Browsing by Subject "RNA, Protozoan"

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Open Access
In vivo architecture of the telomerase RNA catalytic core in Trypanosoma brucei.
(Nucleic acids research, 2021-12) Dey, Abhishek; Monroy-Eklund, Anais; Klotz, Kaitlin; Saha, Arpita; Davis, Justin; Li, Bibo; Laederach, Alain; Chakrabarti, Kausik
Telomerase is a unique ribonucleoprotein (RNP) reverse transcriptase that utilizes its cognate RNA molecule as a template for telomere DNA repeat synthesis. Telomerase contains the reverse transcriptase protein, TERT and the template RNA, TR, as its core components. The 5'-half of TR forms a highly conserved catalytic core comprising of the template region and adjacent domains necessary for telomere synthesis. However, how telomerase RNA folding takes place in vivo has not been fully understood due to low abundance of the native RNP. Here, using unicellular pathogen Trypanosoma brucei as a model, we reveal important regional folding information of the native telomerase RNA core domains, i.e. TR template, template boundary element, template proximal helix and Helix IV (eCR4-CR5) domain. For this purpose, we uniquely combined in-cell probing with targeted high-throughput RNA sequencing and mutational mapping under three conditions: in vivo (in WT and TERT-/- cells), in an immunopurified catalytically active telomerase RNP complex and ex vivo (deproteinized). We discover that TR forms at least two different conformers with distinct folding topologies in the insect and mammalian developmental stages of T. brucei. Also, TERT does not significantly affect the RNA folding in vivo, suggesting that the telomerase RNA in T. brucei exists in a conformationally preorganized stable structure. Our observed differences in RNA (TR) folding at two distinct developmental stages of T. brucei suggest that important conformational changes are a key component of T. brucei development.
Open Access
Single-Cell Analysis Reveals Distinct Gene Expression and Heterogeneity in Male and Female Plasmodium falciparum Gametocytes.
(mSphere, 2018-04-11) Walzer, Katelyn A; Kubicki, Danielle M; Tang, Xiaohu; Chi, Jen-Tsan Ashley
Sexual reproduction is an obligate step in the Plasmodium falciparum life cycle, with mature gametocytes being the only form of the parasite capable of human-to-mosquito transmission. Development of male and female gametocytes takes 9 to 12 days, and although more than 300 genes are thought to be specific to gametocytes, only a few have been postulated to be male or female specific. Because these genes are often expressed during late gametocyte stages and for some, male- or female-specific transcript expression is debated, the separation of male and female populations is technically challenging. To overcome these challenges, we have developed an unbiased single-cell approach to determine which transcripts are expressed in male versus female gametocytes. Using microfluidic technology, we isolated single mid- to late-stage gametocytes to compare the expression of 91 genes, including 87 gametocyte-specific genes, in 90 cells. Such analysis identified distinct gene clusters whose expression was associated with male, female, or all gametocytes. In addition, a small number of male gametocytes clustered separately from female gametocytes based on sex-specific expression independent of stage. Many female-enriched genes also exhibited stage-specific expression. RNA fluorescent in situ hybridization of male and female markers validated the mutually exclusive expression pattern of male and female transcripts in gametocytes. These analyses uncovered novel male and female markers that are expressed as early as stage III gametocytogenesis, providing further insight into Plasmodium sex-specific differentiation previously masked in population analyses. Our single-cell approach reveals the most robust markers for sex-specific differentiation in Plasmodium gametocytes. Such single-cell expression assays can be generalized to all eukaryotic pathogens.IMPORTANCE Most human deaths that result from malaria are caused by the eukaryotic parasite Plasmodium falciparum The only form of this parasite that is transmitted to the mosquito is the sexual form, called the gametocyte. The production of mature gametocytes can take up to 2 weeks and results in phenotypically distinct males and females, although what causes this gender-specific differentiation remains largely unknown. Here, we demonstrate the first use of microfluidic technology to capture single gametocytes and determine their temporal sex-specific gene expression in an unbiased manner. We were able to determine male or female identity of single cells based on the upregulation of gender-specific genes as early as mid-stage gametocytes. This analysis has revealed strong markers for male and female gametocyte differentiation that were previously concealed in population analyses. Similar single-cell analyses in eukaryotic pathogens using this method may uncover rare cell types and heterogeneity previously masked in population studies.