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Functional and phylogenetic analysis of the ubiquitylation system in Caenorhabditis elegans: ubiquitin-conjugating… Jones, Donald; Crowe, Emily; Stevens, Tracy A; Candido, E P M Dec 12, 2001

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http://genomebiology.com/2001/3/1/research/0002.1commentreviewsreportsdeposited researchinteractionsinformationrefereed researchResearchFunctional and phylogenetic analysis of the ubiquitylation systemin Caenorhabditis elegans: ubiquitin-conjugating enzymes, ubiquitin-activating enzymes, and ubiquitin-like proteins				 						 !		" #$	 !"%&"	&'()*+"		!	,		!-"			.	"%#(/00/.!		,123/*4$+					+5	6	!#	+"%+Abstract Background: The eukaryotic ubiquitin-conjugation system sets the turnover rate of manyproteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substraterecognition, respectively. There are also ubiquitin-like proteins with distinct functions, whichrequire their own E1s and E2s for attachment. We describe the results of RNA interference(RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. Wealso present a phylogenetic analysis of UBCs.Results: The C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAishows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homologof yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifierSUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifierRub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog ofyeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis ofC. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18groups, 13 of which include members from all three species. The activating enzymes and theubiquitin-like proteins NED-8 and SUMO are required for embryogenesis. Conclusions: The number of UBC genes appears to increase with developmental complexity, andour results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8and SUMO and their corresponding activating enzymes are required for embryogenesis.Published: 12 December 2001Genome Biology 2001, 3(1):research0002.1–0002.15The electronic version of this article is the complete one and can befound online at http://genomebiology.com/2001/3/1/research/0002© 2001 Jones et al., licensee BioMed Central Ltd (Print ISSN 1465-6906; Online ISSN 1465-6914)Received: 8 June 2001Revised: 20 September 2001Accepted: 24 October 2001Background!	"%7"58"#		%		#"5#!		"				#"5		"9		9##			#%!	3'		:				;(3<=+	"%7"	!			 ! 	 "	 "%7"5# 	>	 :(="%7"58"## 	>	 :$  3= "%7"55	 #	 :*=+ !	 ( 	 "%7"  5			 	 	"#    	>	5%" "%7" !		? 		 "	  	!	   	  ( 	7"		+ !	 3  $ 	 	 "%7"   (  # !		  		 !	 	 !	  !	2 Genome Biology Vol 3 No 1 		 "%7" 	    #"   	"	!	"%		+-!	(!	$  ! 	%	 	7"		  " 	5#		!			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"  !#		 	 # 9			 !"$ +Results and discussionUBC and UEV proteins in C. elegansC !	 %  !	   #		 	7"		 	 !			 3/ "%7"58"## 	>	+ D		 				%5$ 5			!	%		#	  	! :%	 (=+   #					" %	   ;(((3<+!		#				!		!	!#"	#		+   !	!#   !"# !		 				%		  +"!	"5	"	"%	A 0  !	  	"	:(/((		"	!					  !#!		# #		=+ H!			 %	 !	 #	 	  	5  !	 "%	# 	 "	  	 !#?!		 #	 !	   #		 "%	 %			 !				%		%!	5			%				%	!	:		%	=+!		#	7"			%	!	$   %	   	 !	 			  !	$ 	:$ ,$7?				!=!!!				  	5	 	 	"	+ G46"		  !	 3/  $  !	 	# . 	+ !					!!		#				@		%"!		$%	:	 =#			+!		 		 #		 	  ! !	 	#. 	7"		(/33+( ,/(E3+' ! 	 	5		I0AD3+3*	%	" 		 #		  !!  !	 	  	5	    .+ !	 ,/2 *+A #		 	 	     "   $  	 ! !	+!	#		!	.!"#!		 !	 . 		  !	$ +H	 !	!			"	!	$ !+D	!!	#!"	#				%	#		 ! 	  	 % . 	7"		 !" %					"%9	%	@					+	7"		#	!	"	  $ 5	 :		%	=			 "!	 			 !	#			"	%.	7"		+/'3+*		  !	  	 		   		 	 	 	% 		  %9  (0  !   # ! !	 !	 	7"		+  %" !#	 		 	  G46A2(3+A I2AE'+'	##"'33"#				+/'3+*		!	5	 		  	" %  ! !	 	"  G46A/D2+'  !!   	 		+#  !#	 		 	  !		 		#		%		!			7"				"	!	"%	57"	+!		 #			G4603'+(3	  	7"		    %" 9  !	$   !	 	