Characterization And Decay Properties Of The Catalytic Subunits Of RNA Exosome-like Complex In Plasmodium Falciparum

The eukaryotic exosome is an exonuclease complex of 10-11 essential subunits, which is evolutionarily conserved. The exosome, one of the most important 3’→5’ exonucleases, is involved in the 3’-processing and degradation of many RNA species and it plays significant roles in the normal growth and development and regulation in eukaryote. Plasmodium falciparum, the pathogen of severe malaria, belongs to protozoa. And the parasite brings great harm to human health. However, the mechanism of RNA processing and degradation and the RNA exosome in P. falciparum have not been adequately studied until now. This may influence people to comprehensively elucidate the mechanism that how the parasite deals with the complex life cycle and the pressure of host immune response.Proteom-wide bioinformatics analysis revealed that there also existed a RNA exosome-like complex containing at least eight subunits in P. falciparum. Based on the conserved domains analysis and other researches, four catalytic subunits(Pf Rrp4, Pf Rrp41, Pf Dis3, Pf Rrp6) were selected for further studies. Taking P. falciparum 3D7 strain as the research object, quantitative real-time PCR assay was carried out to detect the transcription of these catalytic subunits throughout the asexual stage with the seryl-t RNAsynthetase gene as the internal control. Recombinant expression vectors were constructed and transformed into E. coli. His-tag and GST-tag fusion proteins were expressed and purified in the prokaryotic expression system. His-tag fusion proteins were used to immunize the New Zealand rabbits to prepare polyclonal antibody. Western blot and indirect immunofluorescence assay were performed to determine the expression and subcellular localization of these native proteins in malaria parasite. While the purified GST-tag fusion proteins were used for the characters analysis of RNA degradation in vitro.The quantitative real-time PCR assay indicated that although the transcript levels varied, steady transcription of these genes of the catalytic subunits occurred throughout the erythrocytic stage. It suggested that these subunits may be indispensable for the growth and development. Indirect immunofluorescence analysis revealed the native proteins predominantly expressed in the cytoplasm adjacent to the nuclear membrane in the parasite. Pf Rrp41, Pf Dis3, and Pf Rrp6 exhibited catalytic activity toward single-stranded RNA(ss RNA), whereas Pf Rrp4 and Pf Rrp42 showed no processing activity toward ss RNA or double-stranded RNA(ds RNA). Furthermore, the ss RNA-processing activity was divalent metal ion dependent. And their degradation activity is inversely proportional to the ion concentration within a certain metal ion concentration range. K+ was necessary for the ss RNA cleavage by Pf Dis3 and Pf Rrp6 but not for that by Pf Rrp41. And the RNase activity of Pf Dis3 or Pf Rrp6 achieved their peak under the concentration of about 100 m M K+.In conclusion, there is also a RNA exosome-like complex that participates in the RNA processing and decay in P. falciparum. Three catalytic subunits, termed Pf Rrp41, Pf Dis3, and Pf Rrp6, are believed to possess ss RNA degradation activity. Together, these findings lay the foundation for resolving the architecture of exosome and pave ways for a deeper understanding of RNA metabolism in the malarial parasite P. falciparum.