Structural and functional studies of a noncanonical Dicer from Entamoeba histolytica.
RNaseIII proteins are dsRNA-specific endonucleases involved in many important biological processes, such as small RNA processing and maturation in eukaryotes. Various small RNAs have been identified in a protozoan parasite Entamoeba histolytica. EhRNaseIII is the only RNaseIII endonuclease domain (RIIID)-containing protein in E. histolytica. Here, we present three crystal structures that reveal several unique structural features of EhRNaseIII, especially the interactions between the two helixes (α1 and α7) flanking the RIIID core domain. Structure and sequence analysis indicate that EhRNaseIII is a noncanonical Dicer and it lacks a dsRBD in the C-terminal region (CTR). In vitro studies suggest that EhRNaseIII prefers to bind and cleave longer dsRNAs, generating products around 25 nucleotides in length. Truncation of the CTR or attaching the dsRBD of Aquifex aeolicus RNaseIII can enhance the binding and cleavage activities of EhRNaseIII. In combination with in vitro crosslinking assay, our results suggested that EhRNaseIII functions in a cooperative mode. We speculate that some partner proteins may exist in E. histolytica and regulates the activity of EhRNaseIII through interaction with its CTR. Our studies support that EhRNaseIII plays an important role in producing small RNAs in E. histolytica.
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai, China.|State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China.
Xiang
X
Yu
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China.
Xuhang
X
Li
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China.
Lina
L
Zheng
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai, China.
Jinbiao
J
Ma
State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai, China.
Jianhua
J
Gan
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D020134:Catalytic Domain; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics; D007477:Ions / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D059367:RNA Cleavage; D012330:RNA, Double-Stranded / Q000737:chemistry / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D007477:Ions; D008670:Metals; D012330:RNA, Double-Stranded; D043244:Ribonuclease III