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

!ummar&'''''''''''''''''''''''''''''''''''''''(n'almost'all'eukar&otes0't1e'mature'23end'of't1e'm678'is'generated'b&'an'endoribonu<leol&ti<'<leavage'and'addition'of'a'pol&?8@'tail.'8'large'number'of'protein'fa<tors'are'involved'in'a<1ieving't1e'tBo'steps.'C1e'endoribonu<lease't1at'<arries'out't1e'<leavage'rea<tion'in'1uman'Bas'identified'as't1e'D2EkD'subunit'of'<leavage'and'pol&aden&lation'spe<ifi<it&'fa<tor'?CP!IED2@'and'its'<r&stal'stru<ture'Bas'determined'?Jandel0'C.'6.0'Kaneko0'!.0'Z1ang0'M.0'Nabauer0'D.0'Oet1ant1am0'O.'Janel&0'P'and'Cong0'Q.'7ature.'RRR:952E95V.'2XXV@.'Mere0'a're<ombinant'CP!IED2'1omologue'from't1e'eukar&ote'Giardia lamblia0'an'organism't1at'is'postulated'to'1ave'simple'pol&aden&lation'ma<1iner&0'Bas'ba<teriall&EeYpressed'as'a'glutat1ioneE!Etransferase'?N!C@'fusion'protein'and'purified'b&'affinit&'<1romatograp1&.'Iurt1er0'in vitro'<leavage'assa&'demonstrated't1at't1e'purified'pol&peptide'fra<tion'endonu<leol&ti<all&'<leaves'a'GiardiaEspe<ifi<'model'preEm678'in'an'JgZ2Edependent'manner.'C1ese'results't1erefore'nominate't1e'CP!IED2'1omologue'from'Giardia lamblia to'be't1e'endoribonu<lease'of't1e'pol&aden&lation'rea<tion'in'Giardia lamblia.'''''''''''''''''''''''''''

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(ntrodu<tion''The Polyadenylation reaction ''''''''''''''''''C1e'eukar&oti<'m678'is'<1ara<teri\ed'b&'t1e'presen<e'of'tBo'signs'at't1eir'ends0'a'53'<ap'stru<ture'and'a'23'pol&?8@'tail.'C1e'mature'23end'is'formed'during'a'nu<lear'rea<tion'<alled'pol&aden&lation'and'is'a'ke&'regulator&'step'in'gene'eYpression.'C1e'pol&aden&lation'rea<tion'pro<eeds't1roug1'tBo'major'steps'?Fig.1@:'first't1e'preEm678'is'<leaved'endnu<leol&ti<all&'at'a'spe<ifi<'p1osp1oEdiester'bond0'se<ond'a'pol&?8@'tail'is'added'to't1e'<oding'se^uen<e'b&'pol&meri\ation'of'adenosine'monop1osp1ate'?8JP@'?revieBed'in'ref.'2_@.''

''''''''''''''''''''''''''''''' '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

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2X'nu<leotides'doBnstream'of't1e'<leavage'site0'respe<tivel&.'(n'&east0'<leavage'fa<tor'(8'?CI'(8@0'<leavage'fa<tor'(B'?CI'(B@0'<leavage'fa<tor'(('?CI((@'and'pol&aden&lation'fa<tor'('?PI'(@'parti<ipate'in't1e'first'step'of't1e'rea<tion'B1ile'CI'((0'PI'(0'CI'(80'pol&?8@'binding'protein'?Pab'1p@'and'pol&?8@'pol&merase'?Pap'1p@'are're^uired'for't1e'addition'of't1e'pol&?8@'tail.'C1e'pol&aden&lation'signals'in'&east'are'an'8Eri<1'positioning'element'?PE@'lo<ated'1XE2X'nu<leotides'upstream'of't1e'<leavage'site0'an'upstream'element'?`E@'lo<ated'1XE2X'nu<leotides'upstream'of'PE'and'tBo'`Eri<1'elements'situated'on'an&'side'of't1e'<leavage'site'?revieBed'in'ref.'2D0'29'and'2X@.'''

Table. 1 Polyadenylation machinery in mammals and yeast '

Jammals' eeast'Ia<tor' !ubunit' Ia<tor' !ubunit'CP!I' CP!IE1VX'?1@'

CP!IE1XX'?2@'CP!IED2'?2@'CP!IE2X'?R@'1Iip1'?5@'1V''

CI'((& e11'1pcCft'1p'?1@'ed1'1pcCft'2p?2@'Brr'5pces1'1p'?2@'Pta'1p''

CI'(m CI'(m'EV_'CI'(m'E25'

PI'(' Pfs'1p'Iip'1p?5@'Pfs'2p'et1'1p'?R@''

CstI' CstIEDD'?V@'CstIEVR'?D@'CstIE5X'

CI'(B' 7ab'RpcMrp'1p''

CI'((m 1Clp'?_@' CI'(8' 6na'1Rp'?V@'6na'15p'?D@'P<f'1'1p'Clp'1p'?_@''

