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Article Name

1. Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9).

2. Structural Insight into the Mechanism of Double-Stranded RNA Processing by Ribonuclease III.

Protein Name

Name: 2EZ6

Description (2EZ6) = A. aeolicus ribonuclease III

Number of Amino Acids in (2EZ6) = 218

Number of Nucleic Acids in (2EZ6) = 56

The Type of 2EZ6 PDB = dsRNA

RNA Description of (2EZ6) = Double Stranded RNA (dsRNA) Add scene

2EZ6 is the PDBID code for the Crystal structure of A. aeolicus RNaseIII-dsRBD in complex with dsRNA published in the article listed under the article name and in Figure 6 of the original article 1.

The 2EZ6 seems to be first discovered in the "Aquifex aeolicus", which is a rod-shaped bacterium with a length of 2 to 6 micrometers and a diameter of around half a micrometer. The protein itself (2EZ6) is a 4 chain structure with a sequence form. Interestingly, such a binding mode was observed in the crystal structure of A. aeolicus RNaseIII-dsRBD in complex with short RNA duplex. In this A. aeolicus structure, the bound short RNA duplex is sandwiched between two dsRBD domains on one side and two RNaseIII domains on the other side

Background Information

The PDB 2EZ6 is the crystal structure for the protein RNaseIII. However the complete structure involves the double stranded RNA binding domain binds to RNA, which plays an important rule in the RNA silencing inducing complex (RISC). More specifically, the protein 2EZ6 is a model of the siRNA duplex, which is sandwiched between two dsRBDs in the front and D1 and D2 of RHA helicase core in the back without stereo clashes. The model represents a working model for siRNA duplex recognition and partial unwinding by the full-length RHA protein. The RNA helicase RHA is the loading factor for the RISC complex. RHA has dsRBD1/dsRBD2 domains which facilitates in binding to siRNA. Furthermore,such a binding mode was observed in the crystal structure of A. aeolicus RNaseIII-dsRBD in complex with short RNA duplex.

Although RHA is proposed to facilitate RISC assembly, which enables the binding of siRNA duplex and its interactions with Ago2, TRBP and Dicer. However, the structural and functional features of RHA in dsRNA binding and RISC assembly are largely unknown. Therefore the original study the crystal structures investigated and determined structural insights into siRNA duplex recognition and RISC assembly facilitated by RHA dsRBD domains.

RISC plays an important role as the key cellular machinery in RNAi pathways.Study showed that human RNA helicase A (DHX9) functions as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs).RISC is responsible for slicing or repressing the translation of the mRNA targets in a sequence-specific manner. The two binding domains dsRBD1 and dsRBD2, which stands for double stranded RNA binding domains and they are required for RISC association, and such association is mediated by dsRNA. The crystal structure analysis further revealed that the siRNA is recognized by RHA with the cooperation of dsRBDs. This evidence suggests that RHA functions in the RNA silencing pathway by promoting the formation of active RISC. Interestingly, the two dsRBD domains are indispensable for interaction with RISC while the helicase core is not absolutely needed to facilitate the formation of active RISC in humans.

Protein Function

The PDB 2EZ6 is the Ribonuclease III (RNase III)protein. Members of the ribonuclease III (RNase III) family are double-stranded RNA (dsRNA) specific endoribonucleases characterized by a signature motif in their active centers and a two-base 3' overhang in their productsRNase III represents a highly conserved family of double-stranded RNA (dsRNA) specific endoribonucleases. The primary functions of RNase III involves RNA processing and posttranscriptional gene-expression control. RNase III has helped in understanding the importance of the role of the Dicer in RNA interference. Dicer produces small interfering RNAs. The RNA interference is a range of class of gene-silencing systems initiated by dsRNA

The RNase III family can be divided into four classes based on increasing molecular weight and complexity of the polypeptide chain. The four classes are bacterial RNase III, Saccharomyces cerevisiae Rnt1p, Drosophila melanogaster Drosha, and Homo sapiens Dicer.The bacterial RNase III proteins, such as Escherichia coli RNase III (Ec-RNase III) and Aquifex aeolicus RNase III (Aa-RNase III), are composed of an endonuclease domain (endoND) followed by a dsRNA binding domain (dsRBD).The homodimeric Ec-RNase III was first discovered in 1968 and ever since it has become the most extensively studied member of the family. RNase III has the ability to affect the gene expression in either of two ways: as a processing enzyme or as a binding protein. As a processing enzyme, RNase III cleaves both natural and synthetic dsRNA into small duplex products averaging 10–18 base pairs in length. As a binding protein, RNase III binds and stabilizes certain RNAs, thus suppressing the expression of certain genes. Here the crystal structure of an RNase III-product complex, the first catalytic complex observed for the family.The RNase III-product complex has a 7 residue linker within the protein, which facilitates induced fit in protein-RNA recognition. A pattern of protein-RNA interactions known as four RNA binding motifs in RNase III and three protein-interacting boxes in dsRNA, is responsible for substrate specificity.Meanwhile conserved amino acid residues and divalent cations are responsible for scissile-bond cleavage. Studying the structure of RNase III is important, because it can be extrapolated to other structures of the RNase III family.

This is the scene for the dsRBD1 binding to RNA

Relevance

The major importance of the PDB 2EZ6 is that it is part of the RISC assembly, which codes for RNA silencing. RNA silencing refers to a conserved sequence-specific gene regulation mechanism mediated by small RNA molecules. RNA silencing plays a fundamental role in many important biological processes in eukaryotes, including host gene regulation and defense against invading foreign nucleic acids.More specifically the application of this protein is integral small RNA processing and small RNA-mediated gene regulation. Both RNA processimng and gene regulation are important factors of gene therapy, preventing viral and cancerous mutations in the human body.

Structural Highlights

References

1. Fu, Qinqin, and Y. Adam Yuan. "Structural Insights into RISC Assembly Facilitated by DsRNA-binding Domains of Human RNA Helicase A (DHX9) | Nucleic Acids Research | Oxford Academic." OUP Academic. Oxford University Press, 29 Jan. 2013. Web. 08 Oct. 2017.

2.Gan, J., J. E. Tropea, B. P. Austin, D. L. Court, D. S. Waugh, and X. Ji. "Structural Insight into the Mechanism of Double-stranded RNA Processing by Ribonuclease III." Cell. U.S. National Library of Medicine, 27 Jan. 2006. Web. 08 Oct. 2017.