title: Genome wide screening of RNAi factors of Sf21 cells reveal several novel pathway associated proteins.
abstract: BACKGROUND
  RNA interference (RNAi) leads to sequence specific knock-down of gene expression and has emerged as an important tool to analyse gene functions, pathway analysis and gene therapy. Although RNAi is a conserved cellular process involving common elements and factors, species-specific differences have been observed among different eukaryotes. Identification of components for RNAi pathway is pursued intensively and successful genome-wide screens have been performed for components of RNAi pathways in various organisms. Functional comparative genomics analysis offers evolutionary insight that forms basis of discoveries of novel RNAi-factors within related organisms. Keeping in view the academic and commercial utility of insect derived cell-line from Spodoptera frugiperda, we pursued the identification and functional analysis of components of RNAi-machinery of Sf21 cell-line using genome-wide application.
  
  
  RESULTS
  The genome and transcriptome of Sf21 was assembled and annotated. In silico application of comparative genome analysis among insects allowed us to identify several RNAi factors in Sf21 line. The candidate RNAi factors from assembled genome were validated by knockdown analysis of candidate factors using the siRNA screens on the Sf21-gfp reporter cell-line. Forty two (42) potential factors were identified using the cell based assay. These include core RNAi elements including Dicer-2, Argonaute-1, Drosha, Aubergine and auxiliary modules like chromatin factors, RNA helicases, RNA processing module, signalling allied proteins and others. Phylogenetic analyses and domain architecture revealed that Spodoptera frugiperda homologs retained identity with Lepidoptera (Bombyx mori) or Coleoptera (Tribolium castaneum) sustaining an evolutionary conserved scaffold in post-transcriptional gene silencing paradigm within insects.
  
  
  CONCLUSIONS
  The database of RNAi-factors generated by whole genome association survey offers comprehensive outlook about conservation as well as specific differences of the proteins of RNAi machinery. Understanding the interior involved in different phases of gene silencing also offers impending tool for RNAi-based applications.
authors.forename: Subhanita
authors.initials: S
authors.lastname: Ghosh
authors.forename: Pavan Kumar
authors.initials: PK
authors.lastname: Kakumani
authors.forename: Ajit
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. pawanmal@gmail.com.
authors.forename: Pawan
authors.initials: P
authors.lastname: Malhotra
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Mukherjee
authors.forename: Raj K
authors.initials: RK
authors.lastname: Bhatnagar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D060565:Argonaute Proteins / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics; D002460:Cell Line; D055028:Comparative Genomic Hybridization; D049750:Genome, Insect; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D020365:RNA Helicases / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D043244:Ribonuclease III / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics; D016415:Sequence Alignment; D018411:Spodoptera / Q000145:classification / Q000166:cytology / Q000235:genetics*; D059467:Transcriptome
chemical_list: D060565:Argonaute Proteins; D019476:Insect Proteins; D034741:RNA, Small Interfering; D043244:Ribonuclease III; D020365:RNA Helicases