The endoribonuclease of the eukaryotic nuclear polyadenylation reaction Association with cleavage and polyadenylation specificity factor (CPSF-73) homologue in Giardia lamblia Assad Alhaboub Degree project in biology, 2007 Examensarbete i biologi, 20p, 2007 Biology Education Centre and Department of Cell and Molecular Biology Supervisors: Anders Virtanen and Per Nilsson
20
Embed
The endoribonuclease of the eukaryotic nuclear polyadenylation
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
The endoribonuclease of the eukaryoticnuclear polyadenylation reaction
Association with cleavage and polyadenylationspecificity factor (CPSF-73) homologue in Giardialamblia
AssadAlhaboub
Degree project in biology, 2007Examensarbete i biologi, 20p, 2007Biology Education Centre and Department of Cell and Molecular BiologySupervisors: Anders Virtanen and Per Nilsson
''''''''''''''''''''''''''''''' 'Fig. 1'The major two steps of the polyadenylation reaction.'C1e'm678'is'<leaved'at't1e'pol&aden&lation'site0'aden&late'residues'are'added'to't1e'23end'of't1e'upstream'fragment.'C1e'doBnstream'fragment'is'degraded.'C1e'1ig1l&'<onserved'se^uen<e'88`888'is'one'of't1e'pol&aden&lation'signals'in'mammals.'''''''''''''''''''''''C1e'pol&aden&lation'rea<tion'1as'been'linked'to'ot1er'<ru<ial'intra<ellular'pro<esses'?revieBed'in'ref.'2D'a'2X@.'8<<umulated'eviden<e'indi<ates't1at'pol&aden&lation'fa<tors'and'signals'are're^uired'for'effi<ient'trans<ription'in<luding'initiation0'elongation'and'termination'V022'as'Bell'as'for'spli<ing'501D.'Iurt1ermore0'it'1as'been'suggested't1at't1e'pol&?8@'tail'is're^uired'for'eYport'of'm678'from't1e'nu<leus'1R.'(n'addition0't1e'pol&?8@'provides'a'binding'site'for'pol&?8@'binding'proteins'?P8BP@'t1at'subse^uentl&'intera<t'Bit1'proteins'binding't1e'<ap'at't1e'53end'to'form'a'<ir<ulari\ed'm678't1at'serves'as'a'do<king'site'for't1e'RX!'ribosomal'subunit.'(n't1is'Ba&0't1e'pol&?8@'tail'pla&s'an'important'role'in'initiation'of'translation'2R.'Iurt1ermore0't1e'binding'of'P8BP'to't1e'pol&?8@'tail'also'improves'm678'stabilit&'b&'prote<ting't1e'm678'from'nu<leol&ti<'degradation'_.'Iinall&0't1e'pol&aden&lation'rea<tion'and't1e'pol&?8@'tail'are'knoBn'to'pla&'a'<riti<al'role'for't1e'regulation'of'gene'eYpression'during'earl&'development'2V.''C1e'bio<1emistr&'of't1e'pol&aden&lation'rea<tion'1as'been'eYtensivel&'studied'in'mammals'and'&east'B1ere'it'1as'been's1oBn't1at't1e'pol&aden&lation'ma<1iner&0'B1i<1'is'responsible'for't1e'rat1er'simple'tBoEstep'rea<tion0'is'surprisingl&'<ompleY'and'is'<omposed'of'more't1an'a'do\en'pol&peptides'?revieBed'in'ref.'25@.'C1e'basi<'pro<essing'rea<tion'is'identi<al'in'mammals'and'&east'and't1e'presen<e'of'1omologous'protein'fa<tors'?summari\ed'in'Table 1@'1as'been'reported'1V.'(n'mammals0'<leavage'and'pol&aden&lation'spe<ifi<it&'fa<tor'?CP!I@0'<leavage'stimulation'fa<tor'?CstI@0'<leavage'fa<tors'('a'(('and'pol&?8@'pol&merase'?P8P@'parti<ipate'in't1e'first'step''B1ereas'pol&?8@'tail's&nt1esis're^uires'P8P0'CP!I'and'pol&?8@'binding'proteins'?P8BP2@.'CBo'main'<isEa<ting'pol&aden&lation'signals'1ave'been'identified0't1e'88`888'se^uen<e'B1ere'CP!I'binds'and'`cN`Eri<1'tra<ts'B1ere'CstI'binds0'lo<ated'11E22'nu<leotides'upstream'and'1XE