5	 	 	"	+ ! #						"$ +commentreviewsreportsdeposited researchinteractionsinformationrefereed researchhttp://genomebiology.com/2001/3/1/research/0002.3-   !	 3/  #		 ! 		 !	 		%	%	!	  #		!		#		 ! 	 !	$  %" 9 !	 	5		 	"	 :%	 (=+ !		 #		 !	 %		 	"%7" 3  %%		   ;(*<+ !	 $&5(	7"		  	 % 	@# . . 	7"			#$&5*		%	%			,:,5,=+H				%		!		#		!!		%@5		7"		:!=+!	 #		!	#.	7"		+,		"	"##		;(A5('<	%	"9!	!	  $ $&#			 	@		Table 1RNAi with C. elegans ubiquitin-conjugating enzyme genesCosmid Gene dsRNA Fragment Chromo- Number of Embryonic Secondary Embryonic Embryonic Secondary name source size (bp) some RNAi trials lethality % phenotype lethality (%) lethality (%) phenotypebrood A brood BFeeding method Injection methodUBC proteinsB0403.2 ubc-17 gen PCR 591 X 2 3.4 Normal - - -C06E2.3 ubc-21 gen PCR 1131 X 2 0 Normal - - -C06E2.7 ubc-22 gen PCR 1075 X 2 0 Normal - - -C28G1.1 ubc-23 gen PCR 491 X 2 0 Normal - - -C35B1.1 ubc-1 pR1.7 593 IV 2 0 Normal 0 0 NormalD1022.1 ubc-6 yk640 h7 750 II 2 6.9 Normal - - -F29B9.6 ubc-9 yk312e11 1160 IV 4 60.2 Evl, abnormal 2.7 18.2 Evl, Egl, morphology abnormal morphologyF40G9.3 ubc-20 yk246g3 835 III 4 0 Rare arrest 4.8 0 Some arrest at at L3 stage L3 to L4 stage*F49E12.4 ubc-24 gen PCR 842 II 2 0 Normal - - -F58A4.10 ubc-7 yk486g12 500 III 2 0 Normal - - -M7.1 let-70 cDNA 444 IV - - - - 51 L3 stage arrest(ubc-2)R01H2.6 ubc-18 yk461f2 584 III 2 0 Normal - - -R09B3.4 ubc-12 yk486f10 630 I 6 85.3 Evl, abnormal 5.8 52.3 Evl, morphology abnormalmorphologyY110A2AR.2 ubc-15 yk602e10 644 II 2 0 Normal - - -Y54E5B.4 ubc-16 yk137b4 679 I 2 0 Normal 1.0 1.4 NormalY54G2A.23 ubc-13 yk659d2 499 IV 2 0 Normal - - -Y69H2.6 ubc-19 yk290g1 635 V 2 0 Normal 0.8 1.1 NormalY71G12B.15 ubc-3 yk255b2 1053 I 2 0 Normal 1.6 2.0 NormalY87G2A.9 ubc-14 yk573c8 532 I 6 54.1 Abnormal - - -morphologyY94H6A.6 ubc-8 gen PCR 890 IV 2 0 Normal - - -UEV proteinsF39B2.2 uev-1 yk593d12 499 I 2 0 Normal - - -F56D2.4 uev-2 gen PCR 1074 III 2 0 Normal - - -F26H9.7 uev-3 RT-PCR 660 I 2 0 Normal - - -Gene names have been assigned as indicated in the text. DNA fragments of the indicated sizes were obtained from the cDNA sources shown or fromfragments obtained by genomic PCR (gen PCR), or by reverse transcription from RNA (RT-PCR). Phenotypes include Evl (everted vulva) and Egl (egg-laying defect). Embryonic lethality for broods A and B are shown separately. A dash indicates experiment not done. *Variable penetrance. Two potentialC. elegans ubc genes that have recently come to light are not included in this table. F25H2.8 encodes a protein that is 25% identical to UBC-1 over 100amino acids spanning the active site. This UBC domain is not recognized by Pfam, but is recognized by SMART (UBCc domain). The protein is 56%identical in amino-acid sequence to Drosophila BG:25E8.2 and therefore belongs in group I of Figures 1 and 2. RNAi experiments with F25H2.8 produceparalysis in the adult stage and premature death of the hermaphrodites 4 to 5 days after the beginning of oviposition [85]. YAC clone Y110A2AM maycontain another ubc gene (currently uncurated) encoding a protein 52% identical over 143 amino acids to Y110A2AR.2 (group XVIII)."	  #! + !	  "	 !! 	 		    #		   "			%	 		 ,."#  			+ -#		 !		#			 !	 		 !! !			"	  9 . 	7"		+ -		# "   #		: /A/*+3/'3+*/'3+43BD(+(=! 	 "		  !	 J !	 !	 		 		%	,.	#.!	%				 ;(0<+ H	 	 " 	@		 ,5, "##"		 	#  !	 	7"		   !		 #		 /'3+*  /'3+4 %"  " 		%	+!		 	"	"	 !	% !!		 #		 	 "	 "	 	   !!	,.	" !5	 	+!	.!	@			#			  		 !"#! ! #		  	@		 !"#!"			;((<+	. 	 %	  #			@		!"#!"			;(0('<	 	"!	!	 ;('<   #		 	";(A<+ !	 #		!	"%				@	!"#!			!,.		  !#!	  !	 	% #	+ - 	 		!		 #	!			 #  !	 "!  #	   "#"+ !		 	" "#! 	 !	 "  	9#	!			!!	!#!!		% !				 !"#! !	  #	"!	"!	#			#		5!	"!#	+!	%	."##	!!	#		"	!	!!		#			!	#		#	5			  	@	 		  	 	" 	+E		 		  G46A2(3+A 	#	 	 #	"	!		:!			"!	