P8P' P8P'?9@' Pap'1p' Pap'1p'?9@'P8BP2' P8BP2' Pab'1p' Pab'1p'7umbers'Bit1in'bra<kets'denote'pairs'of'1omologues'in'mammals'and'&east.'CP!I:'<leavage'and'pol&aden&lation'spe<ifi<it&'fa<torf'CI:'<leavage'fa<torf'P8P'and'Pap:'pol&?8@'pol&merase'in'mammals'and'&east'repe<tivel&f'P8BP'and'Pab:'pol&?8@Ebinding'proteins'in'mammals'and'&east'respe<tivel&f'PI:'Pol&aden&lation'fa<tor.'m:'mammalf'&:'&east.'Data'from'ref.29.'''' CPSF-73 '''''''''''''''''''C1e'identit&'of't1e'a<tual'endonu<lease'responsible'for't1e'<leavage'rea<tion'pre<eding'pol&aden&lation'1as'remained'elusive'for'almost'2X'&ears'after't1e'dis<over&'of't1e'<leavage'rea<tion'21.'C1e'large'number'of'proteins'involved'in't1e'rea<tion'and't1e'te<1ni<al'diffi<ult&'in'produ<ing'soluble're<ombinant'protein'1ave'been't1e'main'reasons'for't1is'dela&'in'identifi<ation'22.'MoBever0'a'number'of'arguments'suggested't1at't1e'CP!IED2'pol&peptide'<ould'be't1e'endonu<lease'of't1e'pol&aden&lation'rea<tion.'Iirstl&0'it'is'<onserved'in'man&'organisms'

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in'all't1ree'domains'of'life'2012.'!e<ondl&0'it'1as'been'reported't1at't1e'<&toplasmi<'form'of't1e'multisubunit'fa<tor'CP!I'la<ks't1e'D2kD'subunit'90190'B1i<1'is'<onsistent'Bit1't1e'notion't1at'mature'pol&aden&lated'm678s'are'not'endonu<leol&ti<all&'<leaved'in't1e'<&toplasm.'8't1ird'supporting'argument'<ame'from't1e'identifi<ation'of'CP!IED2'as'a'member'of't1e'metalloE!Ela<tamase'fold'superfamil&0'Zn2ZEdependant'1&drolases'R0'and't1e'later'<lassifi<ation'of'CP!IED2'as'a'member'of't1e'!EC8!P'subfamil&'of'metalloE"!Ela<tamases0'B1ose'members'are't1oug1t'to'be'involved'in'nu<lei<'a<ids'metabolism'D.'8mong't1e'latter'are't1e'D78'repair'proteins'!7J1'a'8rtemis'and'EQ8C1'a'EQ8C20'B1i<1'are'involved'in'preEt678'pro<essing0'and't1e'Bacillus'm678'endnu<leases'P1'a'P2'12.'g1ile'all'proteins'in't1e'metalloE!Ela<tamase'fold'superfamil&'possess'five'motifs'?1E5@0'members'of't1e'!EC8!P'subfamil&'are'<1ara<teri\ed'b&'t1e'presen<e'of't1e'four'<anoni<al'motifs'plus'ot1er't1ree'motifs'80'B'a'C'B1i<1'are'<alled'<olle<tivel&'t1e"!EC8!P'domain'and'repla<e't1e'fift1'motif'in'metalloE"!Ela<tamases.'8ll't1ese'motifs'<ontain'an'a<idi<'residue'or'a'1istidine't1at'<oordinates't1e'<atal&ti<all&'important'tBo'\in<'ions'in't1e'a<tive'site.'8're<ent'stud&'indi<ated't1at'mutations'in't1ese'ke&'residues'in't1e'CP!IED2'&east'1omologue'es11p'resulted'in'<ell'deat1'22.'C1e'same'stud&'also'suggested't1at'CP!IED2'is'likel&'to'be'one'of'tBo'proteins'to'`OE<rossElink'<lose'to't1e'<leavage'site'in'preEm678'and't1at't1e'<leavage'rea<tion'is'a'metalEdependant.'Joreover0'anot1er'stud&'demonstrated't1e'involvement'of'CP!IED2'in'pro<essing'of'1istone'preEm678s0't1e'onl&'m678s't1at'are'<leaved'Bit1out'addition'of'a'pol&'?8@'tail'1X.''8ltoget1er0't1ese'observations'led'to't1e're<ent'suggestion't1at't1e'endonu<lease'a<tivit&'of't1e'pol&aden&lation'rea<tion'is'linked'to't1e'D2'kD'subunit'of't1e'1uman'CP!I'1_.''