'''''''''''''''''''' ''Fig.2'Identification of putative factors of the polyadenylation machinery in Giardia lamblia.'8'genomi<'surve&'revealed'D'1omologues'to'knoBn'fa<tors'of't1e'pol&aden&lation'ma<1iner&.'C1e'figure's1oBs'a'<omparison'betBeen't1e'pol&aden&lation's&stem'in'&east'and'a'putative'one'in'Giardia lamblia.'Ia<tors'from'Giardia'are'represented'as'open'reading'frame'?h6I@'numbers.'Nreen:'&east'fa<tors't1at'1ave'1omologues'in'Giardia.'Iigure'<ourtes&'of'!taffan'!värd.'' GST gene fusion system 'hne's&stem'to'<onsider'for'eYpression'and'purifi<ation'of't1e'CP!IED2'1omologue'from'Giardia lamblia'is'Nlutat1ioneE!Etransferase'?N!C@'gene'fusion's&stem0'a'<ommonl&'used'met1od'to'eYpress'and'purif&'proteins.'EYpression'using'su<1's&stem'in'Escherichia coli &ields'fusion'proteins'Bit1't1e'N!C'at't1eir'amino'terminus'and't1e'protein'at't1e'<arboY&l'terminus.'C1e'fusion'proteins'are'purified'b&'affinit&'<1romatograp1&'using'glutat1ione'immobili\ed'b&'sep1arose'beads.'C1e'glutat1ione'binds'to't1e'N!C'moiet&'?Fig. 3@.'C1e'fusion'proteins'<an't1en'
Fig.3 Principle of affinity chromatography using immobilized glutathione in GST fusion system.'C1e'stru<ture'of'glutat1ione'is'<omplementar&'to'glutat1ioneE!Etransferase'?N!C@'binding'site.'C1e'N!C'tag'<ould'be'removed'b&'en\&mati<'<leavage'in't1e'protease'<leavage'site.''
6esults''Purification of CPSF-73 homologue in Giardia lamblia
''''''''' ''''''''''''Fig. 4'Schematic representation for the purification procedures of GST-glCPSF-73 (T) and glCPSF-73 (NT).'Nlutat1ione'sep1arose'beads'Bere'treated'Bit1'B!8'prior'to'binding'to'N!CEglCP!IED2.'N!CEglCP!IED2'Bas'eit1er'immediatel&'eluted'using'EB'1'or'treated'Bit1'Pre!<ission'protease'to'<leave't1e'N!C'tag'before'elution'b&'EB'2.'B!8:'Bovine'!erum'8lbuminf'PC'miY:'Pre!<ission'ProteaseZ'Pre!<ission'Protease'<leavage'bufferf'EB:'elution'bufferf'C:'taggedf'7C:'nonEtagged.'''''''''''''''''''''''8'previousl&'<ondu<ted'genomi<'surve&'in't1e'Giardia'database'indi<ated'1ig1'similarit&'betBeen'h6I'RR9_'from'Giardia lamblia'and'<leavage'and'pol&aden&lation'spe<ifi<it&'fa<tor'D2'?CP!IED2@'1omologues'from'various'eukar&otes'?!taffan'!värd0'personal'<ommuni<ation@.'C1e'<oding'se^uen<e'of'h6I'RR9_'from'Giardia lamblia ?glCP!IED2@'1ad'been'<loned'to'&ield'a'glutat1ioneE!Etransferase'?N!C@'fusion'protein'before't1is'proje<t'Bas'started.''(n'order'to'get'glCP!IED20'B1i<1'1as'a'mole<ular'Beig1t'of'_R'kD0't1is'se^uen<e'Bas'overeYpressed'in'E. coli'and'purified'b&'affinit&'<1romatograp1&'folloBing't1e's<1eme'in'Iigure'R.'Nlutat1ione'sep1arose'RB'beads'?N'!'beads@'Bere'saturated'Bit1'a<et&lated'bovine'serum'albumin'?B!8@'prior'to't1e'binding'to't1e'l&sate'<ontaining'N!C'tagged'glCP!IED2'?N!CEglCP!IED2@.'C1is'Bas'ne<essar&'in'order'to'blo<k'nonEN!CEspe<ifi<'intera<tions'of'glCP!IED2'Bit1't1e'N'!'beads.'N!CEglCP!IED2'Bas'eluted'from'proteinEbound'beads'using'elution'buffer'?EB'1@'?Fig. 40'purifi<ation'pro<edure'1@.'C1is'elution'buffer'Bas'optimi\ed'to'furt1er'disrupt'unspe<ifi<'binding'and't1ereb&'elute't1e'protein'more'effi<ientl&.'C1is'pro<edure'&ielded'suffi<ient'amount'of'N!CEglCP!IED2'?C@'?Fig. 50'lanes'2'a'2@.'ht1er'N!CEglCP!IED2Ebound'beads'Bere'treated'Bit1'Pre!<ission'Protease'?P@'to'remove't1e'N!C'tag'?Fig. 40'purifi<ation'pro<edure'2@.'C1e'subse^uent'elution'step'of't1e'nonEtagged'glCP!IED2'?7C@'using'EB'2'Bas'satisfa<tor&'?Fig. 60'lane'2@.'''
