Human monoclonal autoantibody fragments from combinatorial antibody libraries directed to the U1snRNP associated U1C protein; epitope mapping, immunolocalization and V-gene usage

\

Molecular Immunology

PERGAMON

Molecular Immunology 24 "0887# 0934Ð0944

Human monoclonal autoantibody fragments from combinatorial antibody libraries directed to the U0snRNP associated U0C protein^ epitope mapping\ immunolocalization and V!gene usage R[M[A[ Hoeta\b\ J[M[H[ Raatsa\ R[ de Wildta\c\ H[ Dumortierd\ S[ Mullerd\ F[ van den Hoogene\ W[J[ van Venrooija\ b

a Department of Biochemistry\ University of Nijme`en\ P[O[B[ 8090\ 5499 HB Nijme`en\ The Netherlands CESAME\ Department of Patholo`y\ University Hospital Maastricht\ P[O[B[ 4799\ 5191 AZ Maastricht\ The Netherlands c MRC Centre for Protein En`ineerin`\ Hills Road\ Cambrid`e\ U[K[ d Institut de Biolo`ie Moleculaire et Cellulaire\ UPR 8910 CNRS\ 04 rue Rene Descartes\ 56999 Strasbour`\ France e Department of Rheumatolo`y\ University Hospital Nijme`en\ P[O[B[ 8090\ 5499 HB Nijme`en\ The Netherlands

Received 6 July 0887^ accepted 05 September 0887

Abstract To study the localization and function of the U0snRNP associated U0C protein\ so far only human sera from systemic lupus erythematosus "SLE# overlap syndrome patients have been used[ Here we report for the _rst time the isolation of human monoclonal anti!U0C autoantibody fragments from IgG derived combinatorial and semi!synthetic human antibody libraries[ Two classes of human monoclonal anti!U0C "auto#antibodies were found] speci_c anti!U0C autoantibodies\ recognizing U0C only\ and cross! reactive antibodies which also react with U0A and Sm!B:B? proteins[ The heavy chains "VH genes# of all _ve antibodies from the semi!synthetic libraries and two of the three U0C!speci_c patient derived autoantibody fragments are encoded by VH2 genes\ in which VH 2!29 "DP!38# was overrepresented[ The heavy chain of the two cross!reactive autoantibodies are derived from the 2!96 "DP!43# gene[ Three epitope regions on the U0C protein are targeted by these antibodies[ "0# Four U0C speci_c antibodies recognize an N!terminal region of U0C in which amino acids 29Ð52 are essential for recognition\ "1# two antibodies recognize only the complete U0C protein\ and "2# two cross!reactive and one U0C speci_c antibody recognize the C!terminal domain in which amino acids 87Ð 015 are critical for recognition[ The two cross!reactive antibodies "K00 and K04# recognize the proline!rich region of the U0C protein "amino acids 87Ð015# and cross!react with proline!rich regions in Sm!B:B? "amino acids 052Ð073# and U0A "amino acids 076Ð 193#[ All 09 antibody fragments are able to immunoprecipitate the native U0snRNP particle[ The two cross!reactive antibodies immunoprecipitate the other Sm containing snRNPs as well[ Using confocal immuno~uorescence microscopy we could show that the major part of the U0C protein is localized within the coiled body structure[ Þ 0888 Elsevier Science Ltd[ All rights reserved[ Keywords] U0C protein^ U0 snRNP^ Autoantibodies^ V!genes^ Coiled bodies^ scFv^ Phage!display^ Combinatorial antibody libraries

0[ Introduction Patients su}ering from systemic lupus erythematosus "SLE# or SLE!overlap syndromes often produce IgG autoantibodies directed to components of the U0 small nuclear ribonucleoprotein particle "snRNP# "for a review\ see Klein Gunnewiek et al[\ 0886#[ Such autoantibodies can be divided into two major groups[ The _rst group encompasses the so!called anti!Sm antibodies which are directed to the common "Sm# proteins and are pre! dominantly found in SLE patients[ This group of autoan!