Giardia lamblia''''''''''''''''''''''''8s'mentioned'above't1e'<ompleYit&'of't1e'pol&aden&lation'rea<tion'in't1e'tBo'BellEstudied's&stems'1as'made'it'te<1ni<all&'diffi<ult'to'reprodu<e't1e'endonu<lease'a<tivit&'in vitro.'MoBever0'in't1e'<ourse'of'finding'a'simpler's&stem'to're<onstitute't1e'rea<tion0't1e'database'of't1e'nearl&'<ompleted'genome'se^uen<e'of't1e'parasite'Giardia lamblia'Bas'sear<1ed'for'proteins'Bit1'1omolog&'to'knoBn'eukar&oti<'pol&aden&lation'fa<tors.'Giardia lamblia0'a'proto\oan'knoBn'to'<ause'a'BaterEborne'enteri<'disease'in'1umans0'belongs'to'a'diverse'group'of'binu<leate'amito<1ondrion'flagellates'<alled'Diplomonads'1.'C1e'la<k'of'mito<1ondria0't1e'presen<e'of'prokar&oti<'features'and'a'model'based'on'smallEsubunits'?!!`@'ribosomal'678'p1&logen&'positioning'Diplomonads'at't1e'base'of't1e'universal'mole<ular'tree'of'life0'1ave'<ontributed'to't1e'vieB't1at'Giardia lamblia'is'a'primitive'and'earl&Ediverged'eukar&ote'?revieBed'in'ref'1@.'MoBever0't1e'dis<over&'of'mito<1ondrial'1omologues'in'Giardi lamblia0't1e'probable'lateral'gene'transfer'from'prokar&otes'and'ot1er're<ent'advan<ed'studies'arguing'against't1e'previous'vieB0'1ave'provided'support'for't1e'<urrent'<lassifi<ation'of'Diplomonads'Bit1in't1e'eukar&oti<'supergroup'EY<avata'2011.'C1e'trans<ription'ma<1iner&'of'Giardia lamblia'1as'similarities'to'bot1'eukar&otes'and'prokar&otes'and't1e'Giardia'lamblia'trans<ripts'are'<1ara<teri\ed'b&'s1ort'23'untranslated'regions'?`C6@'and't1e'absen<e'of'bot1'introns'and'a'<ap'stru<ture'at't1e'53'end'1.'C1e'se^uen<e'8NCPu888P&'1as'been'identified'as'a'putative'pol&aden&lation'signal'2X.'(n'line'Bit1'all't1is0't1e'genome'data'mining'1as'revealed't1at't1is'organism'most'likel&'1as'evolved'strikingl&'simpler'pol&aden&lation'ma<1iner&'t1an'mammals'or'&east'?Fig.2@.'hnl&'seven'open'reading'frames'?h6Is@'Bit1'similarit&'to'knoBn'pol&aden&lation'protein'fa<tors'in'&east'and'mammals'1ave'been'identified'in'Giardia lamblia0'and'among't1e'seven0'a'1omologue'to't1e'long'soug1t'endoribonu<lease'CP!IED2'Bas'found.''

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'''''''''''''''''''' ''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'

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be'eluted'using'redu<ed'glutat1ione.'(t'is'also'possible'to'<leave't1e'N!C'tag'b&'a'siteEspe<ifi<'protease. '

Sepharose Glutathione

GST

Protein

Protease cleavage site

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.''

'' Aims''''''''''''''''''''C1e'bioinformati<'observation'toget1er'Bit1't1e't1ree'arguments'above't1at'indi<ated'CP!IED2'as't1e'a<tual'endoribonu<lease'provided'enoug1'motivation'to'investigate'if't1e'endonu<lease'a<tivit&'<ould'be'linked'to't1e'CP!IED2'1omologue'in'Giardia lamblia.'Conse^uentl&0't1e'proje<t'Bas'set'to'eYpress'and'purif&'t1e'CP!IED2'1omologue'from'Giardia lamblia0'prepare'a'GiardiaEspe<ifi<'preEm678'and'in<ubate't1em'toget1er'in'<leavage'<onditions.'Mere0'('report't1at're<ombinant'CP!IED2'1omologue'from'Giardia lamblia'most'likel&'1as'endonu<leol&ti<'a<tivit&'and'indeed'<leaves'a's1ort'GiardiaEspe<ifi<'model'preEm678'trans<ript.''''''''''''

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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@.'''

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''''''''''''''''''''''''''''''''''''''''' ''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.''''

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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.''

'''''

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Dis<ussion'''''''''''''''''''''(n't1e'attempt'to'eYpress't1e'CP!IED2'1omologue'from'Giardia lamblia0't1e'formerl&'reported'insolubilit&'of't1e'ba<teriall&'eYpressed'1uman'CP!IED2'22'Bas'avoided'b&'folloBing't1e'standard'met1od'des<ribed'in'ref.12.'Iurt1er0't1e'diffi<ult&'of'unspe<ifi<'nonEN!C'intera<tions'en<ountered'during'purifi<ation'Bere'over<ome't1roug1'a'<ombination'of'onE<olumn'purifi<ation'pro<edure0'saturation'of't1e'glutat1ione'sep1arose'RB'Bit1'bovine'serum'albumin'prior'to'purifi<ation'and'modifi<ation'of'elution'<onditions.'''''''''''''''''''''Mere0't1e'results'from'in vitro'<leavage'assa&s's1oBed't1at'a'purified'fra<tion'<ontaining'a're<ombinant'CP!IED2'1omologue'from'Giardia lamblia'possesses'endonu<lease'a<tivit&.'C1e'previousl&'reported'results'Bit1'1uman'CP!IED2's1oBed'onl&'a'nonEspe<ifi<'endonu<lease'a<tivit&1_0'B1ile'in<ubation'of'CP!IED2'1omologue'from'Giardia lamblia'Bit1'a'GiardiaEspe<ifi<'trans<ript'&ielded'tBo'<leaved'produ<ts'Bit1out'intermediates'and'most'likel&'anot1er'eit1er'degraded'or'undete<table's1ort'produ<t0'indi<ating'a'spe<ifi<'endonu<leol&ti<'a<tivit&.'C1e'inabilit&'to'dete<t's1ort'<leaved'fragments'of't1e'trans<ript'<ould'be'be<ause't1e'fragments'1ad'run'out'of't1e'gel'or't1at't1eir'net'radioa<tivit&'Bas'too'small'to'be'dete<ted.'C1erefore0'b&'<ombining't1ese'results'to't1e'proposed'simple'pol&aden&lation'ma<1iner&'?Fig. 2@'in't1e'eukar&ote'Giardia lamblia0'it'is'possible'to'propose't1at'CP!IED2'1omologue'from'Giardia lamblia <ould'potentiall&'be't1e'a<tual'endoribonul<ease'of't1e'pol&aden&lation'rea<tion'in'Giardia lamblia.'''''''''''''''''''''CBo'points'remain'to'be'addressed'before'unambiguousl&'<on<luding't1at't1e'Giardia'CP!IED2'pol&peptide'is'an'endonu<lease.'(t'<ould'be't1at'glCP!IED2'degrades't1e'trans<ript'eYonu<leol&ti<all&'before't1e'first'time'point'?Fig. 12a@'and't1at't1e'released'radiolabeled'nu<leotides'are'not'dete<ted'b&'CQC'be<ause't1e&'do'not'in<lude'a'`.'C1is'is'onl&'possible'if't1e'eYonu<lease'a<tivit&'is'53!23'and't1e'<leaved'produ<t'is'D'nu<leotides'long'sin<e't1ere'is'no'`'present'in't1is'D'nu<leotide'long'fragment.'Co'eYamine't1is0'it'is'possible'to'eit1er'<1e<k'for't1e'possible'appearan<e'of'intermediate'<leaved'produ<ts'before'1X'minutes'of't1e'rea<tion0'or'to'radiolabel'Bit1't1e'nu<leotides'80'C0'C'and'N'and'look'for't1e'released'mononu<leotides'b&'CQC.'C1e'ot1er'point'is't1e'possibilit&'t1at't1e'observed'<leavage'1as'resulted'from'a'<ontaminating'nu<lease'from't1e'ba<teria'used'for'eYpression.'!u<1'a'possibilit&'<an'be'ruled'out'b&'e.g.'mutating't1e'a<tive'site'of'glCP!IED2.'Joreover0'mutagenesis'of't1e'<leavage'site'Bould'bring'interesting'insig1ts'into't1e'issue'of'spe<ifi<it&'of'glCP!IED2'to't1e'<leavage'site.'8not1er'interesting'^uestion'to'address'in't1e'future'is:'Does't1is'endonu<lease'need'ot1er'members'found'in't1e'proposed'ma<1iner&'in'Giardia lamblia0'for'instan<e'CP!IE2X'?Fig. 2@0'to'&ield'<orre<t'23preEm678'<leavagem'C1is'is'of'parti<ular'interest'be<ause'it'Bas's1oBn't1at'depletion'of'CP!IE2X'in't1e'<losel&'related'parasite'Tryanosoma brucei'interrupts'678'pro<essing'15.''''''''''''

