Exportin-5, RanGTP, microRNA complex
Human (Homo sapiens), dog (Canis lupus familiaris)
MicroRNAs, which are a class of non-coding RNA, present in a wide range of eukaryotes, fungi, plants and animals. MicroRNAs play important roles in regulation of biological process, including development, cell proliferation, cell differentiation and apoptosis, transposon silencing and antiviral defense. MicroRNAs are initially transcribed in nucleus as capped and polyadenylated pri-microRNAs with several kilobases in length. The pri-microRNAs are processed by the nuclear RNase III enzyme Drosha to generate ~65nt pre-microRNAs that form stem-loop structures containing 2-nt 3’ overhangs. The pre-microRNAs are translocated from nucleus to cytoplasm through nuclear pore complex by exportin-5 in a RanGTP-dependent manner. In the cytoplasm, the pre-microRNAs are further processed by the cytoplasmic RNase III enzyme Dicer, which excises a ~22 bp RNA duplex. One strand of the duplex is bound to its target mRNA with imperfect complementarity, usually within the 3’–untranslated region, assisted by the RNA-induced silencing complex (RISC).
The crystal structure of the exportin-5:RanGTP:pre-microRNA ternary complex was determined at 2.9 angstrom resolution. The ternary complex defines the structural details of the novel interactions between two proteins and pre-mcroiRNA double strand explains both how exportin-5:RanGTP interacts with such a large set of pre-microRNAs and how exportin-5:RanGTP protects pre-microRNA from digestion by RNase during nuclear translocation (figure 1).
The exportin-5:RanGTP:pre-microRNA complex exhibits an ellipsoidal shape with dimensions 65 x 80 x 110 angstrom. The exportin-5:RanGTP complex has a baseball mitt-like space in which pre-microRNA is packed. A tunnel-like structure at the bottom of the mitt connects the inner space of the mitt with the outer space. A double-stranded stem of the pre-microRNA is packed into the mitt, and the 2-nt 3’ overhang is located in the tunnel. Exp-5:RanGTP binds most regions of the pre-miRNA, protecting it from ribonuclease digestion during travel from the nucleus to the cytoplasm.
The 2-nt 3’ overhang structure is a characteristic feature of pre-microRNA. The protruding unpaired 2-nt 3’ end is inserted deep into the tunnel. The 2-nt 3’ overhang structure in the tunnel is stabilized by a number of hydrogen bonds and salt bridges with amino acid residues of exportin-5. Because all interactions involve atoms of the sugar-phosphate backbone, 2-nt 3’ overhang recognition by exportin-5 is independent of RNA sequence.
Although exportin-5 is an acidic protein with pI=5.6, the protein has localized positive charges from basic residues on the inner surface of the mitt that interact with the negatively charged dsRNA. The stem of the pre-microRNA is roughly recognized through a broad range of positively charged inner surface residues of the Exp-5:RanGTP mitt.
Protein Data Bank (PDB)
author: Eiki Yamashita