 Corresponding author[ Tel[] ¦20!132502545^ fax] ¦20!132439414^ e!mail] W[vanvenrooijÝbioch[kun[nl

tibodies is able to immunoprecipitate not only U0 snRNP but also the U1\ U3:U5 and U4 snRNP particles[ Major targets of anti!Sm antibodies on immunoblot are the Sm!B:B? and Sm!D proteins and the Sm!EFG complex "Brahms et al[\ 0886#[ The second type of autoantibody immunoprecipitates exclusively U0 snRNPs from nuclear extracts and is primarily directed against one or more of the U0snRNP speci_c proteins U0!69K\ U0A and U0C[ In such patients very frequently autoantibodies directed to naked U0 RNA can be found as well "Hoet et al[\ 0881#[ The U0C protein is known to be involved in the binding of the U0snRNP particle to the 4? splice site "Heinrichs et al[\ 0889\ Will et al[\ 0885\ Rossi et al[\ 0885\ Tang et al[\ 0886#[ The N!terminal domain encompassing a CC! HH zinc _nger!like region\ is necessary and su.cient for

S9050Ð4789:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved PII] S 9 0 5 0 Ð 4 7 8 9 " 8 7 # 9 9 9 8 2 Ð 4

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R[M[A[ Hoet et al[:Molecular Immunology 24 "0887# 0934Ð0944

U0C binding to the U0snRNP particle "Nelissen et al[\ 0883#[ In addition\ part of the zinc _nger!like region "amino acids "aa# 12Ð29# has been suggested to be involved in the formation of U0C homodimers "Klein Gunnewiek et al[\ 0884#[ More detailed studies on the function and localization of the U0C protein have been hampered by the lack of speci_c monoclonal antibodies[ Antibody phage!display technology o}ers a valuable tool for generating human variable "V# domain antibody fragments recognizing speci_c antigens[ The genes encoding the V domains of heavy "VH# and light "VL# chains can be cloned into a _lamentous phagemid vector and expressed as a fusion protein of the minor coat protein pIII[ Recently this strategy was used to study a preferential VH!gene usage "DP!54# in U0A speci_c "auto#antibodies "De Wildt et al[\ 0885#[ In this study we employed the technique to generate several speci_c anti!U0C monoclonal antibody fragments\ 4 from semi!synthetic libraries and _ve from patient derived libraries[ Eight of the clones are speci_c for U0C and the epitope regions recognized by these antibodies were mapped[ Two of the patient derived anti! bodies cross!react with the Sm!B:B? and U0A proteins and are directed to a proline!rich region present in these three proteins[

1[ Materials and methods 1[0[ Semi!synthetic and patient libraries Both semi!synthetic libraries S0 and S1 contain 49 functional human VH genes with synthetic CDR2 of vari! able length "3Ð01 aa#[ S0 contains one light chain\ Vl2 "Nissim et al[\ 0883#\ while in S1 all 15 k!light chains and 10 l!light chains were introduced "Gri.ths et al[\ 0883#[ Patient libraries were made from bone marrow cells or peripheral blood cells obtained from 3 SLE!overlap syndrome patients[ Serum samples from these patients were able to immunoprecipitate the U0snRNP complex from a HeLa S099 preparation "data not shown#[ All four patients| sera recognized native and recombinant U0C protein "Klein Gunnewiek et al[\ 0884# in ELISA and on immunoblot[ Construction of the patient libraries was performed essentially as described by Finnern et al[ "0886# except that for the bone marrow libraries the pHENIX vector was modi_ed "the c!myc tag was replaced by a VSV"ves! icular stomatitis virus# tag "VSV^ Kreis et al[\ 0875##[ About 09Ð19×095 lymphocytes from bone marrow or 49×095 lymphocytes from peripheral blood were isolated by Ficoll Paque "Pharmacia 09!A!990!96#[ RNA was iso! lated using RNAzol as described by the manufacturer "Cinna Biotecs\ Houston\ Texas\ U[S[A[# and cDNA syn!

