Transient silencing of Plasmodium falciparum Tudor Staphylococcal Nuclease suggests an essential role for the protein Sujatha Sunil 1 , Manzar J. Hossain 1 , Gowthaman Ramasamy, Pawan Malhotra * International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India article info Article history: Received 2 May 2008 Available online 16 May 2008 Keywords: Tudor Staphylococcal Nuclease RNAi Plasmodium Malaria abstract Plasmodium falciparum Tudor Staphylococcal Nuclease (PfTSN) has a multidomain organization and pref- erentially cleaves single stranded RNAs. PfTSN is quite distinct from its vertebrate homologues both in terms of its primary sequence and functional activity. Here, we analyzed the effect of PfTSN specific siRNA on parasite growth and development. Treatment of parasite culture with PfTSN siRNA at the late ring stage resulted in substantial inhibition in parasite growth. The PfTSN siRNA treated parasite cultures showed significant reduction in specific mRNA and PfTSN expression. Morphological examination of PfTSN siRNA treated parasites showed block in the development of parasite at the trophozoite stage. Treatment of parasites with a specific inhibitor of micrococcal nucleases, 3 0 ,5 0 -deoxythymidine biphos- phate (pdTp) resulted in similar block in parasite development, thereby suggesting that PfTSN plays an important role at the trophozoite stage of the parasite. Collectively, our findings point towards an essen- tial role for the PfTSN in the parasite’s infection cycle. Ó 2008 Elsevier Inc. All rights reserved. Plasmodium parasites cause millions of cases of malaria world- wide every year. The current strategies to reduce mortality and to prevent infection due to malaria involve preventive and thera- peutic uses of anti-malarials and reduction of human-vector con- tact. However, control of malaria in developing countries is still a major public health issue, mainly due to emergence of drug resis- tance parasites and insecticide resistance mosquitoes [1,2]. There- fore, identification of novel drug targets and development of new pharmacophores are urgently needed. Development of newer anti-parasitic drugs relies mostly on their ability to interfere with aspects of metabolism that significantly differ from human host. In this context, novel malaria parasite proteases/nucleases that are both accessible and essential to parasite development are attractive therapeutic targets [3]. Plasmodium Tudor Staphylococcal Nuclease, a member of micrococcal nuclease family possesses four nuclease domains (SN domains), a single Tudor domain and an incomplete SN domain at the C-terminus [4]. The Tudor domain of the protein is a RNA recognition motif. The PfTSN protein is mainly localized in the nu- cleus and shows Ca + dependent endonuclease activity. A human homologue of PfTSN, p100 protein lacks the essential residues for nuclease activity, but functions as a co-activator for signal trans- ducers and activator of transcription (Stats) [5,6]. P100 has been shown to be associated with Epstein-Barr virus infection in human and fungal pathogenesis in mice [7,8]. Homologues of PfTSN are in- volved in a number of cellular functions such as RNA localization and shuttling of RNA [9]. In pea plant, TSN is associated with cyto- skeleton elements [10]. A recent study showed OsTudor-SN as a component of RNA transport particle in rice and provided evi- dences for its role in storage protein RNA transport and localization [11]. TSN has also been shown to be a component of RNA-induced silencing complex (RISC), the effector complex that brings down the cleavage of homologous single-stranded mRNAs [12]. TSN spe- cifically binds hyper-edited double-stranded RNA and promotes its cleavage [13]. Since TSN gene encodes a protein that has been shown to be associated with a number of cellular functions in different organ- isms, we decided to silence the expression of this gene by RNA interference (RNAi) in P. falciparum to know about its potential as a target for the development of new anti-malarials. A number of re- cent studies have shown the usefulness of RNA interference to car- ry-out functional genomic studies in malaria parasites, even though the components of RNAi machinery have not been identi- fied yet in parasite genome database [14–19]. It is possible that RNAi related effects seen in Plasmodium spp. are due to RNAi en- zymes from host cells or due to antisense mechanisms [19]. To delineate the essential role of Plasmodium falciparum TSN in asex- ual blood stages of malaria parasite, we designed siRNA corre- sponding to PfTSN gene and treated the in vitro cultured parasites with siRNAs. Our results demonstrated that PfTSN plays a crucial role in the parasite life cycle and can be an important target for the anti-malarial therapy. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.05.033 * Corresponding author. Fax: +91 11 26162316. E-mail address: pawanm@icgeb.res.in (P. Malhotra). 1 Both the authors contributed equally. Biochemical and Biophysical Research Communications 372 (2008) 373–378 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc