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==Plant Viral Protein p19 Suppression of RNA Silencing==
<StructureSection load='' size='350' side='right' caption='p19 dimer and siRNA [[1rpu]]' scene='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/View1/2'>
==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 [[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 plant's 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.  
<br>
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>.
<br>
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 from carnation and tomato viruses selectively recognizes the double-stranded siRNA. p19 from the carnation virus is presented on this page; however the results are relevant to both p19 examples as the solved structures are very similar, consistent with the high percent of identity (89%) for the homologous proteins<ref name='alignment'>[[Alignment of 1rpu vs 1r9f|Alignment of Carnation italian ringspot virus p19 (in 1rpu) vs. Tomato bushy stunt virus p19 (in 1r9f)]].</ref>.  See [[Alignment of 1rpu vs 1r9f]].
==Results==
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 {{User:Wayne Decatur/Template ColorKey Composition lcprotein}} and {{Template:ColorKey Composition RNA}} in the complex. {{Link Toggle FancyCartoonHighQualityView}}.

The <scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/Twostrandsofrna/3'>RNA in the complex is double-stranded and in A-form</scene>.<br>
The synthetic <font color = '#00FF00'>R</font><font color = '#6060ff'>N</font><font color = '#00FF00'>A</font> 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.<br> 
The duplex region is 19 nts and there are 2-nt 3' overhangs.<br> 
Here is a schematic illustration of the two strands of the silencing RNA (siRNA) in this structure:<br>
<span style="font-weight: bold;font-family: Courier New; font-size: 10pt">
<!-- By setting font to courier, I can use non-proportional font. Whereas by default, rest of Proteopedia is proportional -->
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<span style="color:#00FF00">5'-pCGUACGCGUCACGCGUACGUU-OH-3'</span><br>&nbsp;&nbsp;
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|||||||||||||||||||<br> 
&nbsp;&nbsp;&nbsp;&nbsp;<span style="color:#6060ff">3'-OH-UUGCAUGCGCACUGCGCAUGCp-5'</span></span>
<br>


p19 binds the siRNA as 
<scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/P19isadimer/3'>a dimer</scene>.<br>
Individual monomers of the p19 dimer are colored <font color = '#cbbe8a'>tan</font> and <font color = 'purple'>purple</font>. <br> 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 <font color = '#ff0080'>five alpha-helices</font> and a <font color='#cdcd00'>four-stranded beta sheet</font>.

<scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/8strandcontinuousbetasheet/3'>The p19 dimer</scene> forms a <font color = '#cdcd00'>continuous eight-stranded beta-sheet</font>.

The <font color = '#cdcd00'>continuous eight-stranded beta-sheet</font> 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]].  For more detais see [[P19 complexed with 19-bp small interfering RNA]].


<scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/Specificationforrna/3'>Specification of RNA as the substrate</scene><br>
Several conserved serine and threonine residues in p19 mediate key interactions with <font color = 'red'>2'-hydroxyls</font> 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. 


<!--Dots seemed to crash the jmol window and seems to be due to the dots in views from my tour. (making new scenes with other pdb files and adding dots sing pdb files caused no such problems.) Talked to Eric and he suggested surfaces might look better anyway. However, configuration 1 seemed to cause problems so showing RNA as wireframes; however, I was able start again in sandbox and use lines entered one at time into to Jmol console to get back after doing configuration and for some reason Proteopedia saved it well.  
I first learned more about Jmol's isosurfaces at Bob Hanson's Interactive Scripting documentation at http://chemapps.stolaf.edu/jmol/docs/
and really useful was the info on surfaces in Jmol at
http://jmol.sourceforge.net/docs/surface/ where it clearly illustrated how to make a closed surface around atoms or residues of interest using the ignor command. I had tried it using info from the scripting documentation but 'ignore (not selected)' didn't seem to work for some reason but the info below at Sourceforge helped a lot:
define temp atom expression; isoSurface surf1 select(temp) ignore(not temp) molecular
So i come up with this set of commands:
define endcapaminoacids 39:a,42:a,39:b,42:b;
isoSurface delete surf1 select(endcapaminoacids) ignore(not endcapaminoacids) molecular translucent; #ignore makes surface closed by ignoring contributions from rest to surface
isosurface ID surf1 fill noMesh noDots notFrontOnly frontlit;
  color $surf1 translucent 128 orange;
 -->
<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><br>
p19 specifies the presence of a <font color = 'cyan'>5'-phosphate</font> at the 5'-end of each RNA strand via hydrogen binding between the <font color = 'cyan'>phosphate</font> and a conserved tryptophan (W42).
<br>