!5	  /+(B 	#	 	 " 	@		= !		G4632 2+'  ,/(E3+' ,. 	 	!	 "#!			;(A('<+!	  $ $&			#	!!				!"	 $ 	"#!	"H#;(4<+!	$ 	7"		!		 		 		 %	  G46 	 ;(B<  D		 ;(2< ! 	  !" 	7"		%	% 1	!!	"%!	!"#		"#   154/ :$ 53=  !	 7"	 	7"		+!		!"		 		% !	D-:D		- -		= "%	  !	 !	 !" #			  !	E$DC	"	 ;3/<+ !	.$ (	7"		%	1		 ;3(<			  . 	7"		+ 	5	 	 5	3'!"			"	!	%"!"$ %%%				!	!	 #		 	7"		  " 	%	+ E" 	 	 		  "	  !	 9	 !		5			"	+#	!	$ $&							#			!		#""#!			+!G46#"	 (!	# 		 %   	"	  	 : #		=!:H	=+!		5	5	 	"	 :  	= 		  !	$ %" !	$&	+  /'3+4!		"	!		5		#!	#	!!	!	$ + G46" "	  %	 	7"	  			 ! 	    $ + !	 #	  G46#"	 ( 		#"%!> 	!!#!!	8	,"			%!	!!5#					%	%	+$ !			E.:#	5		5 	"	? G46#"	 (  	= !!   	 #  !	 	5	 	# ;(<+&  !	 E. 		 "  		  !	   #		+ G46 	@	  /A/*+3 ! !		7"		 .. !!  !	 %  !" 3  (A*3/  (A43A:G46#"	 (#"--!#!#!	%"	=+ D" & $  !	 !	 	7"		 E. :G46#"	 (	=+!		@		!	#		 #"  " 	 ! "	   (/33+(I((/3,+3		 D0B3*!".$ (+ !		 	 !	 !	 	7"		 :G=.D,:G46#"	 (#"J&---%"			=!"   $%'+%	"	 !	5		 ! "%#" ! %		 			   5"%7"35.$ ;3(<+!			"!!	""	!		5		#"9+!	 9# 			 # !	 		$   	5	%			!		5			 !#! 		 !  G460BA+(/ :#" J---=I4(D(3 +(0 :#" J-&5$ *=  IB4D3+2 :#" J&5$ 4=+ ! 	     !" 	 	"#!"*A	"	+C!	$ !			7"		 	 :		=  !	 	 	#+!	4 Genome Biology Vol 3 No 1 	Figure 1 (see figure on the next page)ClustalW alignment of the region surrounding the active site of the C. elegans, Drosophila and human UBC proteins. The names of the proteins are color-coded: C. elegans (green), Drosophila (blue), and human (red). Subgroups of UBCs, separated by horizontal lines, have Roman numerals and UBCdesignations that correspond to branches shown on the dendrogram in Figure 2. The UBC designations (right) for the subgroups indicate the mostsimilar S. cerevisiae UBC. The active-site cysteinyl residue (C) is highlighted in red, and an invariant proline (P, green) and tryptophan (W, yellow) are alsoindicated. Other features indicated are the UBC tripeptide motif HPN and variants (in yellow and blue) (see text)commentreviewsreportsdeposited researchinteractionsinformationrefereed researchhttp://genomebiology.com/2001/3/1/research/0002.5Figure 1 (see legend on the previous page)CG5823      KP- PSIYML--------TPNGRFKTNTRLCLSISDFHP----------DTWNPY110A2AR.2  SP- PAITMI--------TPNGRFQTNTRLCLSISDYHP----------ESWNPNCUBE1      KP--PSIILL--------TANGRFEVGKKICLSISGHHP----------ETWQPD1022.1     KP- PNLILL--------TPNGRFELNKKVCLSISGYHP----------ETWLPCG7375      EP--PKVKCATQ-----VYHPNID-LDGNVCLNILR-------------EDWNPUBE2M       DP--PKVKCETM-----VYHPNID-LEGNVCLNILR-------------EDWKPR09B3.4     VP--PVVKCLTK-----VWHPNIN-EDGSICLSILRQNS-------LDQYGWRPUbcD10      KP--PKINFKTR-----IYHPNID-EKGQVCLPIIST------------ENWKPUBE2L3      KP--PKITFKTK-----IYHPNID-EKGQVCLPVISA------------ENWKPUbc84D      MP--PKILFKTK-----IYHPNVD-EKGEVCLPIIST------------DNWKPR01H2.6     KP--PKVAFETK-----IYHPNVD-EEGKFCLPIVTA------------ENWKPUBE2L6      KP--PMIKFTTK-----IYHPNVD-ENGQICLPIISS------------ENWKPCG17030     KP--PRIHINTR-----MYHLNVN-ERGQVCVPILEV------------EHWIPUbcD4       NP--PKARFITR-----IWHPNISSVTGAICLDILK-------------DNWAAHIP2        NP--PKVRFITK-----IWHPNISSVTGAICLDILK-------------DQWAAF40G9.3     SP--PNVKFSTK-----IWHPNVSSQTGVICLDILK-------------DQWAAC06E2.3     EP--PQAKFVTR-----IWHPNISSQTGTICLDILK-------------DKWTACG8188      TP--PKAYFLTK-----IFHPNVA-ANGEICVNTLKKD-------------WKPE2 24kDa    SP--PKGYFLTK-----IFHPNVG-ANGEICVNVLKRD-------------WTACG2257      KS--PSIGFVNK-----IYHPNIDESSGTVCLDVINQA-------------WTAUBE2H       KS--PSIGFMNK-----IFHPNIDEASGTVCLDVINQT-------------WTAY94H6A.6    KS--PSIGFLNK-----IFHPNIDEASGTVCLDVINQVGIGGRSV---WKAWTACG14739     TA--PRVRFVTK-----ILHPNIEFITGLVCMNVLKQA-------------WSScourtless   SP--PKMKFTCD-----MFHPNIF-ADGRVCISILHAPGDDPMGYELSAERWSPUBCJ        SP--PKMRFTCE-----MFHPNIY-PDGRVCISILHAPGDDPMGYESSAERWSPY87G2A.9    SP--PKMRFTCG-----IFHPNIY-ADGRVCISILHAPGDDPTGYELSNERWSPCG9602      