thesis was performed using oligo!dT04 "9[0 nM# and 1 ml "399 U# Super Reverse Transcriptase II "Gibco!BRL# in a total volume of 49 ml[ The cDNA was divided into eight portions to separately amplify VH\ Vl and Vk genes[ All primers were used in 14 pmol per PCR using 4 U Ampli Taq polymerase "Cetus# in a total volume of 49 ml[ In a _rst PCR the di}erent variable heavy chain fam! ilies were ampli_ed individually using a constant IgG! speci_c primer "IgG0!3CH0For# and six VH back primers "Finnern et al[\0886#[ In a second 2?!nested PCR\ six individual PCRs were performed using 0 ml of the _rst PCR reaction mixture\ one VH primer "VH0!5# containing a S_!:NcoI restriction site in combination with a mix of 3 JH forward primers containing a Sal I restriction site "for primer sequences\ see Finnern et al[\ 0886#[ The light chains were ampli_ed as follows] in a _rst PCR k and l light chains were ampli_ed using previously described Vl and Vk primers and l and k constant region primers "Marks et al[\ 0880#[ In a second PCR 0 ml of the _rst PCR mixture was used with Vk and Vl back primers containing an ApaL0 restriction site\ and Jk and Jl for! ward primers containing a Not I restriction site "for primer sequences\ see Finnern et al[\ 0886#[ PCR products resulting from the second PCR of both VH and VL genes were puri_ed by Wizard PCR prep "Promega#[ For the k! and l!light chains 4Ð09 mg puri_ed product was cleaved with Apa L0"Biolabs\ 099 U# and subsequently with Not I "Biolabs\ 099 U#[ Products were phenol:chloroform extracted\ ethanol precipitated\ pur! i_ed from agarose!gel and subsequently used in a ligation reaction with Apa L0:Not I digested and gel!puri_ed pHENIX vector "Finnern et al[\ 0886#[ Approximately 0Ð1 mg puri_ed vector DNA was used in ligations with approximately 9[4 mg puri_ed Vl and Vk DNA fragments using 3999 U T3 DNA!ligase "Biolabs\ 399 U:ml# in a total volume of 149 ml[ Transformation was performed with highly electro!competent TG0 cells[ The libraries were plated out and harvested after overnight growth on TYE plates "1×TY¦0) glucose¦099 mg:ml ampicillin# at 26>C[ All libraries contained over 096 individual clones and more than 74) of the clones contained a full!length insert as shown by PCR _ngerprinting[ DNA containing the light chain repertoire was isolated by the CsCl method "Sambrook et al[\ 0878#[ Fifty micro grams of pHENIX!VL and pHENIX!VK DNA was cut with Nco I and Sal I and gel!puri_ed[ VH fragments were also cut with NcoI and SalI and gel!puri_ed as described above for Vk and Vl DNA fragments[ Subsequent lig! ation and transformation were performed in the same way as described for the light chain cloning[ All libraries contained over 096 individual clones and more than 64) of all clones contained full!length inserts[ From each library 85 clones were analysed by PCR!_ngerprinting "BstN0 digestion# to con_rm that the patterns were highly diverse[

R[M[A[ Hoet et al[:Molecular Immunology 24 "0887# 0934Ð0944

1[1[ Selection and puri_cation of antibody fragments All libraries were panned for binders using immu! notubes "Nunc\ Maxisorp# coated with recombinant U0C protein "Klein Gunnewiek et al[\ 0884#\ 09 mg:ml in 49 mM NaHCO2 pH 8[5 overnight at 3>C[ Panning for the scFv libraries was performed as described by Marks et al[\ 0880[ Panning of the Fab semi!synthetic library "S1# was performed as described by Gri.ths et al[\ 0883[ ScFv sequences were isolated from pHENIX as NcoI! NotI DNA fragments and subcloned into PUC008"H! is#7VSV "J[ Raats\ unpublished#[ Sequencing was per! formed as described by Finnern et al[\ "0886# using the Pharmacia ALF system[ The sequences were compared with the germ!line sequences in the VÐBASE sequence directory "Tomlinson et al[\ MRC Centre for Protein Engineering\ U[K[#[ Individual scFv producing clones were grown in 499 ml cultures[ Expression was induced with 0 mM IPTG[ The cultures were grown for 2 h at 29>C and antibody fragments were harvested from the periplasm as described by De Wildt et al[ "0885#[ 1[2[ ELISA procedures ELISA on recombinant autoantigens was performed as described by De Wildt et al[ "0885#[ Antibody frag! ments were tested as periplasmic fractions\ 09× diluted in PBS[ Antigens were coated at 0 mg:ml[ Recombinant human U0C protein was overexpressed as a C!terminal "his#5!tagged protein in E[ coli and puri_ed as described previously "Klein Gunnewiek et al[\ 0884#[ Peptides of the U0A and Sm!B:B? protein were syn! thesized using Fmoc chemistry as described previously "Halimi et al[\ 0885#[ Peptides were coated overnight at 26>C "099 ml# in 9[94 M carbonate bu}er "pH 8[5# at 1 mM except for peptides 24Ð47 U0A "9[4 mM# and 146Ð171 U0A "9[1 mM#[ After three washing steps with phosphate! bu}ered saline "PBS# containing 9[94) Tween!19 "PBS! T#\ coated plates were incubated with antibody fragment preparations in PBS!T containing 9[4) BSA for 1 h at 26>C[ After washing with PBS!T "2 times#\ anti!VSV tag "P4D3# antibody supernatant was added "Kreis et al[\ 0875# diluted 0 ] 0 in PBS!T:BSA "9[4)# and incubated for 1 h at 26>C[ Wells were washed again with PBS!T and rabbit anti!mouse IgG!HRP conjugate "diluted 0 ] 4999 in PBS!T# was added for 29 min at 26>C[ After washing with PBS!T "1 times# and H1O\ detection of antibody binding was performed with 2\2?\4\4? tetramethyl ben! zidine in the presence of H1O1[ 1[3[ In vitro transcription and translation In vitro transcription\ translation and immu! noprecipitation were performed as described by De Wildt et al[\ "0885#[ U0C and U0A deletion mutants have been