' D
''''''''''''''''''''''''''''''''''''''''' ''Fig. 5 Purification of GST-glCPSF-73 (T) by affinity chromatography. '6e<ombinant'N!CEglCP!IED2'Bas'purified'b&'affinit&'<1romatograp1&'using'glutat1ione'sep1arose'beads'and'eluted'tBi<e'using'elution'buffer'EB'1'?see'materials'and'met1ods@.'C1e'proteins'Bere'anal&\ed'b&'!D!EP8NE'?sodium'dode<&l'sulfate'pol&a<r&lamide'gel'ele<trop1oresis@'and'stained'Bit1'Coomassie'blue.''_'jl'of't1e'first'and'se<ond'eluates'Bere'loaded'in'lanes'2'and'2'respe<tivel&.'_'jl'of't1e'glutat1ione'sep1arose'beads'after'elution'Bere'loaded'in'lane'R.'Jg:'mole<ular'Beig1tf'ZN!C:'Bit1'glutat1ioneE!Etransferasef'C:'tagged.'N'!'Beads:'Nlutat1ione'sep1arose'RB.'''
'''''''''''''''''''''''''''''''''''''''''''''''''' ''''''''''''''''''''''''''''''''''''Fig. 6'Purification of glCPSF-73 (NT) by affinity chromatography.'6e<ombinant'N!CEglCP!IED2'Bas'purified'b&'affinit&'<1romatograp1&'using'glutat1ione'sep1arose'beads0't1e'N!C'tag'Bas'<leaved'b&'Pre!<ission'Protease'and'glCP!IED2'Bas'eluted'using'elution'buffer'EB'2'?see'materials'and'met1ods@.'C1e'proteins'Bere'anal&\ed'b&'!D!EP8NE'?sodium'dode<&l'sulfate'pol&a<r&lamide'gel'ele<trop1oresis@'and'stained'Bit1'Coomassie'blue.'_jl'of't1e'eluate'Bere'loaded'in'lane'2.'_jl'of't1e'glutat1ione'sep1arose'beads'after'elution'Bere'loaded'in'lane'2.'Jg:'mole<ular'Beig1tf'EN!C:'Bit1out'glutat1ioneE!Etransferasef'7C:'nonEtagged.'N'!'Beads:'Nlutat1ione'sep1arose'RB.'P:'Pre!<ission'Protease.''' Preparation of Giardia-specific transcript C1e'23end'and'flanking'se^uen<e'of'!Egiardin'gene0'a'<&toskeletal'gene'in'Giardia lamblia0'Bas'<1osen'as'a'model'm678'to'investigate't1e'<leavage'rea<tion.'C1e'sele<ted'91'nu<leotide'long'fragment'<ontains't1e'putative'pol&aden&lation'signal'8NCC888C'doBnstream'of't1e'translational'stop'<odon'C88'and'V'nu<leotides'upstream'of't1e'<leavage'site.'PC6'amplifi<ation'of't1is'se^uen<e'resulted'in'a'12_'nu<leotides'long'ampli<on'?Fig. 7@'in<luding't1e'CD'promoter'needed'for'in vitro'trans<ription.'C1e'use'of't1is'ampli<on'as'template'for'in'vitro'trans<ription'b&'CD'678'pol&merase'bot1'Bit1'and'Bit1out'purifi<ation'Bas'su<<essful'?Fig. 8@.''''
' _
'''''''''''''''''''''''''''' ''''''''''''''''''''''''''''''''''''''''''''''''''''''''''1'''''''''''''2'''''''''''''''2'Fig. 7 Amplification of !-giardin fragment by PCR.'C1e'template'Bas'prepared'b&'PC6'?Pol&merase'C1ain'6ea<tion@'and'anal&\ed'on'a'1k'agarose'gel.'(n'Qane'2'a'<ontrol'PC6'rea<tion'Bit1out'template'D78'Bas'loaded.'(n'lane'2'a'PC6'rea<tion'Bit1'primers'and'template'Bas'loaded.''''