RP--PKMKFITE-----IWHPNID-KAGDVCISILHEPGDDKWGYEKAEERWLPF58A4.10    KP--PKMKFISE-----IWHPNID-KEGNVCISILHDPGDDKWGYERPEERWLPUBE2G1      RP--PKMKFITE-----IWHPNVD-KNGDVCISILHEPGEDKYGYEKPEERWLPCDC34       SP--PAFRFLTK-----MWHPNIY-ETGDVCISILHPPVDDPQSGELPSERWNPFLJ20419    SP--PTFRFLTK-----MWHPNIY-ENGDVCISILHPPVDDPQSGELPSERWNPCG7656      SP--PSIRFLTK-----VWHPNVY-ENGDLCISILHPPVDDPQSGELPCERWNPY71G12B.15  SP--PSMKFTTK-----VWHPNVY-ENGDLCISILHSPIDDPQSGELACERWNPUBE2A       KP--PTVRFVSK-----MFHPNVY-ADGSICLDILQN-------------RWSPUBE2B       KP--PTVRFLSK-----MFHPNVY-ADGSICLDILQN-------------RWSPC35B1.1     KP--PTVKFISK-----MFHPNVY-ADGSICLDILQN-------------RWSPUbcD6       KP--PTVRFVSK-----VFHPNVY-ADGGICLDILQN-------------RWSPlesswright  SP--PKCKFEPP-----LFHPNVY-PSGTVCLSLLDEEKD-----------WRPUBE2I       SP--PKCKFEPP-----LFHPNVY-PSGTVCLSILEEDKD-----------WRPF29B9.6     TP--PKCKFEPP-----LFHPNVY-PSGTVCLSLLDENKD-----------WKPF56D2.4     IP--PICEFKTP-----LHHPNVD-LRGSIYLKMLEQEH------------WSSDsi/Ubc1    KP--PKVAFTTR-----IYHPNIN-SNGSICLHILR-------------SQWSPeffete      KP--PKVAFTTR-----IYHPNIN-SNGSICLDILR-------------SQWSPLET-70/M7.1 KP--PKVAFTTR-----IYHPNIN-SNGSICLDILR-------------SQWSPUBE2D3      KP--PKVAFTTR-----IYHPNIN-SNGSICLDILR-------------SQWSPUBE2D2      KP--PKVAFTTR-----IYHPNIN-SNGSICLDILR-------------SQWSPUBE2D1      KP--PKIAFTTK-----IYHPNIN-SNGSICLDILR-------------SQWSPUBE2E3      KP--PKVTFRTR-----IYHCNIN-SQGVICLDILK-------------DNWSPUBE2E1      KP--PKVTFRTR-----IYHCNIN-SQGVICLDILK-------------DNWSPUbcD2       KP--PKVTFRTR-----IYHCNIN-SQGVICLDILK-------------DNWSPCG5440      AP--PVVIFRTP-----IYHCNIH-RLGFICLDILK-------------EKWSPCG2574      RA--PRIRFTTR-----IYHCNVD-SRGAICLDVLG-------------ERWSPCG10862     YP--PYLAFLTK-----TYHCNIA-LSGRICLDILG-------------SKWSPBG;DS01486  KA--PKVRFLTK-----IFHPNID-RVGRICLDILK-------------DKWSPbendless    SA--PKVRFITK-----IYHPNID-RLGRICLDVLK-------------DKWSPUBE2N       AA--PKVRFMTK-----IYHPNVD-KLGRICLDILK-------------DKWSPY54G2A.23   AA--PKVRFMTK-----IYHPNID-KLGRICLDILK-------------DKWSPBAA93711    EP--PQIRFLTP-----IYHPNID-SAGRICLDVLKL---PP------KGAWRPCG10682     AA--PVVKFLTS-----CFHPNVD-LQGAICLDILK-------------DKWSAUBE2H10     NA--PTVKFLTP-----CYHPNVD-TQGNICLDILK-------------EKWSACG7220      DS--PEVTFIG---TNIPVHPHVY-SNGHICLSILT-------------EDWSPBAA91954    DS--PQVMFTG---ENIPVHPHVY-SNGHICLSILT-------------EDWSPY54E5B.4    NS--PEVMFVG---ETIPAHPHIY-SNGHICLSILS-------------DDWTPBAB14320    VP--PHFCYLSQCSG--RLNPNLY-DNGKVCVSLLGTWIGKG------TERWTSBAB14724    HP--PRVKLMTTGNNTVRFNPNFY-RNGKVCLSILGTWTGP---------AWSPB0403.2     SP--PKCAFLTTGSGNVRFNPNLY-NDGKICLSILGTWEGRP------EEKWNPF49E12.4    KP--PYLKFCHN-----VYHPNVDPVTCELCSPMLLQ------------ENWKPC28G1.1     KA--PTVKFITK-----IWHPSVSPFDGTICLHDVDN------------GVWPVF39B2.2     EP--PTVRFTTK-----VHMVGVNQSNGVIDKRNLTTLR-----------NWSNEG;25E8.2   EP--PFVRVVHP-----IISGGYVLIGGAICMELLTKQG------------WSSF26H9.7     LI--PRVCFHTF-----IFHPNLG-KYGNWDMRGIQ---------------WERC06E2.7     AL--PQVCQITN-----MYTPFIFIPDQVPNSHLELCGR---------AKHWASY69H2A.9    ASDLPRVIFEQSN----LFHPLICTKSKELCLNRSFP------------EGWKKXVIII UBC6VIIX        UBC12IX      UBC1VIIIXI      UBC8XV    UBC7XIIIXIV   UBC3XVI   UBC2IVVVI     UBC13XVII  UBC11IIIIIIXII    UBC9Yeast%	 "%	  	@ 5 	"	  ! 5	!!	!		5	""	!	$ 	;33<!	#	@			!	"	  !	 	+ 		 $  "#   /A/*+3!"  (A43A!			!	5		!	E.+ !#		  	 "  !	 !"  	 $  	 "# !	 !9#	  # :			 	!= 	#  I'2E3+2  !	 "	   "	 		+I'2E3+2!	"	%$ :G46=#		" ;3*< %	# *'K 	  5 	7"			(2/	"	+-		#!	"	G46 #				 $& !		 I'2E3+2 !  	5	 		"	+  $&5				"	!			%" !	 !		 !"				 + !  :G46#"	 3= ! !   $  !	 !#" 	  !"	%!+.%!		 	%!	 !			   !"  	 	7"		+ G46	@	 !"$ 3E(/ !  !# 	:D(/'B3= %"     :G46#"	 3 #" J&--=+$ 3E(/  	  5	 	# !"#!  ! !	 !	5# 	@ 5	#	 "  $ 3E(/  !! !		5			!#			"			  !	 ;3A<+ !	  	 		 	 	$%((    " !#  !	 	 ;30<+ 5	   ;3'<  	"!			