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| | ==Crystal Structure of the Two Tandem RRM Domains of PUF60 Bound to a Modified AdML Pre-mRNA 3' Splice Site Analogue== | | ==Crystal Structure of the Two Tandem RRM Domains of PUF60 Bound to a Modified AdML Pre-mRNA 3' Splice Site Analogue== |
| - | <StructureSection load='5kw1' size='340' side='right' caption='[[5kw1]], [[Resolution|resolution]] 2.10Å' scene=''> | + | <StructureSection load='5kw1' size='340' side='right'caption='[[5kw1]], [[Resolution|resolution]] 2.10Å' scene=''> |
| | == Structural highlights == | | == Structural highlights == |
| - | <table><tr><td colspan='2'>[[5kw1]] is a 3 chain structure with sequence from [http://en.wikipedia.org/wiki/Human Human]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5KW1 OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=5KW1 FirstGlance]. <br> | + | <table><tr><td colspan='2'>[[5kw1]] is a 3 chain structure with sequence from [http://en.wikipedia.org/wiki/Human Human]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5KW1 OCA]. For a <b>guided tour on the structure components</b> use [http://proteopedia.org/fgij/fg.htm?mol=5KW1 FirstGlance]. <br> |
| - | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene></td></tr> | + | </td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=CL:CHLORIDE+ION'>CL</scene></td></tr> |
| | <tr id='NonStdRes'><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=DU:2-DEOXYURIDINE-5-MONOPHOSPHATE'>DU</scene></td></tr> | | <tr id='NonStdRes'><td class="sblockLbl"><b>[[Non-Standard_Residue|NonStd Res:]]</b></td><td class="sblockDat"><scene name='pdbligand=DU:2-DEOXYURIDINE-5-MONOPHOSPHATE'>DU</scene></td></tr> |
| - | <tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[5kwd|5kwd]], [[5kw6|5kw6]]</td></tr> | + | <tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat"><div style='overflow: auto; max-height: 3em;'>[[5kwd|5kwd]], [[5kw6|5kw6]]</div></td></tr> |
| | <tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">PUF60, FIR, ROBPI, SIAHBP1 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=9606 HUMAN])</td></tr> | | <tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">PUF60, FIR, ROBPI, SIAHBP1 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=9606 HUMAN])</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=5kw1 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5kw1 OCA], [http://pdbe.org/5kw1 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5kw1 RCSB], [http://www.ebi.ac.uk/pdbsum/5kw1 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5kw1 ProSAT]</span></td></tr> | + | <tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://proteopedia.org/fgij/fg.htm?mol=5kw1 FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5kw1 OCA], [http://pdbe.org/5kw1 PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5kw1 RCSB], [http://www.ebi.ac.uk/pdbsum/5kw1 PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5kw1 ProSAT]</span></td></tr> |
| | </table> | | </table> |
| | == Function == | | == Function == |
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| | <div style="background-color:#fffaf0;"> | | <div style="background-color:#fffaf0;"> |
| | == Publication Abstract from PubMed == | | == Publication Abstract from PubMed == |
| - | c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcription of this oncogene is governed by the counterbalancing forces of two proteins on TFIIH--the FUSE binding protein (FBP) and the FBP-interacting repressor (FIR). FBP and FIR recognize single-stranded DNA upstream of the P1 promoter, known as FUSE, and influence transcription by oppositely regulating TFIIH at the promoter site. Size exclusion chromatography coupled with light scattering reveals that an FIR dimer binds one molecule of single-stranded DNA. The crystal structure confirms that FIR binds FUSE as a dimer, and only the N-terminal RRM domain participates in nucleic acid recognition. Site-directed mutations of conserved residues in the first RRM domain reduce FIR's affinity for FUSE, while analogous mutations in the second RRM domain either destabilize the protein or have no effect on DNA binding. Oppositely oriented DNA on parallel binding sites of the FIR dimer results in spooling of a single strand of bound DNA, and suggests a mechanism for c-myc transcriptional control.
| + | Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3'). We found that the PUF60-AdML3' dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60's two central RNA recognition motifs (RRMs). A 1.95 A crystal structure of the two PUF60 RRMs in complex with AdML3' revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3') stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3' splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3' splice sites. |
| | | | |
| - | Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition.,Crichlow GV, Zhou H, Hsiao HH, Frederick KB, Debrosse M, Yang Y, Folta-Stogniew EJ, Chung HJ, Fan C, De la Cruz EM, Levens D, Lolis E, Braddock D EMBO J. 2008 Jan 9;27(1):277-89. Epub 2007 Dec 6. PMID:18059478<ref>PMID:18059478</ref>
| + | Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.,Hsiao HT, Crichlow GV, Murphy JW, Folta-Stogniew EJ, Lolis EJ, Braddock DT PLoS One. 2020 Nov 30;15(11):e0242725. doi: 10.1371/journal.pone.0242725., eCollection 2020. PMID:33253191<ref>PMID:33253191</ref> |
| | | | |
| | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> |
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| | </StructureSection> | | </StructureSection> |
| | [[Category: Human]] | | [[Category: Human]] |
| | + | [[Category: Large Structures]] |
| | [[Category: Albright, R]] | | [[Category: Albright, R]] |
| | [[Category: Braddock, D T]] | | [[Category: Braddock, D T]] |
| Structural highlights
Function
[PUF60_HUMAN] DNA- and RNA-binding protein, involved in several nuclear processes such as pre-mRNA splicing, apoptosis and transcription regulation. In association with FUBP1 regulates MYC transcription at the P2 promoter through the core-TFIIH basal transcription factor. Acts as a transcriptional repressor through the core-TFIIH basal transcription factor. Represses FUBP1-induced transcriptional activation but not basal transcription. Decreases ERCC3 helicase activity. Does not repress TFIIH-mediated transcription in xeroderma pigmentosum complementation group B (XPB) cells. Is also involved in pre-mRNA splicing. Promotes splicing of an intron with weak 3'-splice site and pyrimidine tract in a cooperative manner with U2AF2. Involved in apoptosis induction when overexpressed in HeLa cells. Isoform 6 failed to repress MYC transcription and inhibited FIR-induced apoptosis in colorectal cancer. Isoform 6 may contribute to tumor progression by enabling increased MYC expression and greater resistance to apoptosis in tumors than in normal cells. Modulates alternative splicing of several mRNAs. Binds to relaxed DNA of active promoter regions. Binds to the pyrimidine tract and 3'-splice site regions of pre-mRNA; binding is enhanced in presence of U2AF2. Binds to Y5 RNA in association with TROVE2. Binds to poly(U) RNA.[1] [2] [3] [4] [5] [6]
Publication Abstract from PubMed
Pre-mRNA splicing is critical for achieving required amounts of a transcript at a given time and for regulating production of encoded protein. A given pre-mRNA may be spliced in many ways, or not at all, giving rise to multiple gene products. Numerous splicing factors are recruited to pre-mRNA splice sites to ensure proper splicing. One such factor, the 60 kDa poly(U)-binding splicing factor (PUF60), is recruited to sites that are not always spliced, but rather function as alternative splice sites. In this study, we characterized the interaction of PUF60 with a splice site from the adenovirus major late promoter (the AdML 3' splice site, AdML3'). We found that the PUF60-AdML3' dissociation constants are in the micromolar range, with the binding affinity predominantly provided by PUF60's two central RNA recognition motifs (RRMs). A 1.95 A crystal structure of the two PUF60 RRMs in complex with AdML3' revealed a dimeric organization placing two stretches of nucleic acid tracts in opposing directionalities, which can cause looping of nucleic acid and explain how PUF60 affects pre-mRNA geometry to effect splicing. Solution characterization of this complex by light-scattering and UV/Vis spectroscopy suggested a potential 2:1 (PUF602:AdML3') stoichiometry, consistent with the crystal structure. This work defines the sequence specificity of the alternative splicing factor PUF60 at the pre-mRNA 3' splice site. Our observations suggest that control of pre-mRNA directionality is important in the early stage of spliceosome assembly, and advance our understanding of the molecular mechanism by which alternative and constitutive splicing factors differentiate among 3' splice sites.
Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60.,Hsiao HT, Crichlow GV, Murphy JW, Folta-Stogniew EJ, Lolis EJ, Braddock DT PLoS One. 2020 Nov 30;15(11):e0242725. doi: 10.1371/journal.pone.0242725., eCollection 2020. PMID:33253191[7]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Liu J, He L, Collins I, Ge H, Libutti D, Li J, Egly JM, Levens D. The FBP interacting repressor targets TFIIH to inhibit activated transcription. Mol Cell. 2000 Feb;5(2):331-41. PMID:10882074
- ↑ Page-McCaw PS, Amonlirdviman K, Sharp PA. PUF60: a novel U2AF65-related splicing activity. RNA. 1999 Dec;5(12):1548-60. PMID:10606266
- ↑ Liu J, Akoulitchev S, Weber A, Ge H, Chuikov S, Libutti D, Wang XW, Conaway JW, Harris CC, Conaway RC, Reinberg D, Levens D. Defective interplay of activators and repressors with TFIH in xeroderma pigmentosum. Cell. 2001 Feb 9;104(3):353-63. PMID:11239393
- ↑ Matsushita K, Tomonaga T, Shimada H, Shioya A, Higashi M, Matsubara H, Harigaya K, Nomura F, Libutti D, Levens D, Ochiai T. An essential role of alternative splicing of c-myc suppressor FUSE-binding protein-interacting repressor in carcinogenesis. Cancer Res. 2006 Feb 1;66(3):1409-17. PMID:16452196 doi:http://dx.doi.org/10.1158/0008-5472.CAN-04-4459
- ↑ Liu J, Kouzine F, Nie Z, Chung HJ, Elisha-Feil Z, Weber A, Zhao K, Levens D. The FUSE/FBP/FIR/TFIIH system is a molecular machine programming a pulse of c-myc expression. EMBO J. 2006 May 17;25(10):2119-30. Epub 2006 Apr 20. PMID:16628215 doi:http://dx.doi.org/7601101
- ↑ Hastings ML, Allemand E, Duelli DM, Myers MP, Krainer AR. Control of pre-mRNA splicing by the general splicing factors PUF60 and U2AF65. PLoS One. 2007 Jun 20;2(6):e538. PMID:17579712 doi:http://dx.doi.org/10.1371/journal.pone.0000538
- ↑ Hsiao HT, Crichlow GV, Murphy JW, Folta-Stogniew EJ, Lolis EJ, Braddock DT. Unraveling the mechanism of recognition of the 3' splice site of the adenovirus major late promoter intron by the alternative splicing factor PUF60. PLoS One. 2020 Nov 30;15(11):e0242725. doi: 10.1371/journal.pone.0242725., eCollection 2020. PMID:33253191 doi:http://dx.doi.org/10.1371/journal.pone.0242725
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