''''''''''''''''''''''''''''''''''''''' 'Fig. 8 In vitro transcription'of"!-giardin fragment.'Bot1'unpurified'and'purified'PC6'produ<ts'Bere'used'as'templates'for'in vitro'trans<ription'b&'CD'678'pol&merase.''C1e'678'trans<ripts'Bere'anal&\ed'on'1Xk'denaturing'a<r&lamide'gel'?19:1'a<r&lamidecbisa<r&lamide@0'DJ'urea.''''
' 9
The purified Giardia lamblia CPSF-73 homologue fraction has nuclease activity ''''''''''''''''''''Co'investigate'if'nu<lease'a<tivit&'<ould'be'asso<iated'Bit1't1e'ba<teriall&'eYpressed'glCP!IED2'pol&peptide0'an'in vitro'<leavage'assa&'Bas'performed.'(n<ubation'of't1e'!Egiardin'substrate'Bit1'up'to'1XXX'nJ'of't1e'glCP!IED2'fra<tion'?7C@'for'2'1'in'<leavage'<onditions'did'not'result'in'an&'nu<lease'a<tivit&'?data'not's1oBn@.'MoBever0't1e'previousl&'reported'metalEdependen<e'of'1uman'CP!IED2'22'tempted'me'to'tr&'t1e'addition'of'different'divalent'metal'ions'to't1e'rea<tion'miY.'(ndeed0't1e'addition'of'Jg2Z'indu<ed'a'nu<lease'a<tivit&'?Fig. 9@.'''''''''
''''''''''''''''''''''' 'Fig. 9'Mg2+-dependent'cleavage of the !-giardin substrate with glCPSF-73 (NT).'C1e'!Egiardin'substrate'Bas'in<ubated'Bit1'glCP!IED2'?7C@'at'2X!C'for'11'under'<onditions'for'678'<leavage.'C1e'678'Bas'anal&\ed'on'1Xk'denaturing'a<r&lamide'gel'?19:1'a<r&lamidecbisa<r&lamide@0'DJ'urea.'Zn2Z'and'Jg2Z'Bere'present'to't1e'<on<entrations'indi<ated'above't1e'lanes.'''''''''''''''''''''''Co'investigate'if't1e'observed'nu<lease'a<tivit&'Bas'a'uni^ue'propert&'of't1e'glCP!IED2'sample'?7C@'t1at'Bas'used'in't1e'rea<tions's1oBn'in'Iigure'9'or'if'it'Bas'a'propert&'t1at'<ould'also'be'linked'to't1e'purified'protein'sample'N!CEglCP!IED2'?C@0't1e'tagged'protein'sample'Bas'tested'for'nu<lease'a<tivit&.'8s's1oBn'in'Iigure1X'N!CEglCP!IED2'possessed'nu<lease'a<tivit&0'suggesting't1at't1e'N!C'tag'did'not'interfere'Bit1't1is'fun<tion'of'glCP!IED2.''''''''