%	#		%		$ +	!#			#	!9  		5		   !	 !" 	 $ 3( $ 3*#	!	!!			 	:G46#"	 3&=+!		3	""		!		$%AG0		7"		:#"-&=%"	!#E.		  !		5	 	# :		G46#"	 (&= %!				5		@	! ! 		"	+G46	@		 !	3/	"	$ 3*			+$ 3(	!!	E5 	%	'5;34<!!	E 	 ,0 ;3B<+ '5     #		@		 "%7"  0* !	 	 ,0 "	.	A		"%7"5   	  	5"	 	 ! 	 "%	57"	 	#	  !	 	+ - 	  %				  !	 		5! 	#  #" & 3 		!!5			#"3"5"##		%"!	9 ;32<+!		$ 3E(/	%	%	!	"$ 3($ 3* " %	 	 " % !	 $  	%	  +%!	G46#"	 3!			  	5			 	7"		+ 		# 	 ! "	   $ "  %!J&--- 	# 	 $%'+ $%' !  	%	   %@5	 	@	 ! ! !		  !		%	  !	 	 	"" :,=+ !	!	 3 "  !	 "%7"  		!	 	%9" !	,;*/<+!	  $ #	%	 %@5	 	@	  (/33+(+ E		I((/3,+3  	 		  (/33+( %" !  ! %@5	 	@	  9 !		%	!+"#	"	!		!#"!	 :G=.D,G46   !	 	5	 	#:G46#"	 (J&---=+		%	!					$%'+	  I((/3,!		%	!#"9#!		%	!+!	%!#!"E-3	 $%A:G46#"	 3-J=!		  	!!:/'3+*3BD(+(=			%#				%.	7"		+C!		!		  5	G46A/D2+*$ "%7"5	 ;*(<+ !  "   !	 !" 	 $   !	 %! !"#!  #		"	+!	$ " /'3+4!!	!				#"&--	+!	 *A/5	"		@	3BD(+(	7"		 !	 %@ 	"   G465	  (:G46G46(=!!		1	;*3<+ #"  G46#"	 3  	 --$ 	! %@5	 	@	 	  #			#$ 		>+-#"J- :$ B5	= 	 !	 "%	   	"	 	 ! !  #" J-& :$ *5	= 	+ G46	@	I2AE'+':$ B5	=!5#(4	"		!!	#"	"	+ I4(D(3 +(0 :$ *5	= !		 !  #  *3 	"	 3/ !! 	 + - 	$%*G*A  	  !	 "%7"  			5	5			"#3:3=*;**<+ C!	 #	  *A5		 "%7" !			 %		 		 "# 		  5"	;*A<!		 5%;*0< # !	+"! 	  9 %" !	 $ B5	 	 !"#!   		 ! ! 	$%B 	#"	 !	 "%7"  !	 #"	#		>	"	5('5%!!	;*'<+  $ 5(:#"J&-=  !   %@5	 	@	;((< %" !" 	 !#  ! 	9!	+Inhibition of ubc and uev genes in C. elegans by double-stranded RNA!	 "    $   $& 	 			@	%,.+G46	 ;*4<!	!!"%	5	 :=,.	#  	7"		 #		6 Genome Biology Vol 3 No 1 	commentreviewsreportsdeposited researchinteractionsinformationrefereed researchhttp://genomebiology.com/2001/3/1/research/0002.7Figure 2Phylogenetic relationship of the UBC proteins of C. elegans (green), Drosophila (blue), and human (red). The dendrogram was prepared using the Phylippackage of programs as described in Materials and methods. The major branches are separated by dotted lines and assigned Roman numerals thatcorrespond to the numbering system on Figure 1. Some branches have a UBC label that indicates the most similar S. cerevisiae UBC. C. elegans UEVproteins are indicated with an asterisk.F39B2.2*EG;25E8.2BAB14724B0403.2BAB14320BAA91954CG7220Y54E5B.4UBE2D2UBE2D3LET-70/M7.1effeteDsi/Ubc1UBE2D1UBE2E1UBE2E3UbcD2CG5440CG2574CG10862BG;DS01486.1UBE2NY54G2A.23bendlessBAA93711C06E2.7UbcD10Ubc84DCG17030UBE2L3/UBE2L1R01H2.6UBE2L6E2 24kDaCG8188HIP2UbcD4F40G9.3C06E2.3C28G1.1R09B3.4CG7375UBE2MY94H6A.6UBE2HCG2257CG14739F26H9.7*UBE2IF29B9.6lesswrightF56D2.4*F58A4.10CG9602UBE2G1Y71G12B.15CG7656CDC34FLJ20419UBE2G2courtlessY87G2A.9UbcD6UBE2BUBE2AC35B1.1UBE2H10CG10682CG5823Y110A2AR.2D1022.1NCUBE1F49E12.4Y69H2.60.1IIIIIIYeastIV     UBC4/5VVI     UBC13VIIVIIIIX     UBC1X      UBC12XI      UBC8XII     UBC9XIIIXIV    UBC3XV     UBC7XVI    UBC2XVII  UBC11XVIII  UBC6					"#	#						 ! #		  %! !	 		    		#	+ &  ! 	! !	 %		 	  #		"	#		5	"		 " ;*B*2<+E		 	 "	 	!  !!"!	,."	'		 	!	 !	 	 		 ;A/<+ - !	 "5	$ "	%	8	,. "# " !	!	 :		 	 	!  %! 	!7"	=+ !	 #	  !	 				 		 	@	  	% 	!  !				%	+!		#			#		% 	 	"#  				 ! 	@	5	!#		!%	 (+#	!!!	"	"			 		  !	" #	%						">	%	 (	!		+!	!		   (   ! ,. 	% 	! !	 	 !		 %%	  " ! 		 !	 	% 	 %	 	"	+ # !		 !		 	 				%!	"	!	43!",. 		% !	 		#	!!	 !			 "  	 	 " $  	!! !	 %"	  		# 	%+ 	!						%",.8		@		+G46"!	3/   #				"%			5:%	 (=( !+,.8		@		:%"		#	@		="##	!%						+.	!	 #				+LET-70 (UBC-2)				!			9  )*  +,( ;A(<+%"	% ,.5										#"!		5#	+!!						!!			!	!		!		!! 		  !	 	  !  #	 :13 1*=+!		   	!			  	"			%#"			#	5	"+ - %	 !			  !	 #	 "	  	 	"	  !	 			 %9 	%#		+ 		  %! 		  " !	   ! !	 		 " !	5	+ 		 	  !	   !	5	  %	 			 		!		 :+++  ++++""%!		"=+  154/:$ 53="!#	$%A  $%0 ;(3<+ !		 	 	 	 		"	 	  ;A3<+ !	 !" $%A !#$ 33  	  !	 "%7"  -  ;A*<%%!	!5		+!		$%A	#"!!5		9	%	"	!!	G46	@;AA<+UBC-9!	 		 $ 52 	   "   $   !	   %	   #	 . :9*(3	((=!!		%!	+E				!	