Wayne Decatur: New page: 300px
'''3D structure of p19 complexed to siRNA''' ==Background== [http://en.wikipedia.org/wiki/RNA_Silencing RNA silencing] i...

2009-04-22T08:47:09Z

New page: left|300px '''3D structure of p19 complexed to siRNA''' ==Background== [http://en.wikipedia.org/wiki/RNA_Silencing RNA silencing] i...

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[[Image:Unremediated1rpuWithPolyview3D.png|left|300px]] 

'''3D structure of p19 complexed to siRNA'''

==Background==
[http://en.wikipedia.org/wiki/RNA_Silencing RNA silencing] is a gene inactivation system in many eukaryotes that relies on tiny RNAs as the targeting molecules. One function of RNA silencing, which is also called [http://en.wikipedia.org/wiki/Post-transcriptional_gene_silencing post-transcriptional gene silencing (PTGS)] or [http://en.wikipedia.org/wiki/RNA_interference RNA interference (RNAi)], is to act in surveillance against molecular parasites, such as viruses. Double-stranded RNA triggers the RNA silencing pathway and most plant viruses use a double-stranded RNA to replicate their genome. Various plant viruses have developed evasion techniques to circumvent this surveillance system. In one such evasion strategy, the plant viral protein p19 suppresses a plants anti-viral RNA silencing response. p19 binds with high affinity to the double-stranded RNA silencing mediator, called [http://en.wikipedia.org/wiki/Small_interfering_RNA siRNA], and this binding sequesters the siRNA, preventing its participation in later steps of RNA silencing.
 
siRNAs are generally characterized by their short length (21–26 nt), 2 nt, 3′ overhanging ends, and 5′ phosphate groups. The most efficient silencing is obtained with siRNA duplexes composed of 21-nt sense and 21-nt antisense strands, paired in a manner to have a 2-nt 3' overhang (see [http://www.rockefeller.edu/labheads/tuschl/sirna.html the Tuschl lab's guide] for designing siRNAs)<ref>Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate., Elbashir SM, Martinez J, Patkaniowska A, Lendeckel W, Tuschl T, EMBO J. 2001 Dec 3;20(23):6877-88. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/11726523 11726523]</ref>.
 
Structural studies <ref>Size selective recognition of siRNA by an RNA silencing suppressor., Vargason JM, Szittya G, Burgyan J, Tanaka Hall TM, Cell 2003 Dec 26;115(7):799-811. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/14697199 14697199]</ref><ref>Recognition of small interfering RNA by a viral suppressor of RNA silencing., Ye K, Malinina L, Patel D J, Nature 2003 426(6968):874-878. Epub 2003 Dec 3. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/14661029 14661029]</ref> have revealed how p19 selectively recognizes the double-stranded siRNA.

==Results==
{{Structure
|PDB= 1rpu |SIZE=400|SCENE=User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/View1/2|CAPTION= p19 and siRNA (1rpu), resolution 2.50Å (<scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/View1/2'>initial scene</scene>)
|SITE=
|LIGAND=
|ACTIVITY=
|GENE=
|DOMAIN=
|RELATEDENTRY=
|RESOURCES=[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=1rpu FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=1rpu OCA], [http://www.ebi.ac.uk/pdbsum/1rpu PDBsum], [http://www.rcsb.org/pdb/explore.do?structureId=1rpu RCSB]
}}
The X-ray crystal structure of <scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/Rnaprotein1rpu/3'>p19 complexed with double-stranded siRNA</scene> includes both {{Template:ColorKey Composition Protein}} and {{Template:ColorKey Composition RNA}} in the complex.

The {{Template:ColorKey Composition RNA}} in the complex is <scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/Twostrandsofrna/3'>double-stranded and in A-form</scene>. 
The synthetic RNA substrate mimics the 21-nt double-stranded [http://en.wikipedia.org/wiki/Small_interfering_RNA siRNAs] that occur in the double-strand RNA-induced RNAi silencing pathway. 
The duplex region is 19 nts and there are 2-nt 3' overhangs. 
Here is a schematic illustration of the two strands of the silencing RNA (siRNA) in this structure: 


        5'-pCGUACGCGUCACGCGUACGUU-OH-3'   
         ||||||||||||||||||| 
    3'-OH-UUGCAUGCGCACUGCGCAUGCp-5'
 

p19 binds the siRNA as
<scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/P19isadimer/3'>a dimer</scene>. 
Individual monomers of the p19 dimer are colored tan and purple. The apparent breaks seen in the monomer chains internally are due to an inability to model this region because of poor electron density here, presumably as a result of flexibility in the linker.

