title: Elucidation of RNA binding regions of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) to transcripts of a chromatin remodeling protein essential for spermatogenesis.
abstract: BACKGROUND
  Gonadotropin-regulated testicular RNA helicase (GRTH) is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig and germ cells. It is a transport protein of mRNAs from nucleus to cytoplasmic sites and is essential for posttranscriptional regulation and completion of spermatogenesis. Transition protein 2 (Tp2), which associates with GRTH and is required for spermatid elongation, failed to express in GRTH null mice with impaired mRNA nuclear export. The present study determines GRTH binding motifs/regions that associate with Tp2 mRNA transcripts.
  
  
  METHODS
  RNA-protein interaction was analyzed using biotin-labeled electrophoretic mobility gel shift assays (EMSA). 3'-biotin-labeled RNA (Tp2) was incubated with mGRTH protein (full length/sequential deletion of specific and conserved RNA helicase motifs of GRTH) expressed from in vitro TNT coupled reticulocyte lysate system. Binding specificity was further elucidated by mutagenesis and antibody supershift analysis.
  
  
  RESULTS
  RNA-EMSA revealed that the 3' UTR of Tp2 RNA (127 nt from TGA) was retarded in presence of full length GRTH. Nucleotide sequences downstream of TGA of the Tp2 transcript (1-47 and 78-127 nt) are important for binding to GRTH. Sequential deletions/point mutations in GRTH revealed region(s) of conserved binding motifs of RNA helicases (Ia and V) essential for GRTH binding to Tp2 mRNA.
  
  
  CONCLUSIONS
  Our studies provide insights into the association of Tp2 expression via binding to the conserved RNA binding motifs of GRTH protein and the basis for understanding GRTH in the regulation of the genes essential for germ cell elongation and completion of spermatogenesis.
authors.forename: Ruifeng
authors.initials: R
authors.lastname: Yang
authors.forename: Chon-Hwa
authors.initials: CH
authors.lastname: Tsai-Morris
authors.forename: Jung Hoon
authors.initials: JH
authors.lastname: Kang
authors.forename: Maria L
authors.initials: ML
authors.lastname: Dufau
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D042002:Chromatin Assembly and Disassembly; D053487:DEAD-box RNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D005854:Germ Cells / Q000378:metabolism; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D013091:Spermatogenesis
chemical_list: D020413:3' Untranslated Regions; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; C489505:Tnp2 protein, mouse; C487765:Ddx25 protein, mouse; D053487:DEAD-box RNA Helicases