.			!	$ 52+H	!		5		#	,.">	!	"$ 52	#!	#	7"		!	$   	 # %! !	 $   !	!	!				+!	,.	"		 	  ! %! "+ - 	 %	!!						!	+,.!( 	"		%		#"5%"%		"			+!		7"		% 	  "! !#!	 !	 !	 ,. 	%!			#	!:%	 (=+19$ 52	"	  	 		   			  ! 		 	%#		 :%% "	 	 	"	=+ !	   % "		  !	 "!  #	 :1A=+!				5		"!		"		!		##5#		 :#=!		  ""	 !	""# !	 1A55" +   		#	  (,.				+%!!9	%	9					!(,. :G46#"	 *=+ -  	 " !	 #  !		 :	 #	  !	 "	 	@	# !		#!  !	 		 %	 !	 	 	 	? 		G46#"	 *=+  $%2  		  #!  % 	 !	"%7"59		$C"%	"%	 "# -  ;A0< ,5D( ;A'<  0*;A4<+ !	 "  $C   	 %""#			"##	!	! 	#"# !	   	 "	 	 ;AB<+	 ("-	 " 	 			 	!!		!#	"	"%	!%5	#	;A2<+!	"(   		"	@	+UBC-12%  ,.5		 		 		 !	   	 #	 ! 	"	5		5	 		 ;0/<+ !	 	7"	  	  B0K %		#  03K % 8	   ,. :%	 (=+			"	% ,.!	%			5"	;0/<+19$ 5(3	"				"!	1A#	!!	"%	7"	""	!	  "# !	 1A55" + $ 5(35		"!		%	!	#	"		  !	 	#" #>	 	# 	# 8 Genome Biology Vol 3 No 1 	commentreviewsreportsdeposited researchinteractionsinformationrefereed researchhttp://genomebiology.com/2001/3/1/research/0002.9Figure 3RNAi phenotypes of C. elegans ubc and related genes. Nematodes were treated with RNAi by the feeding method. (a) Reduced and bent tail spikeproduced by ubc-9 RNAi. (b) Curled and branched tail spike produced by ubc-12 RNAi. (c,d) ubc-14 RNAi produces a blunt and rounded tail region witha protrusion near or surrounding the anus. Both specimens are young larvae. (e) Normal alae on an untreated adult. (f) Branched alae (arrows)produced by ubc-9 RNAi on an adult specimen. (g) Irregular alae that diverge around a granular deposit induced by ubc-12 RNAi. (h) Typicalmorphologically abnormal L1 seen with ubc-9 RNAi. (i) Arrested embryogenesis produced by ubc-14 RNAi with advanced development of the pharynx.Most specimens treated with ubc-14 RNAi arrest at an earlier stage, without pharyngeal development. (j) Arrested embryogenesis produced by sumoRNAi. (k) Vulval eversion on sumo RNAi-treated young adult. (l) Everted vulva and egg-laying defect produced by uba-2 RNAi. The scale bars indicate 25m. Magnification: (a,b,k) 400; (c-j) 1,000; (l) 200.(a) (b) (c) (d)(e)(i) (j) (k) (l)(f) (g) (h)#"	+E9	%			!	5 ,.:G46#"	 *%=+  $%(3%  	 !	"%7"59		,"%(9.	B .5B +	$ $ 5(3 !#! 	 .5B		"%7"  !			;0/<+!	9"%	,"%(G.	B5:	=	!	"	!!	# 	  * "%7"5#	 	 5	@	 ;0(<+ L9"    	 !"#! 		  !	 !#" #		  !	 	 	.	   	! ;03<+ ,		"	!	"##		!	!""(	7"		#34L( ;0*<-  "%7"% !	G46 , :,5"		5#	=;0A<+UBC-14% 		 		!! ,. 	5#" %" %		  "	 		+ "5# 	 ! ,.5		 	% 			 	5#>	 !@ 		 !	  ! !	  !		 		  !	  #	 :G46#"	 *=+ !	!			 "	%! ,. 		%"%!	#!	!""	 %  % "  "	 "!		#:G46#"	 *=+!	 	 !#  	$ $ 5(A "	 "  !! 	"  % 	"! %	!  	 ;00<+ !	  !#$%4	"	!	"	!	,%!		%	5%" 	 "	(!		  "  !		#  % , 	 ;0'<+  $%'!!  	%	5%"	   5	  !	 	#  % , 	 ;*/<+E		!	  !#(/33+(		 " ,. 	@		+ !	 	 		 	I((/3,+3:		%!J&---G46#"	 3=!!9!		%			+!	9"	"!		  		 	 "  8	 	@		 "# @"	 ,. 			# !	  	7"		+ !  !	 !		 "##	# !   $ '  	7"	"	#!+UBC-20H!				 #		!		"%	%!	,.		#	!%,.8			5  	%"	+ -8	 :G46A/D2+*G46#"	 3 #" -J= ,. 	"	   %	  	#	 	9 		 			 	  !	 1*551A#	 :%	 (=+.	%			+,.		%!			#	!!	!	!!	!		+!			#	@		!		!									9!		!%			9	   	  	7"	  				!	1*#			:		!(K!	#					=+		   !#	$  :G46#"	 3 #" -J= 	"   #!				#!#	#	 ;04<+ C		@	  	    5 		  !"# 99" "##	# !!		!		$ !		#";04<+/01!	!"!#	$		 5!% 		"#!"#% "% %	 	  !	 			 	#  !"5# ;0B<+ 	$   	 		 /'3+*:G46#"	 (#"-J=	!				99"%!!		##		"5	"+#,.			#%!#				@		%8	+!			"  		  !	 	7"	 	+!	# !		 "	 % ,. !  		  !	 	  !	 	,.  "  !	 ,. :% @# !  	 ,.= %!  				+ -  	" "	!	"##		!		#	!			5	 ,. !%+  ;02<	 !!	   		 ""	 ""    %	 	@	# #! :%5 = ,.	 1(  13 #	 	 !	 	5 	5	!			"#		+ !	 9  				   ,. % !			# 	! #! !			 %	 	@	 % "! 		,.+Ubiquitin-activating enzymes of C. elegans!	  #		"		#			#"%7"55#	>	5$ (:%	 3=+!		#		!	%			#!	"	   !	 %   1 	! 	+ C	  !		#			$  		!	>	!	!#  	 	 "%7" ;'/<+ !	 !	 "#		 		  !			 (?  	 $%3 ( #	!	 	 !	 "%7"59	 	 $C!	$%*$(#	!		,"%(:		;'(<		=+ - !		 !			 	>	 !	 	 	5			!	$ 		+dsRNAi with E1 genes!	