' 1X
'''''''''''''''''''''''''''''''''''''''''''''''''''''' 'Fig. 10'Cleavage of !-giardin substrate with glCPSF-73 (NT) and GST-glCPSF-73 (T).'C1e'!Egiardin'substrate'Bas'in<ubated'Bit1'glCP!IED2'?7C@'and'Bit1'N!CEglCP!IED2'?C@'at'2X!C'for'11'under'<onditions'for'678'<leavage.'2'mJ'JgCl2'Bere'added'to't1e'rea<tion'miY.'C1e'678'Bas'anal&\ed'on'1Xk'denaturing'a<r&lamide'gel'?19:1'a<r&lamidecbisa<r&lamide@0'DJ'urea.'Q282X'substrate'is'_R'nu<leotides'long'and'Bas'used'as'a'si\e'marker.'7C:'nonEtaggedf'C:'tagged.''''''''''''''''''''''g1en't1e'trans<ript'Bas'in<ubated'in't1e'presen<e'of'Jg2Z'but'in't1e'absen<e'of'glCP!IED2'for'1VX'minutes0'no'<leavage'of't1e'678'resulted'?Fig. 11a0'lane'_@0'<onfirming't1at't1e'observed'Jg2ZEdependant'a<tivit&'Bas'not'a<1ieved'b&'a'nu<lease't1at'Bas'a<<identall&'present'in't1e'Jg2Z'solution'added'to't1e'rea<tion'miY'nor'due'to'a'Jg2ZEindu<ed'<leavage.''!in<e'no's1orter'<leavage'produ<ts'Bere'dete<ted'furt1er'doBn't1e'gel0'an'eYonu<leol&ti<'a<tivit&'mig1t'be'present.'Co'test't1is'possibilit&0't1e'kineti<s'of't1e'<leavage'Bas'eYamined'taking'samples'after'X0'1X0'2X0'RX0'_X'and'1VX'minutes'of'in<ubation.'C1e'rea<tion'produ<ts'Bere'anal&\ed'b&'gel'ele<trop1oresis'to'dete<t'a<<umulation'of'678'fragments'Bit1'time'and'b&'t1in'la&er'<1romatograp1&'?CQC@'to'dete<t'an&'released'mononu<leotides.'C1e'<leavage'pattern'observed'in'Iigure'11a'provided'no'eviden<e'for'eYonu<leol&ti<'a<tivit&'sin<e'a'gradual's1ortening'of't1e'trans<ript'<ould'not'be'seen.'7otabl&0'all'of't1e'<leaved'produ<ts'at't1e'
' 11
indi<ated'time'points'1ad'similar'si\es0'a'<riti<al'propert&'of'an'endonu<leol&ti<'rea<tion.'Jost'importantl&0'no'released'`JPs'<ould'be'dete<ted'?t1e'678'Bas'internall&'radioa<tivel&'labeled'Bit1"#$E22Pl'`CP@'in't1e'CQC'<1romatogram'?Fig. 11b@.'C1ese'tBo'observations'strongl&'implied't1at't1e'observed'<leavage'Bas'due'to'an'endonu<lease'a<tivit&'t1at'is'asso<iated'Bit1't1e'purified'glCP!IED2'fra<tion.'8dditionall&0'<leavage'in't1e'predi<ted'<leavage'site'Bould'1ave'&ielded'tBo'<leavage'produ<ts'of'about'5XEVX'nu<leotides'long.'C1e'presen<e'of'tBo'<leavage'produ<ts'Bit1'si\es'greater't1an'_R'nu<leotides'?Fig. 10@'suggested't1at't1e'<leavage'did'not'take'pla<e'at't1e'eYpe<ted'<leavage'site.'C1is'also'implies't1at't1e'endonu<leol&ti<'<leavage'o<<urred'eit1er'simultaneousl&'or'<onse<utivel&'at'tBo'different'positions'Bit1in't1e'678'substrate'?Fig. 10 and 11a@.''
(a)' ''''''''''(b)' 'Fig. 11 (a)'Time course analysis of'cleavage of !-giardin substrate by glCPSF-73.'C1e'!Egiardin'substrate'Bas'in<ubated'Bit1'glCP!IED2'at'2X!C'under'<onditions'for'678'<leavage.'2'mJ'JgCl2'Bere'added'to't1e'rea<tion'miY.'C1e'678'Bas'anal&\ed'on'1Xk'denaturing'a<r&lamide'gel'?19:1'a<r&lamidecbisa<r&lamide@0'DJ'urea. (b)'1jl'from'ea<1'rea<tion'Bas'removed'and'anal&\ed'b&'CQC'?t1in'la&er'<1romatograp1&.'!'is't1e'origin'of'separation'of'nu<leotidesf'P'is't1e'lo<ation'of't1e'eYpe<ted'released'nu<leotides'?7o'`JP'Bas'available'to'use'as'positive'<ontrol@f'J'is'si\e'marker.'EJg2Z'indi<ates't1e'absen<e'of'Jg2Z'and'EglCP!ID2'indi<ates't1e'absen<e'of'glCP!IED2.''
V. BuratoBski0'!.'2XX5.'Conne<tion'betBeen'm678'23end'pro<essing'and'trans<ription'termination.'Current'hpinion'in'Cell'Biolog&.'1D:25DE2V1.'''
D. Callebaut0'(.0'Jos1ous0'D.0'Jornon0'P.P.0'and'de'Oillarta&0'P.P.'2XX2.'JetalloE!Ela<tamase'fold'Bit1in'nu<lei<'a<ids'pro<essing'en\&mes:'C1e'!EC8!P'famil&.'7u<lei<'8<ids'6es.'2X:2592E2VX1.''