Each <scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/Secondarystructureofmonomer/3'>monomer of p19</scene> is made of five alpha-helices and a four-stranded beta sheet.

<scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/8strandcontinuousbetasheet/3'>The p19 dimer</scene> forms an continuous eight-stranded beta-sheet.

The continuous eight-stranded beta-sheet of <scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/Sheetcradlesminorgroove/4'>the p19 dimer cradles the minor groove of the duplex region</scene> of the double stranded siRNA. This is unusual because most proteins use loops and helices to bind double-stranded RNA, for example, see [[1di2]], [[2zi0]], [[2hvy]] or [[2az0]].

<scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/Specificationforrna/3'>Specification of RNA as the substrate</scene> 
Several conserved serine and threonine residues in p19 mediate key interactions with 2'-hydroxyls specifying RNA as the substrate, rather than DNA. This view only shows part of the network of interactions with the ribose sugar 2'-hydroxyls of the RNA. Other interactions with the 2'-hydroxyls involve water-mediated contacts and the sidechains of p19. Additionally, the phosphate backbone of the duplex is extensively contacted in a specific manner by p19. As expected to insure recognition of any anti-viral siRNAs, there are no base-specific contacts.


<scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/Phosphatespeci/1'>Specification of the 5' phosphate on the siRNA strands</scene> 
p19 specifies the presence of a 5'-phosphate at the 5'-end of each RNA strand via hydrogen binding between the phosphate and a conserved tryptophan (W42).
 

<scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/Molecular_caliper_perfected/2'>p19 is a molecular caliper</scene> 
Most unprecedented is the method of size selection where p19 acts as a molecular caliper reading the size of the substrate. The aromatic rings of two tryptophan residues (W39 and W42) project from the 'reading' helix near the N-terminus of each of the two p19 monomers, stacking on the terminal base-pairs and symmetrically bracketing the ends of the duplex region of the siRNA. Thus the p19 'caliper' measures and specifically selects siRNA based on the size of the duplex region. The ability of p19 to efficiently accommodate binding of duplexes ranging from 19 to 21 bp could originate in structural plasticity of the distance separating the two 'reading head' helices. Each 'reading' helix is connected to the structured core of p19 by a short flexible loop and several side-chain interactions this presumably allows some flexibility in the positioning of the RNA end-capping tryptophan residues. 

==Conclusions==
The X-ray crystal structure shows the size of the duplex as the major determinant of the siRNA recognized by p19 and biochemical experiments support this assessment. In contrast, the 2-nt 3' overhang seems to have little specific contact with the protein, consistent with Vargason et al. finding comparable binding affinities of p19 for a blunt-ended 19-bp RNA duplex, lacking the typical 2-nt single-stranded 3' overhang. 
Thus, p19 seems adequately generalized to bind to any host siRNAs, the highest affinity for those with 19 bps of duplex with 5' phosphates. p19 accommodates the the 2 nts overhang at the 3' end that are produced as part of the generation of the siRNAs, and biochemical analysis show binding is best for the 21 nt siRNAs. The structural basis for the size selective interaction of p19 with siRNAs, where an extended beta-sheet spans the length of the helix positioning flanking helices mediating end-capping stacking interactions with the RNA, has not yet been seen in other interactions of protein with double-stranded RNAs.

==About this Structure==
1rpu is a [[Protein complex]] structure of sequences from [http://en.wikipedia.org/wiki/Carnation_italian_ringspot_virus Carnation italian ringspot virus]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1RPU OCA]. The molecular weight of p19 protein chain designated 'a' seen in the solved structure is 15.6 kDa (138 residues visible). The molecular weight of p19 protein chain designated 'b' seen in the solved structure is 15.2 kDa (135 residues visible). Total size of the p19-RNA complex seen in the structure is 52.1 kDa. Biological Carnation italian ringspot virus p19 is 172 amino acids and full-length p19 was expressed and purified to generate the crystals in this structure.