0936

described previously "Nelissen et al[\ 0880^ De Wildt et al[\ 0885#[ 1[4[ Western blotting and LIA procedures HeLa nuclear extract "Habets et al[\ 0878# from 1×095 cells was fractionated by SDS!02) PAGE and blotted onto a nitrocellulose _lter[ Immunodetection of scFvs and Fabs\ after incubation with a 09× diluted per! iplasmic fraction in PBS\ was performed as described previously "De Wildt et al[\ 0885#[ LIA strips were purchased from Innogenetics "Ghent\ Belgium# and used according to the manufacturers proto! col[ 1[5[ Immuno~uorescence confocal microscopy Periplasmic fractions were dialysed overnight against PBS "pH 6[3#[ HEp!1 cells "human epithelioma cells^ ATCC CCL!12# were grown on coverslips and _xed "09 s# in methanol "−19>C# and subsequently rinsed with acetone "3>C#[ Rabbit anti!p79 coilin antibodies "R177# were a kind gift of Dr E[ Tan "Scripps Research Institute\ La Jolla\ U[S[A[#[ Double immuno~uorescence stainings were performed as follows] "0# 29 min incubation with anti!p79 "0 ] 099 diluted#\ "1# 29 min incubation with Texas Red labelled goat!anti!rabbit antibodies "Dak! opatts dilution 0 ] 099#\ "2# scFv "dilution 0 ] 4# incubation 0 h\ "3# 0 h incubation with mouse anti!VSV tag P4D3 "0 ] 099\ Boehringer Mannheim#\ "4# 29 min incubation with FITC!labelled goat anti!mouse antibodies "0 ] 49\ Dakopatts#[ Each individual step was followed by three washings with PBS "4 min each#[ Finally\ cells were stored in mowiol and examined under a confocal microscope[ Z!scans were recorded and superimposed for double staining[ 2[ Results 2[0[ Selection of human anti!U0C antibody fragments Six patient derived libraries as well as two semi!syn! thetic libraries were screened for the presence of human antibody fragments directed to the U0C protein[ An IgG speci_c primer was used in the _rst PCR reaction to eliminate ampli_cation of IgM derived cDNAs\ and all VH families were individually ampli_ed to avoid primer biases "see Materials and methods#[ The diversity of the patient libraries was con_rmed by the BstNI restriction enzyme pattern of 85 clones from each library[ The size of the libraries and the number of selected clones are given in Table 0[ Anti!U0C antibodies were isolated from the patient! derived libraries by two rounds of selection and from the semi!synthetic libraries by four rounds of selection\ using

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Table 0 ScFv libraries derived from IgG repertoire of SLE patients Patient code

Library size

Starting material

U0C clones

D07 D07 Z4 Z4 D94 O00 * *

0[9×096 0[9×097 0[9×096 0[5×097 0[8×097 2[7×096 ½097 5[4×0909

PBL BM PBL BM BM BM S0 S1

* * G7 K00\ K04\ K25 K18 * C0G0\ C0E6\ C0A4\ C0C5 C0R

 PBL  peripheral blood lymphocytes^ BM  bone marrow derived lymphocytes^ S  semi!synthetic libraries[ "S0 see] Nissim et al[\ 0883^ S1 see] Gri.ths et al[\ 0883#[