<scene name='User:Wayne_Decatur/Plant_Viral_Protein_p19_Suppression_of_RNA_Silencing/Molecular_caliper_perfected/2'>p19 is a molecular caliper</scene><br>
Most unprecedented is the method of size selection where p19 acts as a molecular caliper reading the size of the substrate. The <font color = 'orange'>aromatic rings of two tryptophan residues (W39 and W42)</font> 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. Many other structures use aromatic rings to stack on nucleotide bases and cap strands in helices, see [[1gm5|F204 and Y208 of 1gm5]], [[1msw|W639 of 1msw]] and [[1qln]].<br>

==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. <br>
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).<!--Calculated using http://www.expasy.org/tools/protparam.html--> 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]

==3D structures of protein p19==

Updated on {{REVISIONDAY2}}-{{MONTHNAME|{{REVISIONMONTH}}}}-{{REVISIONYEAR}}

[[1rpu]], [[6bjv]] – CirvP19 + SiRNA – Carnation italian ringspot virus<br />
[[6bjh]], [[6bjg]] - CirvP19 (mutant) + SiRNA<br />
[[4j39]], [[4j5v]],  [[4jgn]], [[4jnx]], [[4knq]], [[4ktg]] - TbsvP19 + SiRNA - Tomato bushy stunt virus <br />
[[1rpf]], [[4jk0]], [[4kq0]] - TbsvP19 (mutant) + SiRNA<br />
[[3lzl]],[[3lzn]],[[3lzo]],[[3lzp]],[[3lzq]],[[3lzr]], [[5i0w]], [[5i0v]]  – P19 – ''Campylobacter jejuni''<br />
[[5dof]] - TtP19 – ''Tetrahymena thermophila''<br />
[[5doi]] - TtP19 + P45 <br />
[[6cus]] - RsvP19 M-domain (mutant) + inositol-hexakisphosphate – Rous sarcoma virus - NMR<br />
[[6cv8]], [[6cw4]] - RsvP19 M-domain (mutant) + inositol-trisphosphate – Rous sarcoma virus - NMR<br />

==Related Structures and Topics==
*[[Suppression of RNA Silencing by Viruses|Suppression of RNA Silencing by Viruses]]
*[[Tomato aspermy virus protein 2b Suppression of RNA Silencing|Tomato aspermy virus protein 2b Suppression of RNA Silencing]]
*[[Flock house virus B2 protein Suppression of RNA Silencing|Flock house virus B2 protein Suppression of RNA Silencing]]
*[[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
*[[RNA Interference|RNA interference]]

==Notes and Literature References== 
<references/>

==Additional Literature and Resources==
*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]<br>
*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]<br>
*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]<br>
*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]<br>
*Sizing up small RNAs., Jabri E, Nature Structural & Molecular Biology 2004 11: 112. PMID:[http://www.ncbi.nlm.nih.gov/pubmed/14749769 14749769 ]<br>
*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]<br>
*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]<br>
*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]<br>
*[http://www.nature.com/focus/rnai/ Nature Reviews RNAi collection]<br>
*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"/> 
</StructureSection>

[[Category: RNA Silencing]]
[[Category: post-transcriptional gene silencing (PTGS)]]
[[Category: Small Interfering RNA]]
[[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]]
[[Category:Topic Page]]
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