Functional Roles Of Intermediate-size NcRNA(RUF6-15) In The Growth Process Of P. Falciparum 3D7 In Intraerythrocytic Stages

Malaria is a kind of very severe tropical diseases. The most responsible for the deadliest form human malaria is caused by P. falciparum (P.falciparum). The P. falciparum has very complex gene regulation system to adapt to the changing lifestyle and the complexity of life environment, needing two types of host-female anopheles and people. There are two stages including erythrocytic stage and liver stage in the human body, the clinical symptoms of malaria are caused by the intraerythrocytic stages of the parasite, which multiply inside the host’s red blood cells (RBCs).The completion of whole genome sequencing and subsequent chip technology, the rapid development of high-throughput sequencing technology, noncoding RNA (ncRNA) of P. falciparum is becoming more and more aroused concern. It can be divided into three classifications according to the length:less than 50 nt、50-500 nt、more than 500 nt(Long non-coding RNA, lncRNA). The homologous regions of Dicer, Piwi, PAZ or RdRp are not found in the database data of P. falciparum through comparative genomics analysis. So that there is no existence of endogenous small RNAs in P. falciparum. Therefore, medium length ncRNA has caused widely attention. The study of constitutive medium length ncRNA is fairly mature, such as tRNA and rRNA, which not translated into proteins, but involved in the translation process; In addition to snRNA and snoRNA, which involved in RNA splicing and RNA modification, etc. Chakrabarti found there were six new RNA coding genes of unknown function(RUFs), which falled into known RNA functional categories. More interesting, RUF5 and RUF6 was identified only in the P. falciparum and P. reichenowi. In P. falciparum, these RUF6 clusters had 15 highly homologous transcripts. These clusters are arranged flanking malarial surface antigens, namely RIFIN or VAR genes at the middle or end of the chromosomes. PfEMPl was a family of proteins encoded by the var multi-gene family. It’s a parasite protein family of key importance in P. falciparum malaria immunity and pathogenesis, mediated antigen variation and the key adhesion molecular of the red blood cell binding to microvascular. The proteins involved in the knobs formation of infected red blood cells surface, and then made the malaria parasite escape the host immune attack by adhesion in internal organs capillaries, and cause fatal pathological damage. Very little is known about the RUF6. Here, we focused on the RUFs involved in the regulation of Var gene and the growth process of P. falciparum3D7 in intraerythrocytic stages.Here, by Northern Blot、FISH、Electransfection、RNA-Seq、qRT-PCR、 Dual-luciferase reporter gene assay, we firstly reveal the biological function of 15th in the RUF6 homologous clusters (RUF6-15). The level of transcription of RUF6-15 is more and more highly along with the growth of the growing development in the wildtype 3D7 strain, which paly regulatory role in the nuclei after the transcription. Transfection overexpressing of RUF6-15 strain grow faster compared with control strain and RUF6-15 knockdown strain grow slower compared with the control strain. High-throughput sequencing data and experiment verification shows that RUF6-15 target Var gene families. Dual-luciferase reporter gene assay reveals RUF6-15 activate 5’UTR region of var genes to regulate the var gene transcription. Cytoadherence assay data shows that the overexpression strain have obvious stronger ability of adhesion than control, while the adhesion ability of knockdown strain slightly decreased.Here, we research the function of RUF6 is significant to understand the pathogenesis of malaria and to develop new prevention and control measures.