recombinant U0C protein as antigen[ The speci_city of the antibodies was determined by ELISA using a panel of recombinant autoantigens[ Eight of the 09 antibody clones reacted speci_cally with U0C\ while two antibodies showed cross!reactivity with U0A "Fig[ 0#[ 2[1[ V!gene sequences of anti!U0C antibodies Sequencing of the 09 antibody clones showed that the VH genes of nine clones were derived from the VH2 family "Table 1#[ Four antibodies\ three from the semi!synthetic and one from the patient library D94 use the germline VH gene VH 2Ð29 "DP!38#[ One patient!derived clone G7 is related to P0\ a polymorphism of a gene related to VH 2Ð29\ while another clone "K25# appeared most hom! ologous to 0Ð07 "DP!03#\ a VH0 gene[ The two cross!reactive antibody clones "K00 and K04# are related to VH 2!96 "DP!43#[ K00 contains two somatic

mutations in its heavy chain while K04 is completely germline encoded[ The light chains of all _ve patient derived clones are of the k!type[ 2[2[ Reactivity of antibodies on Western blot containing HeLa nuclear extract To obtain more insight in their speci_city and reac! tivity\ all clones were tested on an immunoblot containing a nuclear extract of HeLa cells "Fig[ 1A#[ Two anti!U0C antibodies from the semi!synthetic libraries "C0E6 and C0R# as well as four of the _ve patient derived antibodies "K18\ K25\ K00\ K04# reacted with U0C on immunoblot[ The two cross!reactive clones "K00 and K04# reacted not only with U0C but also "and even more strongly# with U0A and Sm!B:B? "and K00 also with a band with the same molecular weight as the U1snRNP associated A? protein#[ The cross!reaction between U0C\ U0A and Sm! B:B? was con_rmed by incubating a LIA blot "Inno! genetics# with K00 "Fig[ 1B#[ 2[3[ Antibody fragment epitope mapping To map the epitope regions recognized by the antibody fragments we performed immunoprecipitations with 24S! labeled deletion mutants of U0C made by in vitro trans! lation "Fig[ 2A#[ The antibody fragments were indirectly bound to protein A agarose via anti!c!myc tag or anti! VSV tag antibody[ Typical immunoprecipitation results obtained with antibodies K04 and K25 are shown in Fig[ 2A and the overall results for all ten anti!U0C antibodies are summarized in Fig[ 2B[ The antibodies can be gro! uped into three distinct epitope classes] "0# antibodies which recognize only U0Cwt suggesting

Fig[ 0[ Speci_city of selected anti!U0C antibody fragments determined by ELISA[ Antibody fragments "periplasmic fractions\ 09× diluted in PBS# selected from patient!derived combinatorial libraries "K00\ K04\ K18\ K25 and G7# and from semi!synthetic antibody libraries "C0R\ C0G0\ C0E6\ C0A4\ C0C5# were tested on various recombinant autoantigen substrates\ i[e[ La\ U0A\ U0C\ CENP!B\ Ro41\ Jo0\ Ro59[ All antigens were used at a concentration of 0 mg:ml[

0938

R[M[A[ Hoet et al[:Molecular Immunology 24 "0887# 0934Ð0944 Table 1 VH and VL gene sequences of anti!U0C antibody fragments VH genes Clone

Library

Germline VH!gene "family#

CDR2

D!segment

Mutations aa "nt#

C0C5 ClGl C0R C0A4 C0E6:G00 G7 K18 K25 K00 K04

S0 S0 S1 S0 S0 Z94\ PBL D94\ BM Z94\ BM Z94\ BM Z94\ BM

2Ð29:DP!38 "H2# 2Ð29:DP!38 "H2# 2Ð29:DP!38 "H2# 2Ð42:DP!31 "H2# DP!34 "H2# P0 "H2# 2Ð29:DP!38 "H2# 0Ð07:DP!03 "H0# 2Ð96:DP!43 "H2# 2Ð96:DP!43 "H2#

DLAKMTNND DVHYMSGED RSVKH NPESRQS EQYR VGNAAYDD EGELRAFDI ENQGMTTGYYYYNGMD VDEYGMDV DDSYGLDY

* * * * * * * D4Ð13 * D4Ð4

9 9 9 9 9 03 "08# 9 7 "01# 1 "5# 9

Clone

Library

Germline VL!gene "family#

CDR2

Mutations aa "nt#

C0C5 ClG0 C0R C0A4 C0E6:G00 G7 K18 K25 K00 K04

S0 S0 S1 S0 Sl Z94\ PBL D94\ BM Z94\ BM Z94\ BM Z94\ BM

2I:DPL05 "l2# 2I:DPL05 "l2# Al 6:DPK07 "k1# 2I:DPL05 "l2# 2I:DPL05 "l2# L01 "k0# B2:DPK13 "k3# A08:DPK04 "k1# A06:DPK07 "k1# A06:DPK07 "k1#