(			%,. 				:%	 3=+,.! 	"					!		 ! 		  	% %	# "	 %!			"+-!			"		A	@"	!	%		@	# ,.+,.99" * "				%#		!	!		  !			!,. $,  23 	 " 		 :=  #10 Genome Biology Vol 3 No 1 	!		:G46#"	 *=+,.! "		%5 	 %" !	 		  	  	5!		!			! ,.+Ubiquitin-like proteins in C. elegans #		    		 "%7" 4 		"%7";'3<4 		""%7"  % 	 ;'*<+  !	  #				""%7"59	%	 :E/('-/A+' ;'A<=  * :3'G46(+A=+ 	",. 	@		 !	 ! ! 4  4 			 #		 ;*2<+E		   * 		" 	 ! "	# 5 	#		"%7"59	%	,. 	  	 	@  !	 		 "5:=  !	 "%7"59	 + !"  			@	!		#		"!	%,.+E		!	   #		 		  	 (4 !	 	#  "%7"59	  !  	!	 		"	!			7"		+H	!		@		#!!			7"		"! !	 "%7"59	 	 .5B  $C 			  	%#		 :%	 3=! 	!	5	!		!	"	%	5# !	 	# 8"##  # 	>	+G46	@	!"#! ,."		%	(//K#	:G46#"	 *8="!			%! 			 "	 :G46#"	 *9=  %	  !	5!	  !#+  	" ! ,.!	%			"%		;*B<+,.!$,			%		#	!%	"	!	>	!	#	"	:(0/%=!"#!!		#	>				!	"	!	8		!;0/<+ConclusionsCommon ubc knockout phenotypes in C. elegans	9%		"""!		!	5	 "	 %,.! 		 		 #		+%				!	!	!	5"	%( ! ,.			%	!	!		 		 !	  	5#"ECJ#		 :%9	;'0<=+- 		"!*	"	!	1(#	!			5#>		 	+ #> !		5 	  !	   "	 %" 		 !	 ECJ #		 # ;''<  !	 !	 	5	##		 ;'4< ;'B<+	@#5#! #%!		#		#"	 	  	#"	 !	   !	 		# +!	"	""##	!!		!	  #		 	 		  ! 	 	! %	#	7"	!					#	#		#"+				!	1(#	!	"	!	"%"	%	!	!	#	;'2<+"5  !	   #		 "!  	  "5!	 #		  	"   #  !	 	 ;4/<+G46!		!	"		"!	"!	"	#		!!"	!	1Acommentreviewsreportsdeposited researchinteractionsinformationrefereed researchhttp://genomebiology.com/2001/3/1/research/0002.11Table 2RNAi with C. elegans ubiquitin-activating enzyme genes and ubiquitin-like genesCosmid Gene name dsRNA source Chromosome Number of RNAi trials      Embryonic lethality (%) Secondary phenotypeUbiquitin-activating enzymesF11H8.1 uba-3 gen PCR III 2 41 EvlC26E6.8 ula-1 yk433e12 III 2 6 EvlW02A11.4 uba-2 yk632h1 V 2 40 EvlC47E12.4 uba-1 yk9g12 IV 2 100 Low brood size, deathC08B6.9 aos-1 - V Not done - -Ubiquitin-like fusion proteinsB0303.4 yk490f10 III 2 0 NoneC16C8.4 yk262b4 II 2 0 NoneF49C12.9 yk162f5 IV 2 0 NoneZK688.5 yk16d10 III 2 2.5 NoneSmall ubiquitin-like proteinsF45H11.2 ned-8 T01966 I 1 2 Evl, abnormal morphologyK12C11.2 sumo yk222g1 I 2 100 Evl, abnormal morphologyF46F11.4 gen PCR I 2 0 NoneF32H5.3 yk534f3 V 2 0 NoneDNA fragments used to generate the dsRNA were obtained from the indicated sources, some fragments were generated by genomic PCR (gen PCR).Phenotypes include Evl (everted vulva). A dash indicates experiment not done.;'2<	%		!%!	!			#!	%		;4(<+!"##	!!		%			!(  ,.%	"		!	%		!	"		!	1A  % !	 %		  		  " 	+,		"	!		!!	"%7"58"#			%	";43<"		 "	  			  	5	 " 		 	%!	%			!	( +Comparison of UBC function in C. elegans and otherorganisms!			"#			%			!	!		"	%99"!	  		#		  !	!				!	!#"#			99	"%,. +.%				"!		#		*!" * !	"%7"59		5	##		5 +!	  !#  *!" ** :I4(D(3 +(0= 	 %					!"#!*! 			;4*<+#	"%7"%*A	9"5#!	D(3;**<*;4A<!	5!	5L!%(;40<+!	*A!		#!		!	"%			%!			*A!	G46+G46"!			 !	  %@5	 	@	  *A 	7"	;4'<!	3"%"	*A  !			  "	 !	*A%@	@	 ;44<+ -  	! !	 !		 $  !!	  %@ 	@	 $ 5( $ 5*  $ 5B	"	"+	  !	 ,"%(G.	B 8"# ! 			 ;4B<!"#!* ""!!		!!	5%	 ! 		"	5		" *"$*! ;42<+-  ,.  !	 	#.5B 8"#55! 	 	"  	% 	! 						%	+!			%5% 		  	!	 	  .5B  		@#!"#!.5B  	7"		%!		 ;03<+!	"%	$ 5##		 	"9		 		!	#				@(*$    3/     30  	+!			%%	@	*/$ !	!"5		!"#!3'		"	!		! "+ "!  !	 		  	  $  !"	%!	!	$ !!		5%	 !#  	  	@	 %	# !	 $  !		5!5		@	:#"&G46#"	 (=+!		#	#		"%7"58"##	>				@!				7"		"$  			  	 	"	5	+	!!			"%	#		!	"	" 	"   !	 	  			@!#		"$ 	!		#			   	    	+ - !		 !	 		  !		   	 !	$ #		  "%	"		"!1$1H#"#%""M59N%				%			#"#+Materials and methodsNematode culture    :.3=  				 ""	 % 	!7"	 ;B/<+.D# 	 "	 ,.	@		 :		 %	= 	 (   55!#	30 #G%	+Polymerase chain reaction		 ! 	 :,=  			 ,:,5,=			"	"	%	;'A<+RNAi assaysFeeding method!	,.			%!			#	!L!+ ;A/<!!  !		5"				%+;02<+!	.	!#		:!		%	=.#	#			%,:%	 (=		"%	(32+*'"			,."+!				 	  		 	@		+ !		 "!  :1A=#	 		 		 "	  	 	 !,.5# %	  	  		 "+ 	 43 !  (0O " 		 " 				!		!,.5#%			##	#!(0O+!	#!	"				!		