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Jaterial'and'Jet1ods'''''''''''''''''''''C1e'Giardia'Database'?1ttp:ccBBB.mbl.educNiardia@'Bas'sear<1ed'for't1e'open'reading'frame'?h6I@'RR9_'previousl&'identified'as'1&pot1eti<all&'en<oding'for'CP!IED2'from'Giardia lamblia.'C1e'obtained'se^uen<e'Bas'used'for't1e'<al<ulation'of't1e't1eoreti<al'mole<ular'Beig1tcp('from't1e'EYP8!&'?EYpert'Protein'8nal&sis'!&stem@'proteomi<s'server'of't1e'!Biss'institute'of'bioinformati<s'?!(B@'?1ttp:ccBBB.eYpas&.orgctoolscpintool.1tml@.'''''''''''''''''''''C1e'pNEoEVP'plasmid'?8mers1am'Bios<ien<es@'Bit1't1e'se^uen<e'<orresponding'to't1e'h6I'RR9_'from'Giardia lamblia'<loned'in'frame'Bit1't1e'glutat1ioneE!Etransferase'?N!C@'gene'and'transformed'into't1e'proteaseEdefi<ient'strain'Eschericia coli'BQ21'?DE2@0'Bas'kindl&'provided'b&'!taffan'!värd'?Department'of'Cell'and'Jole<ular'Biolog&0'`ppsala'`niversit&0'!Beden@.' Protein'eYpression'and'purifi<ation''Expression of recombinant glCPSF-73 '''''''''''''''''''PlasmidE<ontaining''E.coli'Bas'groBn'at'2D!C'in'QuriaEBertani'brot1'?QB@'<ontaining'ampi<illin'1XX'jgcml'to'a'<ell'densit&'?8VXX@'of'1.XE1.2'?8VXX:'absorban<e'at'Bave'lengt1'VXX'nm@.'C1e'eYpression'of't1e'insert'Bas'under't1e'<ontrol'of tac'promoter'and'indu<ed'for'R'1'b&'adding'isoprop&l'!ED't1iogala<tosidase'?(PCN@'to'X.1'mJ'final'<on<entration.'C1e'<ells'Bere'pelleted'b&'<entrifugation'at'5XXX'g'for'2X'minutes.'C1e'pellets'Bere'reEsuspended'in'l&sis'buffer'<ontaining'1X'mJ'CrisEMCl'pM'_.X0'15X'mJ'EDC80'1XX'jgcml'l&so\&me0'5'mJ'dit1iot1reitol'?DCC@'and'1.5k'!arkos&l'?7Elaur&lsa<osine@0'soni<ated'on'i<e'at'amplitude'5'for'RY2X'se<.'C1e'l&sate'Bas'<larified'b&'<entrifugation'at'1X'XXX'g'for'15'minutes'at'RpC0'Critono'1XX'Bas'added'to't1e'l&sate'to'2k'?vcv@'final'<on<entration'12.''''''Purification of recombinant glCPSF-73 '''''''''''''''''''8ll'steps'Bere'performed'at'R˚C.'8'medium'of'5Xk'slurr&'of'glutat1ione'sep1arose'RB'?N'!'beads@'?NE'Mealt1<are@'Bas'prepared'b&'adding'1'ml'of'1Y'PB!'?1RX'mJ'7aCl0'2.D'mJ'KCl0'1X'mJ'7a2'MPhR0'1._'mJ'KM2PhR0'pM'D.2@'to'1.22'ml'N'!'beads.'C1e'5Xk'slurr&'N'!'beads'Bere'first'saturated'in'a'pol&Eprep'<1romatograp1&'<olumn'?BioE6ad@'Bit1'X.1'mgcml'of'a<et&lated'bovine'serum'albumin'?B!8@0'Bas1ed'and'e^uilibrated'Bit1'1o'PB!'as'binding'buffer.'6e<ombinant'N!CEtagged'CP!IED2'?N!CEglCP!IED2@'Bas'alloBed'to'bind'to't1e'N'!'beads'b&'passing't1e'l&sate't1roug1't1e'<olumn.'C1e'protein'bound'beads'Bere'Bas1ed'b&'1o'PB!.''''Purifi<ation'pro<edure'1''''''''''''''''''''C1e'N!CEglCP!IED2'Bas'obtained'b&'eluting'using'elution'buffer'EB'1'?5X'mJ'CrisEMCl0'2X'mJ'glutat1ione0'2'J'7aCl0'X.5kCritono'1XX0'pM'_.X@.'''Purifi<ation'pro<edure'2''''''''''''''''''''Iirst0't1e'N'!'beads'<ontaining'N!CEglCP!IED2'Bas'Bas1ed'Bit1'Pre!<ission'<leavage'buffer'PCB'?5X'mJ'CrisEMCl0'15X'mJ'7aCl0'1'mJ'EDC80'1'mJ'DCC0'pM:'D.5@'and'in<ubated'over'nig1t'in'Pre!<ission'<leavage'miY'?R9X'jl'of'PCB'and'1X'jl'?2X'units@'of'Pre!<ission'Protease'?P@@'in'order'to'<leave'off't1e'N!C'tag.'!e<ond0'glCP!IED2'Bas'eluted'using'elution'buffer'EB'2'?5X'mJ'CrisEMCl0'2'J'7aCl0'Critono'X.5k0'pM'_.X@.''