==Reference for the Structure==
Size selective recognition of siRNA by an RNA silencing suppressor., Vargason JM, Szittya G, Burgyan J, Tanaka Hall TM, Cell 2003 Dec 26;115(7):799-811. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/14697199 14697199]

==Related Structures and Topics==
*[[User:Wayne Decatur/Suppression of RNA Silencing by Viruses|Suppression of RNA Silencing by Viruses]]
*[[User:Wayne Decatur/Tomato aspermy virus protein 2b Suppression of RNA Silencing|Tomato aspermy virus protein 2b Suppression of RNA Silencing]]
*[[User:Wayne Decatur/Flock house virus B2 protein Suppression of RNA Silencing|Flock house virus B2 protein Suppression of RNA Silencing]]
*[[1rpu]] Carnation italian ringspot virus p19 bound to siRNA
*[[1r9f]] Tomato bushy stunt virus p19 bound to siRNA
*[[2zi0]] Tomato aspermy virus protein 2b bound to siRNA
*[[2az0]] Flock house virus B2 protein bound to double-stranded RNA (dsRNA)
*[[2b9z]] Flock house virus B2 protein solution structure

==Notes and Literature References==
<references/>
==Additional Literature and Resources==
*[http://www-unix.oit.umass.edu/~wdecatur/p19tutorial/ Tour of p19 bound to an siRNA] by Wayne Decatur, in an exploration-friendly interface that is adapted from Eric Martz's [http://molvis.sdsc.edu/fgij/index.htm FirstGlance in Jmol] 
*Structural basis for RNA-silencing suppression by Tomato aspermy virus protein 2b., Chen HY, Yang J, Lin C, Yuan YA, EMBO Rep. 2008 Aug;9(8):754-60. Epub 2008 Jul 4.PMID:[http://www.ncbi.nlm.nih.gov/pubmed/18600235 18600235] 
*The structure of the flock house virus B2 protein, a viral suppressor of RNA interference, shows a novel mode of double-stranded RNA recognition., Lingel A, Simon B, Izaurralde E, Sattler M. EMBO Rep. 2005 Dec;6(12):1149-55. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16270100 16270100] 
*Dual modes of RNA-silencing suppression by Flock House virus protein B2. Chao JA, Lee JH, Chapados BR, Debler EW, Schneemann A, Williamson JR, Nat Struct Mol Biol. 2005 Nov;12(11):952-7. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16228003 16228003] 
*Recognition of small interfering RNA by a viral suppressor of RNA silencing., Ye K, Malinina L, Patel D J, Nature 2003 426(6968):874-878. Epub 2003 Dec 3. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/14661029 14661029] 
*Sizing up small RNAs., Jabri E, Nature Structural & Molecular Biology 2004 11: 112. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/14749769 14749769 ] 
*Crystal structure of p19--a universal suppressor of RNA silencing., Baulcombe DC, Molnar A, Trends Biochem Sci. 2004 29(6):279-281. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/15276178 15276178] 
*Plant viral suppressors of RNA silencing., Roth BM, Pruss GJ, Vance VB, Virus Res. 2004 Jun 1;102(1):97-108. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/15068885 15068885] 
*Novel modes of protein-RNA recognition in the RNAi pathway. Lingel A, Sattler M, Curr Opin Struct Biol. 2005. 15(1):107-115. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/15718141 15718141] 
*[http://www.nature.com/focus/rnai/ Nature Reviews RNAi collection] 
*The Tombusvirus-encoded P19: from irrelevance to elegance. Scholthof HB, Nature Reviews Microbiology. 22006 May;4(5):405-11. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/16518419 16518419]
<ref group="xtra">PMID:16419274</ref>
<references group="xtra"/>
[[Category: RNA Silencing]]
[[Category: post-transcriptional gene silencing (PTGS)]]
[[Category: Virus]]
[[Category: Plant Virus]]
[[Category: double-stranded RNA (dsRNA)]]
[[Category: siRNA]]
[[Category: Carnation italian ringspot virus]]
[[Category: Burgyan, J.]]
[[Category: Hall, T M.T.]]
[[Category: Szittya, G.]]
[[Category: Vargason, J M.]]
[[Category: Protein-rna complex]]
[[CAtegory: Protein complex]]
[[Category: Rna double helix]]
[[Category: A-form helix]]
[[Category: Rna length recognition]]
[[Category: Rnai]]

User:Wayne Decatur/Sandboxp19test - Revision history

Wayne Decatur: New page: 300px '''3D structure of p19 complexed to siRNA''' ==Background== [http://en.wikipedia.org/wiki/RNA_Silencing RNA silencing] i...

Wayne Decatur: New page: 300px
'''3D structure of p19 complexed to siRNA''' ==Background== [http://en.wikipedia.org/wiki/RNA_Silencing RNA silencing] i...