##"	+!			5#	  	% 	  			 % "# !	"%	"!!		##	#3A !	!		!			"!			+Injection method!	,.		% !		!G46	 ;*4<!;0/<+-8		"			 	##  0 !  	  "#	 "		 	%+ !	"		!			!					##@	(4 !+!%	##:%=%		@		!	5	"	!		;0/<+!	 " 		 !	 		  !	 	  	!			##	#!!"+! %  	## :%  =  #		 		 		"			+Microscopy-"		 	##		9	 	3K #	  # (/  " >	  	5!	!#!	"#)	@3	!						+12 Genome Biology Vol 3 No 1 	Bioinformatics methods		 	7"				 %	% 1 	!	 !	"%%	 ;B(<+	""		>	 "# 	%5%	 	  !	 # ;B3<  , ;*(<+ 	 	7"		 #	 		#			 !  	%5%	 	  !	 1$1H# 	  !	 "	   -"		;B*<"#!		"	#+G46!#		5!	""!	#		!5>	 ! !	 ! 9#	  # ;BA< "# !		#!%58#	!"		(///%5		!		+!	#C.."	#			!		"		+!	""!	!9#			"#!	!	5 # 			 % +D+ D%	 - $	5 #$+Accession numbersUBCs /A/*+3 P((/4'? /'3+* (0A*3? /'3+4 (0A*(?3BD(+((0'2(?*0 (+(*3202?(/33+(*A(20?G4632 2+'32232? G46A/D2+* **'32? G46A2(3+A 33AA2? G460BA+(/A/2B3?4+(3*B3/?,/(E3+'(''A'?,/2 *+A3A/'2?I((/3,+3 G46'/A((? I0A0 +A 34('4? I0AD3+3*L2*B'A? I'2E3+'  '*A/*? I4(D(3 +(0 3(A*2?IB4D3+2 '/A*(?I2AE'+'G46'/B2(UEV proteinsG46*2 3+33(2BA?G460'3+A('A42?G463'E2+4G46'/B2(E1sG46((EB+( ('/*4? 3''+B 3(('3? H/3((+A /AB2(?A4(3+A3//(A?/B '+2(2/B3Ubiquitin-like fusion proteins /*/*+A *A30'? ('B+A 32A/A? G46A2(3+2 33A3(?)L'BB+0AA23/Small ubiquitin-like proteinsG46A0E((+3333A2?L(3((+3L(B2'2?G46A'G46((+A304'*?G46*3E0+*3('BAAdditional data files !		 "	 !	 #"		 	 	7"		 "	 ,!				#		  #	"	  !	,.	@		+ !	 !	 		#	!		7"		>	!!	!	%!	#				G46#"	 (+AcknowledgementsThis work was supported by a grant from the Canadian Institutes ofHealth Research to E.P.M.C. We thank Y. Kohara for cDNA clones and A.Fire for vector pPD129.36 and bacterial strain HT115 used in the RNAiexperiments. We also thank M. Groombridge for helpful discussions andfor additional observations on the ubc-2 phenotype.References1. Haas AL, Siepmann TJ: Pathways of ubiquitin conjugation. FASEBJ 1997, 11:1257-1268.2. Hershko A, Ciechanover A, Varshavsky A: The ubiquitin system.Nat Med 2000, 6:1073-1081.3. Scheffner M, Smith S, Jentsch S: The ubiquitin-conjugationsystem. In Ubiquitin and the Biology of the Cell. Edited by Peters J-M,Harris JR, Finley D. New York: Plenum Press; 1998, 65-98.4. Palombella VJ, Rando OJ, Goldberg AL, Maniatis T: The ubiquitin-proteasome pathway is required for processing the NF-B1precursor protein and the activation of NF-B. Cell 1994,78:773-785.5. 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Arnason T, Ellison MJ: Stress resistance in Saccharomyces cere-visiae is strongly correlated with assembly of a novel type ofmultiubiquitin chain. Mol Cell Biol 1994, 14:7876-7883.43. Chen ZJ, Parent L, Maniatis T: Site-specific phosphorylation ofIB by a novel ubiquitination-dependent protein kinaseactivity. Cell 1996 84:853-862.44. Strack P, Caligiuri M, Pelletier M, Boisclair M, Theodoras A, Beer-Romero P, Glass S, Parsons T, Copeland RA, Auger KR, et al.:SCFTRCP and phosphorylation dependent ubiquitination ofIB catalyzed by Ubc3 and Ubc4. Oncogene 2000, 19:3529-3536.45. Desterro JM, Rodriguez MS, Hay RT: SUMO-1 modification ofIB inhibits NF-B activation. Mol Cell 1998, 2:233-239.46. Matunis MJ, Coutavas E, Blobel G: A novel ubiquitin-like modifi-cation modulates the partitioning of the Ran-GTPase-acti-vating protein RanGAP1 between the cytosol and thenuclear pore complex. J Cell Biol 1996, 135:1457-1470.47. 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Altmann ME: Ein gen für ein neues ubiquitin-konjugierendes enzym:genomische organisation, expression und funktion (A gene for a new ubiq-uitin-conjugating enzyme: genomic organisation, expression and function).Doctoral dissertation. Georg-August Universität, Göttingen; 2001[http://webdoc.gwdg.de/diss/2001/altmann/]commentreviewsreportsdeposited researchinteractionsinformationrefereed researchhttp://genomebiology.com/2001/3/1/research/0002.15

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