' 1R

'''''''''''''''''''C1e'purit&'of't1e'purified'proteins'Bas'anal&\ed'on'!D!EP8NE'?sodium'dode<&l'sulfate'pol&a<r&lamide'gel'ele<trop1oresis@'?see'polyacrylamide gel'electrophoresis@0'stained'Bit1'Coomassie'Brilliant'Blue.'C1e'purified'fra<tions'Bere'dial&\ed'overnig1t'against'dial&sis'buffer'?1X'mJ'MEPE!'?pM:'D.9@0'1Xk'?vcv@'gl&<erol0'5X'mJ'KCl'and'X.25'mJ'DDC@.'Protein'<on<entrations'Bere'determined'b&'Bradford'8ssa&'?BioE6ad@'using'bovine'gamma'globulin'as'referen<e.'''Preparation'of'D78'templates'and'in vitro trans<ription'''''''''''''''''''''!tandard'pol&merase'<1ain'rea<tion'?PC6@'Bas'performed'as'des<ribed'b&'t1e'manufa<turer'using'Pu6e'Ca^'6ead&ECoENo'PC6'Beads'?NE'1ealt1<are@'to'amplif&'t1e'91'nu<leotides'long'fragment'<orresponding'to't1e'23end'and't1e'flanking'se^uen<e'of't1e'!Egiardin'gene'from'Giardia lamblia.'C1e'fragment'Bas'available'in'a'pNEJEC've<tor'?Promega@'?kindl&'provided'b&'!taffan'!värd@'immediatel&'doBnstream'a'CD'promoter.'PC6'Bas'<ondu<ted'using't1e'oligonu<leotides'of't1e'CD'promoter'region'?underlined@'as'forBard'primer'5'3C88C8CN8CCC8CC8C8NNN'23'and'53CCN8NCCCNCNNCNC8N8CNCNNCNCNNC'23'as'reverse'primer'?(nvitrogen@.'C1e'si\e'of't1e'produ<t'Bas'<onfirmed'b&'running'1k'agarose'gel'stained'Bit1'X.5'jgcml'et1idium'bromide'in'X.5o'CBE'buffer'?CriscboratecEDC8:'R5.5'mJ'Cris0'R5.5'mJ'bori<'a<id0'1'mJ'EDC80'pM'_.X@'at'11X'O'and'visuali\ed'under'`O'lig1t.'''''''''''''''''''''''(n'vitro'trans<ription'Bit1'CD'678'pol&merase'Bas'performed'using'eit1er'purified'?PC6'purifi<ation'kit0'q(8NE7@'or'nonEpurified'PC6'produ<t'as'a'template'in't1e'presen<e'of'"#$E22Pl'`CP'using'J8o(s<ript'CD'Kit'as'des<ribed'b&'t1e'manufa<turer'?8mbion@.'C1e'trans<ription'produ<t'Bas'resolved'in'1Xk'denaturing'a<r&lamide'gel'?see'polyacrylamide gel electrophoresis@0'eYposed'to'oEra&'film'and'developed.'C1e'bands'<orresponding'to't1e'trans<ript'Bere'<ut'out'and'eluted'overnig1t'in'2XX'jl'678'elution'buffer'?1X'mJ'CrisEMCl0'X.2'mJ'sodium'a<etate0'1X'mJ'EDC80'X.2k'!D!0'pM:'D.V@.'678'substrates'Bere'purified'b&'using'pa<ked'!ep1adeY'NE5X'?NE'Mealt1<are@'<olumns.'8'2'ml's&ringe'Bas'filled'Bit1'!ep1adeY'NE5X'in'1X'mJ'CrisEMCl'pM'D.90'1'mJ'EDC8'Bit1'a'g1atman'filter'NIc8'in't1e'bottom'of't1e's&ringe.'2XX'ul'of'eluted'678'Bas'applied'to't1e'pa<ked'<olumn.'C1e'<olumn'Bas'<entrifuged'for'5'min'at'5XX'g.'Eluted'678'Bas'et1anolEpre<ipitated'b&'adding'X.1'volume'of'2J'sodium'a<etate'pM'5.20'2'volumes'of'1XXk'et1anol.'C1e'sample'Bas'kept'at'E2X'˚C'for'2X'minutes.'C1e'678'Bas't1en'<entrifuged'at'1X'XXX'g'for'2X'minutes'at'R˚C.'C1e'supernatant'Bas'removed'and't1e'pellet'Bas'Bas1ed'Bit1'DXk'et1anol'and'<entrifuged'for'1X'XXX'g'for'2'minutes.'C1e'supernatant'Bas'removed'and'pellets'Bere'dried'and'resuspended'in'nu<leaseEfree'Bater'?(nvitrogen@.'C1e'<on<entration'of'678'Bas'measured'in'a's<intillation'<ounter'?Be<kman@'using'"#$E22Pl'`CP'as'referen<e.'C1e'<on<entration'of't1e'678'Bas'<al<ulated'a<<ording'to't1e'formula:''CPJ'of'"#$E22Pl'`CPc'r"#$E22Pl'`CPl's'CPJ'in'678'samplec'r678l''C1e'<al<ulated'<on<entration'of't1e'678'Bas'adjusted'for't1e'total'<on<entration'of'`CP'?'#$E22Pl'`CP'plus'`CP0'ratio'1:2XXX@'during'in vitro'trans<ription0'also'taking'into'a<<ount't1e'number'of'ura<ils'in't1e'678'substrate'?21'ura<ils@.'CPJ'is'<ounts'per'minute.''''''

