel loci neighbored genes involved in parasite survival or virulence pathways. Remarkably, upon further analysis of intergenic lncRNA properties, we discovered a family of twenty-two telomere-associated lncRNAs encoded in the telomere-associated repetitive element (TARE) region of P. falciparum chromosome ends. We found that each lncRNA-TARE was encoded adjacent and divergent to a subtelomeric var virulence gene. Moreover, we found that lncRNA-TARE expression was sharply induced between the parasite DNA replication and cell division cycles, that lncRNA-TARE loci contained numerous transcription factor binding sites only otherwise found in subtelomeric var promoter regions, and that the GC content and evolutionary sequence conservation of lncRNA-TAREs was similar to that of P. falciparum ribosomal RNA.

Next, we set out to assemble P. falciparum intergenic lncRNA and antisense RNA transcript structures using state-of-the-art deep sequencing and computational tools. Towards this end, we harvested an unprecedented sample set that finely maps temporal changes across 56 hours of P. falciparum blood stage development, and developed and validated strand-specific, non-polyA-selected RNA sequencing methods. This enabled the annotation of over one thousand high-confidence, bona fide lncRNA transcript models, and their comprehensive global analysis. We discovered an enrichment of negatively correlated, tail-to-tail overlapping sense-antisense transcript pairs, suggesting a conserved role for antisense-mediated transcriptional interference in P. falciparum gene regulation. We also discovered a highly correlated spliced antisense counterpart to a gene required for sexual commitment, that the expression of an intriguing subset of antisense transcripts significantly dropped during parasite invasion, and that lncRNA-TARE and 'sterile' var virulence gene transcription was markedly up-regulated during parasite invasion. Lastly, we predicted over one thousand circular RNAs (circRNAs), and validated six circRNA transcript structures. Importantly, this thesis work represents the first focused investigation of lncRNAs in P. falciparum malaria, with the characterization of a compelling family of telomere-associated lncRNAs and numerous antisense RNAs. The data, methods, and results herein offer exceptional technological advancements coupled with compelling insights into the biology of the devastating human pathogen P. falciparum malaria. It is my hope that this work will facilitate future P. falciparum lncRNA functional studies and the strand-specific profiling of additional P. falciparum samples.