6bjh: Difference between revisions
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==CIRV p19 mutant T111S in complex with siRNA== | |||
<StructureSection load='6bjh' size='340' side='right' caption='[[6bjh]], [[Resolution|resolution]] 2.58Å' scene=''> | |||
== Structural highlights == | |||
<table><tr><td colspan='2'>[[6bjh]] is a 4 chain structure. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6BJH OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6BJH FirstGlance]. <br> | |||
</td></tr><tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=6bjh FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6bjh OCA], [http://pdbe.org/6bjh PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=6bjh RCSB], [http://www.ebi.ac.uk/pdbsum/6bjh PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=6bjh ProSAT]</span></td></tr> | |||
</table> | |||
== Function == | |||
[[http://www.uniprot.org/uniprot/P19_CIRV P19_CIRV]] Acts as a suppressor of RNA-mediated gene silencing, also known as post-transcriptional gene silencing (PTGS), a mechanism of plant viral defense that limits the accumulation of viral RNAs. Binds to short interfering RNAs (siRNAs) with high affinity. Acts as a molecular caliper to specifically select siRNAs based on the length of the duplex region of the RNA.<ref>PMID:14697199</ref> <ref>PMID:19254536</ref> | |||
<div style="background-color:#fffaf0;"> | |||
== Publication Abstract from PubMed == | |||
Tombusviruses express a 19 kDa protein (p19) that, as a dimeric protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The p19 protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the p19 binding surface with the aim of creating p19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe p19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant p19, p19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type p19. Overall, our results suggest that p19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The p19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver. | |||
Enhanced specificity of the viral suppressor of RNA silencing protein p19 toward sequestering of human microRNA-122.,Cheng J, Danielson DC, Nasheri N, Singaravelu R, Pezacki JP Biochemistry. 2011 Sep 13;50(36):7745-55. doi: 10.1021/bi2008273. Epub 2011 Aug, 17. PMID:21819044<ref>PMID:21819044</ref> | |||
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | |||
[[Category: | </div> | ||
[[Category: Pezacki, J | <div class="pdbe-citations 6bjh" style="background-color:#fffaf0;"></div> | ||
[[Category: Schirle, N | == References == | ||
[[Category: | <references/> | ||
[[Category: | __TOC__ | ||
</StructureSection> | |||
[[Category: Foss, D V]] | |||
[[Category: MacRae, I J]] | |||
[[Category: Pezacki, J P]] | |||
[[Category: Schirle, N T]] | |||
[[Category: High affinity for human mirna-122]] | |||
[[Category: Rna binding protein]] | |||
[[Category: Rna silencing sirna]] | |||
[[Category: Viral suppressor]] | |||
Revision as of 15:01, 16 January 2019
CIRV p19 mutant T111S in complex with siRNACIRV p19 mutant T111S in complex with siRNA
Structural highlights
Function[P19_CIRV] Acts as a suppressor of RNA-mediated gene silencing, also known as post-transcriptional gene silencing (PTGS), a mechanism of plant viral defense that limits the accumulation of viral RNAs. Binds to short interfering RNAs (siRNAs) with high affinity. Acts as a molecular caliper to specifically select siRNAs based on the length of the duplex region of the RNA.[1] [2] Publication Abstract from PubMedTombusviruses express a 19 kDa protein (p19) that, as a dimeric protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The p19 protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the p19 binding surface with the aim of creating p19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe p19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant p19, p19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type p19. Overall, our results suggest that p19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The p19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver. Enhanced specificity of the viral suppressor of RNA silencing protein p19 toward sequestering of human microRNA-122.,Cheng J, Danielson DC, Nasheri N, Singaravelu R, Pezacki JP Biochemistry. 2011 Sep 13;50(36):7745-55. doi: 10.1021/bi2008273. Epub 2011 Aug, 17. PMID:21819044[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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