' 15

! 1#

Cleavage!assa+s!!!!!!!!!!!!!!!!!!!!Cleavage!assa+s!,ere!performed!in!a!15!6l!rea7tion!mi9ture!t;at!7ontained!<.>!n?!labeled!ABC!substratesD!15!m?!EEPEH!IpE:!>.9LD!15M!IvNvL!gl+7erolD!55!m?!PClD!5.25!m?!DDSD!55!6?!CaCl2D!ABCguardS?!ABase!in;ibitor!IPor7ineL!ITE!Eealt;7areL!2!unitsD!555!ng!BHC!and!555V1555!n?!of!re7ombinant!protein!unless!ot;er,ise!indi7ated.!ZnCl2!and!?gCl2!,ere!separatel+!added!to!t;e!rea7tion!in!t;e!indi7ated!7on7entrations.!S;e!rea7tions!,ere!in7ubated!at!X5!C!for!1!;!or!as!indi7ated!in!t;e!timeV7ourse!assa+!IYig.!12LD!stopped!b+!adding!ABC!loading!buffer!I99M!formalde;+deD!25!m?!Sris!>.9D!15!m?!EDSCD!5.55M!9+lene!7+anol!YYD!5.55M!bromop;enol!blueL!!and!anal+Zed!on!15M!denaturing!a7r+lamide!gels!I19:1!a7r+lamideNbisa7r+lamideLD!>?!urea!Isee!L.!Tels!,ere!e9posed!to!a!P;ospoV[mager!s7reen!I?ole7ular!D+nami7sL.!!Cleavage!rea7tions!,ere!anal+Zed!b+!t;in!la+er!7;romatograp;+!IS\CL!for!t;e!release!of!]?P!produ7ts!b+!spotting!1!6l!of!t;e!rea7tion!onto!a!pol+et;+leneimine!7ellulose!Y!plate!I?er7kL!and!using!5.>5!?!PE2P_<D!pE!X.5!I;XP_<L!as!a!solvent.!S\C!plates!,ere!e9posed!to!a!P;ospoV[mager!s7reen!I?ole7ular!D+nami7sL.!!!!Pol+a7r+lamide!gel!ele7trop;oresis!!!"!#$%&'()*'&(+,-./0.&/.&.)1*%$2%*.3,3/4!"!#56789/!!!!!!!!!!!!!!!!!!!!HDHVpol+a7r+lamide!Ia7r+lamide:bisa7r+lamide!29:1L!gelsD!5M!in!sta7king!gel!I12.5M!of!`9!sta7king!buffer!I5.92!?!SrisVECl!pE!#.`D!5.`M!HDHLD!1#.#M!of!X5M!29:1!a7r+lamide!solution!IBioVAadLD!5.99M!of!15M!ammonium!pero9odisulp;ate!CPH!and!5.599M!of!tetramet;+let;+leneVdiamine!SE?EDL!and!15M!in!separation!gel!I25M!of!<9!separation!buffer!I1.<<!?!SrisVECl!pE!`.`D!5.<M!HDHLD!XX.XM!of!X5M!29:1!a7r+lamide!IBioVAadLD!5.5M!of!15M!CPH!and!5.55M!of!SE?EDL!,ere!prepared!using!gel!apparatus!IBioVAadL.!`!6l!of!purified!protein!samplesD!155!m?!of!DSS!and!1!volume!of!2a!sample!buffer!I55!m?!SrisVECl!pE!#.`D!1M!I,NvL!HDHD!`M!IvNvL!gl+7erolD!5.525M!I,NvL!bromop;enol!blueL!,ere!loaded!to!t;e!gel!after!being!;eated!for!at!95!C!for!X!minutes.!S;e!gel!,ere!run!in!1a!HDH!running!buffer!I5.XM!SrisD!1.<<M!gl+7ineD!5.1M!HDHL!on!155!b.!S;e!resulting!gels!,ere!stained!,it;!Coomassie!Brilliant!Blue.!!!".:(1;*,:0/$%&'()*'&(+,-./0.&/.&.)1*%$2%*.3,3/!!!!!!!!!!!!!!!!!!!S;e!ABC!s+nt;esiZed!b+!,:/<,1*%!trans7ription!and!t;e!rea7ted!ABC!from!t;e!,:/<,1*%!7leavage!assa+s!,ere!anal+Zed!in!15M!denaturing!pol+a7r+lamide!gel!I<5M!19:1!a7r+lamideNbisa7r+lamideIBioVAadLLV>?!urea!after!adding!ABC!loading!buffer!to!t;e!!sample.!S;e!gels!,ere!run!in!5.5a!SBE!ISrisNborateNEDSC:!<5.5!m?!SrisD!<5.5!m?!bori7!a7idD!1!m?!EDSCD!pE!`.5L!!at!15!c.!!!!!

