title: Helical defects in microRNA influence protein binding by TAR RNA binding protein.
abstract: BACKGROUND
  MicroRNAs (miRNAs) are critical post-transcriptional regulators of gene expression. Their precursors have a globally A-form helical geometry, which prevents most proteins from identifying their nucleotide sequence. This suggests the hypothesis that local structural features (e.g., bulges, internal loops) play a central role in specific double-stranded RNA (dsRNA) selection from cellular RNA pools by dsRNA binding domain (dsRBD) containing proteins. Furthermore, the processing enzymes in the miRNA maturation pathway require tandem-dsRBD cofactor proteins for optimal function, suggesting that dsRBDs play a key role in the molecular mechanism for precise positioning of the RNA within these multi-protein complexes. Here, we focus on the tandem-dsRBDs of TRBP, which have been shown to bind dsRNA tightly.
  
  
  RESULTS
  We present a combination of dsRNA binding assays demonstrating that TRBP binds dsRNA in an RNA-length dependent manner. Moreover, circular dichroism data shows that the number of dsRBD moieties bound to RNA at saturation is different for a tandem-dsRBD construct than for constructs with only one dsRBD per polypeptide, revealing another reason for the selective pressure to maintain multiple domains within a polypeptide chain. Finally, we show that helical defects in precursor miRNA alter the apparent dsRNA size, demonstrating that imperfections in RNA structure influence the strength of TRBP binding.
  
  
  CONCLUSIONS
  We conclude that TRBP is responsible for recognizing structural imperfections in miRNA precursors, in the sense that TRBP is unable to bind imperfections efficiently and thus is positioned around them. We propose that once positioned around structural defects, TRBP assists Dicer and the rest of the RNA-induced silencing complex (RISC) in providing efficient and homogenous conversion of substrate precursor miRNA into mature miRNA downstream.
authors.affiliation: Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Roderico
authors.initials: R
authors.lastname: Acevedo
authors.affiliation: Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Nichole
authors.initials: N
authors.lastname: Orench-Rivera
authors.affiliation: Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Kaycee A
authors.initials: KA
authors.lastname: Quarles
authors.affiliation: Department of Chemistry and Center for RNA Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Showalter
mesh_terms: D001665:Binding Sites; D002942:Circular Dichroism; D006801:Humans; D035683:MicroRNAs / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism; D000099253:Trans-Activation Responsive RNA-Binding Protein
chemical_list: D035683:MicroRNAs; D016601:RNA-Binding Proteins; D043244:Ribonuclease III; D000099253:Trans-Activation Responsive RNA-Binding Protein