NSRDSSGNHVV NSRDSSGNHVV MQGTHWLYT NSRDSSGNHVV NSRDSSGNHVV QQANSFPPT MQATHWPPT MQALQPNT MQGTHWPPT MQGTHWPLT

9 9 9 9 9 6 "05# 9 9 "1# 0 "2# 1 "2#

VL genes

 S0  semi!synthetic library "Nissim et al[\ 0883#\ S1  semi!synthetic library "Gri.ths et al[\ 0883#[ Z94 and D94 are patient derived antibody libraries "PBL  peripheral blood lymphocytes^ BM  bone marrow#[  aa "nt#  number of amino acid "nucleotide mutations# in VH:VL gene compared to germline genes[

recognition of a conformation which requires the whole protein "C0E6 and C0R#\ "1# antibodies which recognize the fragments 0Ð59 and 29Ð038 suggesting that the region between aa 29Ð59 "C0C5\ C0G0\ C0A4\ G7 and K18# is important for antibody binding\ and "2# antibodies which recognize a region spanning aa 29Ð 015 in which aa 87Ð015 appear to be essential "K00\ K04 and K25#[ The 87Ð015 region contains a proline! rich sequence which is also found in the U0A "aa 052Ð194# and Sm!B:B? "aa 076Ð139# sequences[ Both K00 and K04 recognize U0A and Sm!B:B? on immu! noblot "Fig[ 1A and B#[ To investigate whether the antibodies of class 2 recognize the proline!rich sequence shared by these three proteins\ a few deletion mutants of U0A were tested "Fig[ 2C#[ Both K00 and K04 recognized Awt and A192 "aa 0Ð192#\ while the deletion mutant A "027Ð192 "in which aa 027Ð192 are lacking# was not recognized e.ciently[ Since the proline!rich sequences are lacking in the latter mutant\ this result indeed suggests that the epitope domain recognized by K00 and K04 includes

the proline!rich region of the U0A protein[ To inves! tigate this possibility in more detail the antibodies K00\ K04 "and K25 as a negative control# were tested with synthetic peptides derived from U0A and Sm! B:B? "Table 2#[ Both K00 and K04 recognized U0A "aa 052Ð073# and Sm!B:B? "aa 076Ð193#[ Antibody K04 reacted also with U0A "aa 079Ð194# and K00 with Sm B:B? "aa 100Ð117# and Sm B:B? "aa 115Ð 139#[ We conclude that although K00 and K04 both recognize the proline!rich sequence present in U0C\ U0A and Sm!B:B?\ there are some _ne!tuned di}er! ences in speci_city between these antibodies[ K25 did not react with any of the synthetic peptides[ 2[4[ Immunoprecipitation of "U#snRNP complexes To test whether the antibodies were able to recognize the U0snRNP particle\ we used an S099 extract of 21PO3! labeled HeLa cells "Habets et al[\ 0874# in immu! noprecipitation experiments[ Human monoclonal anti! body fragments were indirectly bound to protein A agarose via either the c!myc or the VSV!tag[ A typical

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K18 are able to speci_cally immunoprecipitate the U0snRNP particle "data not shown#[ Combining the data from the RNA precipitation stud! ies "Fig[ 3# and epitope mapping "Fig[ 2 and Table 2# one can conclude that antibodies from all three subgroups "Fig[ 2B# are able to immunoprecipitate the U0snRNP particle and therefore recognize the U0C protein within the U0 snRNP particle[ Since antibodies directed to the region containing aa 29Ð59 "antibody K18# and the region containing aa 29Ð015 "antibody K25# are able to immunoprecipitate the U0snRNP particle it can be concluded that residues within these two regions of the U0C protein are accessible in the U0snRNP particle "see also Dumortier et al[\ 0887#[ 2[5[ The U0C protein colocalizes with p79 coilin