8<knoBledgements'''''''''''''''''''''('Bould'like'to'eYpress'm&'gratitude'for'Per'7ilsson'for'1is'<losed'guidan<e't1roug1'out't1e'Bork.'('Bould'also'like'to'deepl&'t1ank'Professor'8nders'Oirtanen'for'a<<epting0'supervising'and'providing'me'Bit1'1is'valuable'suggestions.'J&'t1anks'eYtends'to'7i<klas'Menriksson0'Qei'Con\e'and'K&riakos'Kokkoris'from't1e'pol&?8@'group'for't1eir'1elp'and'for'<reating'su<1'a'friendl&'Bork'spa<e0'!taffan'!värd0'Emma'and'Manna'from't1e'Giardia'group'for'providing'and'1elping'me'Bit1't1e'plasmids'for'Giardia'and'Karin'Carlson'for'1er'1elpful'advi<es'during't1e'proje<t.'Iinall&'('Bould'like'to't1ank'm&'brot1er'8s1raf'for'1is'<ontinuous'support'and'en<ouragement.''''''''''''''''''''''''''''''

' 1D

6eferen<es''1. 8dam0'6.D.'2XX1.'C1e'biolog&'of'Giardia lamblia.'Ji<robiol'6ev.'1R:RRDERD5.'2. 8nant1araman0'O.0'koonin0'E.O.'and'8rvind0'Q.'2XX20'Comparative'genomi<s'and'evolution'of'proteins'involved'in'678'metabolism.'7u<lei<'8<ids'6es.0'2X:1R2DE1RVR.''

2. 8ndersson0'P.'h.0'!jogren0'8.'J.0'Davis0'Q.'8.0'Emble&0'C.'J.'6oger0'8.'P.'2XX2.'P1&logeneti<'anal&ses'of'diplomonad'genes'reveal'fre^uent'lateral'gene'transfers'affe<ting'eukar&otes.'Current'Biolog&.'12:9RE1XR.'

R. 8ravind0'Q.'1999.'8n'evolutionar&'<lassifi<ation'of't1e'metalloE!Ela<tamase'fold'proteins.'(n'sili<o.'Boil.'1:V9E91.'''

5. Bird0'N.'Zorio.'D8'and'Bentle&0'DQ.'2XXR.'678'pol&merase'(('<arboY&Eterminal'domain'p1osp1or&lation'is're^uired'for'<otrans<riptional'preEm678'spli<ing'and'2tEend'formation.'Jole<ular'Cell'Biolog&. 2R:_9V2E9.'

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.''

_. Coller0'P.'J.0'Nra&'7.'K.'and'gi<kens'J.'P.'199_.'m678'stabili\ation'b&'pol&?8@'binding'protein'is'independent'of'pol&?8@'and're^uires'translation.'Nenes'Dev.'12:222VE2225.'

9. Di<kson0'K.!.0'Bilger0'8.0'Ballant&ne0'!.0'and'gi<kens0'J.P.'1999.'C1e'<leavage'and'pol&aden&lation'spe<ifi<it&'fa<tor'in'Xenopus laevis'oo<&tes'is'a'<&toplasmi<'fa<tor'involved'in'regulated'pol&aden&lation.'Jol.'Cell'Biol.'19:5DX5E5D1D.''''

1X. Dominiski0'Z.0'eang0'o.C.'and'Jar\luff0'g.I.'2XX5.'C1e'pol&aden&lation'fa<tor'CP!IED2'is'involved'in'1istoneEpreEm678'pro<essing.'Cell'122:2DER_.'

11. Emble&0'C.J.0'Jartin0'g.2XXV.'Eukar&oti<'evolution0'<1anges'and'<1allenges.'7ature.'RRX:V22EV2X.'

12. 'Even0'!.0'Pellegrini0'h.0'Zig0'Q.0'Qabas0'O.0'Oin10'P.0'Bre<1emmierEbae&0'D.0'and'Put\er0'M.'2XX5.'6ibonu<leases'P1'and'P2:'tBo'novel'endoribonu<leases'in'B.subtilis'Bit1'fun<tional'1omlg&'to'E.coli'67ase'E.'7u<lei<'a<ids'6es.'22:21R1E2152.''

