Biology subjects

Merrick, C. J.

Publications and source records attributed to Merrick, C. J..

5 recordsLinked to original sources

bioRxiv2021

DNA replication dynamics during erythrocytic schizogony in the malaria parasites Plasmodium falciparum and Plasmodium knowlesi

Malaria parasites are unusual, early-diverging protozoans with non-canonical cell cycles. They do not undergo binary fission, but divide primarily by schizogony. This is a syncytial mode of replication involving asynchronous production of multiple nuclei within the same cytoplasm, culminating in a single mass cytokinesis event. The rate and efficiency of parasite replication is fundamentally important to malarial disease, which tends to be severe in hosts with high parasite loads. Here, we have studied for the first time the dynamics of schizogony in two human malaria parasite species, Plasmodium falciparum and Plasmodium knowlesi. These differ in their cell-cycle length, the number of progeny produced and the genome composition, among other factors. Comparing them could therefore yield new information about the parameters and limitations of schizogony. We report that the dynamics of schizogony differ significantly between these two species, most strikingly in the gap phases between successive nuclear replications, which are longer in P. falciparum and shorter, but more heterogenous, in P. knowlesi. In both species, gaps become longer as schizogony progresses, whereas each period of active replication grows shorter. In both species there is also extreme variability between individual cells, with some schizonts producing many more nuclei than others, and some individual nuclei arresting their replication for many hours while adjacent nuclei continue to replicate. The efficiency of schizogony is probably influenced by a complex set of factors in both the parasite and its host cell.

microbiology↗

bioRxiv2021

Dynamics of DNA replication during male gametogenesis in the malaria parasite Plasmodium falciparum

Malaria parasites undergo a single phase of sexual reproduction in their complex lifecycle, during which they cycle between mosquito and vertebrate hosts. Sexual reproduction occurs only at the point when parasites move into the mosquito host. It involves specialised, sexually committed cells called gametocytes, which develop very rapidly into mature gametes and then mate inside the mosquito midgut. The gamete development process is unique, involving unprecedentedly fast replication and cell division to produce male gametes. A single male gametocyte replicates its [~]23Mb genome three times over to produce 8 genomes, segregates these into newly-assembled flagellated gamete cells and releases them to seek out female gametes, all within [~]15 minutes. Here, for the first time, we use fluorescent labelling of de novo DNA synthesis to follow this process at the whole-cell and single-molecule levels, yielding several novel observations. Firstly, we confirm that no DNA replication occurs before gametogenesis is triggered, although the origin recognition complex protein Orc1 is abundant even in immature gametocytes. Secondly, between repeated rounds of DNA replication there is no detectable karyokinesis - in contrast to the repeated replicative rounds that occur in asexual schizonts. Thirdly, cytokinesis is clearly uncoupled from DNA replication, and can occur even if replication fails, implying a lack of cell cycle checkpoints. Finally the single-molecule dynamics of DNA replication are entirely different from those in asexual schizonts.

microbiology↗

bioRxiv2021

Plasmodium falciparum GBP2 is a telomere-associated protein that binds to G-quadruplex DNA and RNA

In the early-diverging protozoan parasite Plasmodium, few telomere-binding proteins have been identified and several are unique. Plasmodium telomeres, like those of most eukaryotes, contain guanine-rich repeats that can form G-quadruplex structures. In model systems, quadruplex-binding drugs can disrupt telomere maintenance and some quadruplex-binding drugs are potent anti-plasmodial agents. Therefore, telomere-interacting and quadruplex-interacting proteins may offer new targets for anti-malarial therapy. Here, we report that P. falciparum GBP2 is such a protein. It was identified via Proteomics of Isolated Chromatin fragments, applied here for the first time in Plasmodium. In vitro, PfGBP2 binds specifically to G-rich telomere repeats in quadruplex form and it can also bind to G-rich RNA. In vivo, PfGBP2 partially colocalises with the known telomeric protein HP1 but is also found in the cytoplasm, probably due to its affinity for RNA. Consistently, its interactome includes numerous RNA-associated proteins. PfGBP2 is evidently a multifunctional DNA/RNA-binding factor in Plasmodium.

microbiology↗

bioRxiv2021

The in vivo RNA structurome of the malaria parasite Plasmodium falciparum, a protozoan with an A/T-rich transcriptome

Plasmodium falciparum, a protozoan parasite and causative agent of human malaria, has one of the most A/T-biased genomes sequenced to date. This may give the genome and the transcriptome unusual structural features. Recent progress in sequencing techniques has made it possible to study the secondary structures of RNA molecules at the transcriptomic level. Thus, in this study we produced the in vivo RNA structurome of a protozoan parasite with a highly A/U-biased transcriptome. We showed that it is possible to probe the secondary structures of P. falciparum RNA molecules in vivo using two different chemical probes, and obtained structures for more than half of all transcripts in the transcriptome. These showed greater stability (lower free energy) than the same structures modelled in silico, and structural features appeared to influence translation efficiency and RNA decay. Finally, we compared the P. falciparum RNA structurome with the predicted RNA structurome of an A/T-balanced species, P. knowlesi, finding a bias towards lower overall transcript stability and more hairpins and multi-stem loops in P. falciparum. This unusual protozoan RNA structurome will provide a basis for similar studies in other protozoans and also in other unusual genomes.

microbiology↗

bioRxiv2021

G-quadruplex RNA motifs influence gene expression in the malaria parasite Plasmodium falciparum

G-quadruplexes are non-helical secondary structures that can fold in vivo in both DNA and RNA. In human cells, they can influence replication, transcription and telomere maintenance in DNA, or translation, transcript processing and stability of RNA. We have previously showed that G-quadruplexes are detectable in the DNA of the malaria parasite Plasmodium falciparum, despite a very highly A/T-biased genome with unusually few guanine-rich sequences. Here, we show that RNA G-quadruplexes can also form in P. falciparum RNA, using rG4-seq for transcriptome-wide structure-specific RNA probing. Many of the motifs, detected here via the rG4seeker pipeline, have non-canonical forms and would not be predicted by standard in silico algorithms. However, in vitro biophysical assays verified the formation of non-canonical motifs. The G-quadruplexes in the P. falciparum transcriptome are frequently clustered in certain genes and associated with regions encoding low-complexity peptide repeats. They are overrepresented in particular classes of genes, notably those that encode PfEMP1 virulence factors, stress response genes and DNA binding proteins. In vitro translation experiments and in vivo measures of translation efficiency showed that G-quadruplexes can influence the translation of P. falciparum mRNAs. Thus, the G-quadruplex is a novel player in post-transcriptional regulation of gene expression in this major human pathogen.

microbiology↗