Fig[ 1[ A] Reactivity of anti!U0C antibody fragments on immunoblot containing HeLa nuclear extract[ HeLa nuclear extract from 1[095 cells was fractionated by SDS!02) PAGE and blotted onto a nitrocellulose _lter "Habets et al[\ 0874#[ Immunodetection of scFvs and Fabs was performed as described by "De Wildt et al[\ 0885#[ Strips were incubated with] "Z4#^ anti!RNP:Sm serum^ "0# K1^ "1# K00^ "2# K04^ "3# K18 ^ "4# K25^ "5# Control anti!VSV^ "6# G7^ "7# Control anti!c!myc[ 0 Note in lane 2 a band between SmB:B? and U0A with the same molecular weight as the U1A? "20[4 KD#[ 1 Note] all recombinant antibody frag! ments contain a VSV!tag except G7 which contains a c!myc tag[ B] LIA reactivity of recombinant human autoantibody fragments[ LIA strips "Innogenetics B[V[\ Ghent\ Belgium# containing a set of autoantigens were incubated with three recombinant autoantibody fragments K1\ K00 and K25 "periplasmic fractions\ 09× diluted#[ Antibody reactivity was subsequently detected using anti!VSV MoAb and peroxidase!con! jugated rabbit!anti!Mouse IgG[ Lane 0^ K1\ lane 1^ K00\ lane 2^ K25[

result of this type of experiment is shown in Fig[ 3[ The two cross!reacting clones K00 and K04 are able to immu! noprecipitate very e.ciently all Sm!B:B? containing "U#snRNP particles "U0\ U1\ U3ÐU5# while the U0C speci_c antibody fragment K25 precipitates only the U0snRNP particle[ Also antibody clones C0E6\ C0R and

Because the U0C protein is thought to be directly involved in pre!mRNA binding "Heinrichs et al[\ 0889\ Will et al[\ 0885\ Rossi et al[\ 0885\ Tang et al[\ 0886# and is the only U0snRNP speci_c protein not directly bound to the U0 RNA "Nelissen et al[\ 0880#\ it was interesting to analyse the subcellular localization of this protein[ Four anti!U0C antibodies "K18\ K25\ K00 and K04# were used in these confocal immunolocalization experi! ments[ As can be seen in Fig[ 4\ staining with anti!U0C antibodies "K00 and K25# resulted in either a coiled body staining pattern or in a combined coiled body: nucleoplasmic speckles pattern[ The appearance of nucleoplasmic speckles appeared to be dependent on the concentration of the antibody fragment preparation[ When using relatively low concentrations of antibody\ the anti!U0C antibodies decorated relatively large dots in the nucleus reminiscent of coiled bodies "antibody K25\ Fig[ 4A#[ To prove the presence of U0C in the coiled bodies we performed a double labeling experiment using an antibody directed to p79!coilin[ Indeed\ the U0C stained by K25 antibody "Fig[ 4A# colocalized with p79 coilin protein "Fig[ 4B and colocalization Fig[ 4C#[ When relatively high concentrations of antibody were used the U0C antibodies produced a more speckled pattern "K00\ Fig[ 4D# very similar to that obtained with anti!U0A monoclonal antibodies "data not shown#[ Double immu! no~uorescence staining with antibody K00 and anti!p79 coilin antibody showed only partial co!localization "Fig[ 4D\ E\ F#[ The other U0C!speci_c clones gave an identical staining pattern as K00 and K25 "not shown#[ These results suggest that in the nucleus a major part of the U0C protein is located within the coiled bodies[ 3[ Discussion For the _rst time the isolation and characterization of human U0C!reactive monoclonal antibody fragments "Fabs and scFvs# have been described[ Eight antibodies

R[M[A[ Hoet et al[:Molecular Immunology 24 "0887# 0934Ð0944

0940

Fig[ 2[ Epitope mapping of anti!U0C antibody fragments A] Immunoprecipitations are shown for K04 and K25 using 24S!methionine labelled in vitro translates of U0C deletion mutants[ I  09) input translate\ P  immunoprecipitate[ B] Overall results of epitope mapping studies of anti! U0C reactive autoantibody fragments using immunoprecipitations with in vitro translates of U0C deletion mutants[ Epitope regions are indicated "bottom#[ C] Immunoprecipitations are shown for K00\ K04 and K25 using 24S!methionine labelled in vitro translates of U0A deletion mutants[ I  09) input translate\ P  immunoprecipitate[ Note] immunoprecipitations are performed with 099 ml periplasmic fraction as described by De Wildt et al[ "0885#[

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Table 2 Reactivity of monoclonal autoantibody fragments"scFvs# with peptides of U0A and Sm!B:B? Antigen