12. Irangioni0'P.'O.'and'7eel0'B.'1992.'!olubili\ation'and'purifi<ation'of'en\&mati<all&'a<tive'glutat1ione'!Etransferase'?pNEo@'fusion'proteins.'8nal'Bio<1em.'21X:1D9E1_D.'

1R. Maung0'e.'and'Carmi<1ael0'N.N.'199V.'6ole'of'pol&aden&lation'in'nu<leo<&toplasmi<'transport'of'm678.'Jole<ular'Cell'Biolog&.'1V:152RE15R2.''

15. Mendriks0'E.'I0'8bdulE6a\ak0'8.0'Jatt1eBs0'K.'6.'2XX2.'tbCP!I2X'Depletion'b&'678'(nterferen<e'Distupts'Pol&si<troni<'678'Pro<essing'in'Cr&panosoma'Bru<ei.'C1e'Pournal'of'Biologi<al'C1emistr&.'2D_:2V_DXE2V_D_.'

1V. Kaufmann'(.0'Jartin'N.0'Iriedlein'8.0'Qangen'M.0'and'Keller'g.'2XXR.''Muman'Iip1'is'a'subunit'of'CP!I't1at'binds'to'`Eri<1'678'elements'and'stimulates'pol&?8@'pol&merase.'EJBh'P.'22:V1VEV2V.'

1D. Qi0'e.'C1en'Ze0'gang'g.0'Baker'C.C.0'Krug'6.J.'2XX1.'C1e'2tEendEpro<essing'fa<tor'CP!I'is're^uired'for't1e'spli<ing'of'singleEintron'preEm678s'in'vivo. D:92XE921.''

1_. Jandel0'C.'6.0'Kaneko0'!.0'Z1ang0'M.0'Nabauer0'D.0'Oet1ant1am0'O.'Janel&0'P'and'Cong0'Q.'2XXV.'Pol&aden&lation'fa<tor'CP!IED2'is't1e'preEm678'23EendEpro<essing'endonu<lease.'7ature.'RRR:952E95V.''

' 1_

19. Jende\0'6.0'Jurt1&0'K.N.0'6&an0'K.'Janle&0'P.Q.0'and'6it<1er0'P.D.'2XXX.'P1osp1or&lation'of'CPEB'b&'Eg2'mediates't1e're<ruitment'of'CP!I'into'an'a<tive'<&toplasmi<'pol&aden&lation'<ompleY.'Jol.'Cell.'V:1252E1259.''

2X. Jeng0'C.C0'8le&0'!.B.0'!vard0'!.N.0'!mit10'J.'g.0'Muang0'B.0Kim0'P.0'Nillin0'I.D.'199V.'(mmunolo<ali\ation'and'se^uen<e'of'<altra<tinc<entrin'from't1e'earl&'bran<1ing'eukar&ote'Niardia'lamblia.'Jol'Bio<1em'Parasitol.D9:1X2E1X_.'

21. 7evins0'P.'6.0'Darnell0'P.'E.'19D_.'!teps'in't1e'pro<essing'of'8d2'm678:'pol&?8@Z'nu<lear'se^uen<es'are'<onserved'and'pol&?8@'addition'pre<edes'spli<ing.'Cell'15:1RDDE92.'

22. Proudfoot0'7.'P.'2XXR.'7eB'perspe<tives'on'<onne<ting'messenger'678'23end'formation'to'trans<ription.'Current'hpinion'in'Cell'Biolog&.'1V:2D2E2D_.'

22. 6&an0'K.0'Calvo0'h.0'and'Janle&0'P.'2XXR.'Eviden<e't1at'pol&aden&lation'fa<tor'CP!IED2'is't1e'm678'23pro<essing'endonu<lease.'678.'1X:'5V5E5D2.''

2R. !a<1s0'8.'B.0'!arnoB0'P.'and'Ment\e0'J.'g.'199D!tarting'at't1e'beginning0'middle0'and'end:'translation'initiation'in'eukar&otes.'Cell.'_90'_21E_2_.''

25. !1atkin0'8.'P.0'and'Janel&0'P.'Q.'2XXX.'C1e'ends'of't1e'affair.'7ature'!tru<tural'Biolog&.'D:_2_E_R2.''

2V. Cakagaki0'e.0'!eipelt'6.'Q.0'Peterson'J.'Q.0'and'Janel&0'P.'Q.'199V.'C1e'pol&anden&lation'fa<tor'CstIEVR'regulates'alternative'pro<essing'of'(gJ'1eav&'<ain'preEm678'pro<essing'and'pol&aden&lation.'Cell'_D:9R1E952.''

2D. ga1le0'E'and'6uegsegger0'`.'1999.'23EEnd'pro<essing'of'preEm678'in'eukar&otes.'IEJ!'Ji<robiolog&'6evieBs.'22:2DDE295.'

2_. ga1le0'E.'and'Keller0'g.'199V.'C1e'bio<1emistr&'of'pol&aden&lation.'Crends'Bio<1em.!<.'21:2RDE25X.'

29. Zarudna&a0'J.'(.0'Kolomiets0'(.'J.0'and'Movorun0'D.'J.'2XX2.'g1at'7u<lease'Cleaves'PreEm678'in't1e'Pro<ess'of'Pol&aden&lationm'(`BJB'life.'5R:2DE21.''

2X. Z1ao0'P.0'M&man0'Q.0'and'Joore0'C.'1999.'Iormation'of'm678'23ends'in'eukar&otes:'me<1anism0'regulation0'and'interrelations1ips'Bit1'ot1er'steps'in'm678's&nt1esis.'Ji<robiol.'Jol.'Biol.'6ev.'V2:RX5ERR5.''

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