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You may include any references to papers as in: the use of JSmol in Proteopedia ^[1] or to the article describing Jmol ^[2] to the rescue.

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(RNaseIII)

Number of Amino Acids in (2EZ6) = 218

Number of Nucleic Acids in (2EZ6) = 56

Catalytic Valley Length and Width = 50Å, 20Å

The Type of 2EZ6 PDB = dsRNA

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

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 ^[3]

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 the 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

As mentioned earlier that 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 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

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, which represents a highly conserved family of double-stranded RNA (dsRNA) specific endoribonucleases. The primary functions of RNase III involves RNA processing and post-transcriptional gene-expression control^[4]. 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. 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. Here the crystal structure of an RNase III-product complex is displayed in scenes.

Relevance

The major importance of the PDB 2EZ6 is that it is part of the RISC assembly ^[5], 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.

Furthermore, researchers examined the change in gene expression in relationship to-disease to predict its effect on the disease based on early changes in gene expression after irradiation^[6].Therefore, gene silencing and the regulation of certain genes could be an easy solution to the prevention of deadliest diseases.

(eQTL) Definition: Expression quantitative trait loci (eQTLs) are genomic loci that contribute to variation in expression levels of mRNAs.

In another study eQTL mapping was applied to identify associations between gene dysregulation and single nucleotide polymorphism (SNP) genotypes in glioblastoma multiform (GBM). A set of 532,954 SNPs was evaluated as predictors of the expression levels of 22,279 expression probes. The final number of significant probes was reduced to 9257 after identifying SNPs associated with fold change in expression level rather than raw expression levels in the tumor and making some adjustments for confounding variables in the experiment. Essentially, the expression probes were involved in signal transduction, transcription regulation, membrane function, and cell cycle regulation. These methods and results indicate that there are many fixed position on the loci that can serve as key part of regulatory pathways associated with GBM.

^[7].

Structural Highlights

The is the complete crystal structure of the(RNase III)

This is the scene for the dsRBD1 binding to RNA

This is the scene for the side chain and including all residues

The scene is the list of 8 Residues makes up large part of the(RNase III) active

This scene shows the

RNaseIII

The binding of dsRBD to RNA through H-bonding

References

1. ↑ Hanson, R. M., Prilusky, J., Renjian, Z., Nakane, T. and Sussman, J. L. (2013), JSmol and the Next-Generation Web-Based Representation of 3D Molecular Structure as Applied to Proteopedia. Isr. J. Chem., 53:207-216. doi:http://dx.doi.org/10.1002/ijch.201300024
2. ↑ Herraez A. Biomolecules in the computer: Jmol to the rescue. Biochem Mol Biol Educ. 2006 Jul;34(4):255-61. doi: 10.1002/bmb.2006.494034042644. PMID:21638687 doi:10.1002/bmb.2006.494034042644
3. ↑ Fu Q, Yuan YA. Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9). Nucleic Acids Res. 2013 Jan 29. PMID:23361462 doi:http://dx.doi.org/10.1093/nar/gkt042
4. ↑ Weinheimer I, Haikonen T, Ala-Poikela M, Moser M, Streng J, Rajamaki ML, Valkonen JP. Viral RNase3 Co-Localizes and Interacts with the Antiviral Defense Protein SGS3 in Plant Cells. PLoS One. 2016 Jul 8;11(7):e0159080. doi: 10.1371/journal.pone.0159080., eCollection 2016. PMID:27391019 doi:http://dx.doi.org/10.1371/journal.pone.0159080
5. ↑ Gan J, Tropea JE, Austin BP, Court DL, Waugh DS, Ji X. Structural insight into the mechanism of double-stranded RNA processing by ribonuclease III. Cell. 2006 Jan 27;124(2):355-66. PMID:16439209 doi:10.1016/j.cell.2005.11.034
6. ↑ Port M, Herodin F, Valente M, Drouet M, Lamkowski A, Majewski M, Abend M. First Generation Gene Expression Signature for Early Prediction of Late Occurring Hematological Acute Radiation Syndrome in Baboons. Radiat Res. 2016 Jul;186(1):39-54. doi: 10.1667/RR14318.1. Epub 2016 Jun 22. PMID:27333084 doi:http://dx.doi.org/10.1667/RR14318.1
7. ↑ Shpak M, Hall AW, Goldberg MM, Derryberry DZ, Ni Y, Iyer VR, Cowperthwaite MC. An eQTL analysis of the human glioblastoma multiforme genome. Genomics. 2014 Apr;103(4):252-63. doi: 10.1016/j.ygeno.2014.02.005. Epub 2014 Mar, 4. PMID:24607568 doi:http://dx.doi.org/10.1016/j.ygeno.2014.02.005