K00

K04

K25

rU0A

¦¦

¦¦

*

U0A peptides 24Ð47 020Ð042 038Ð055 052Ð073 079Ð194 192Ð114 146Ð171

* * * ¦ * * *

* * * ¦ ¦ * *

* * * * * * *

Sm!B:B?peptides 076Ð193 ¦¦ 100Ð117 2 115Ð139 ¦

¦¦ * *

*

rU0C

¦¦

¦¦

¦¦

*

Pro!rich sequence

PVPGMPP PPYMPP PPPGMIPPP PPGAMPP

PPPGMRPPMGPP PPPGMRPPPP PPPPGMRPPRP 007 097

PAPGMRPP014 PPPPGMMP004

Cut!o} ELISA signals] "OD!C#] OD ³ 1×"OD control antibody# ¦:−] 3×"OD!C# × OD × 1×"OD!C# ¦] 7×"OD!C# × OD × 3×"OD!C# ¦¦] OD × 7×OD!C

are speci_c for U0C while two patient!derived antibodies cross!react with the U0A and Sm!B:B? proteins[ Most of the patient!derived antibodies were isolated from the bone marrow libraries[ This might be caused by the num! ber of plasma cells which is much higher in bone marrow compared to peripheral blood[ 3[0[ Epitope regions Recent B!cell epitope mapping studies indicate that most human "patient# anti!U0C antibodies are directed to the native U0C protein "Satoh et al[\ 0885#[ Two of our anti!U0C antibodies "C0E6 and C0R# recognize only complete recombinant U0C "Fig[ 2B#\ suggesting that also in this case the conformation of the whole protein is necessary for antibody binding[ Using U0C deletion mutants\ Misaki et al[ "0882# found that 099) of the anti!U0C positive sera tested "n  06# reacted with a region comprising residues 091Ð 014[ Some sera also reacted with the region containing aa 22Ð36[ This region 22Ð36 contains an amino acid sequence similar to that of the herpes simplex virus type 0 ICP3 protein[ These results were con_rmed by Halimi et al[ "0885# who reported similar epitope regions\ i[e[ aa 4Ð37 and aa 86Ð022[ James and Harley "0884#\ using an assay with overlapping octapeptides of U0C\ found that the region spanning amino acids 006Ð014\ which shows sequence similarity with EBNA!0\ contained a major epi! tope[ Ten other\ less frequently used U0C epitopes\ most of them localized in the regions 04Ð39\ 45Ð57 and 67Ð83

Fig[ 3[ Immunoprecipitation of "U#snRNPs using human anti!U0C reactive monoclonal autoantibody fragments[ Recombinant antibody fragments "099 ml of periplasmic fraction# were bound to protein A agarose via anti!VSV or anti!c!myc monoclonal antibody "De Wildt et al[\ 0885# and incubated with 21PO3!labelled S099 extract "Habets et al[\ 0874#[ The RNA from the selected ribonucleoprotein antigens was separated on a 7) urea!PAGE\ dried and exposed to Kodak!X!Omat _lm[ "0#^ K00 "1#^ K04 "2#^ K25 "3# ^ anti!BSA scFv "c#^ anti!VSV MoAb[

were also identi_ed[ Thus most studies indicate that the immunodominant regions are in the domain between aa 4Ð37 and the C!terminal part "aa 86Ð022# of the protein[ In agreement with this\ _ve of our antibodies "C0C5\ C0G0\ C0A4\ K18\ G7# recognize the region in which aa 29Ð59 are essential while three antibodies recognize the C!terminal domain in which aa 87Ð015 appear to be essential "Fig[ 2B#[ It is interesting that both regions are

) R[M[A[ Hoet et al[:Molecular Immunology 24 "0887# 0934Ð0944

0942

Fig[ 4[ Immunolocalization of U0C protein with U0C reactive human autoantibody fragments using confocal microscopy[ HEp!1 cells were _rst incubated with "A# K25 and "C# K00 and bound antibody fragments were detected with anti!VSV monoclonal antibody and FITC!conjugated goat! anti!mouse IgG[ Subsequently anti!p79 coilin "serum R177# was added and bound rabbit antibodies were detected with Texas Red conjugated goat! anti!rabbit Ig ð"B# and "E#Ł[ Z!scans were superimposed on each other in "C# and "F#[

used and which mutations are present in autoantibody fragments[ We focused on the heavy chain genes because these are thought to be primarily responsible for the speci_city of an antibody "fragment#\ while the light chain is thought to only in~uence the a.nity\ and in some cases the _ne!tuned speci_city of an antibody "Ohlin et al[\0885#[ Four out of the _ve patient autoantibody frag! ments originate from the VH2 gene family[ In addition it was found that all the antibody fragments from the semi! synthetic libraries used a VH2 gene[ Although the VH2 family is the largest heavy chain family\ it is interesting to note that four antibodies\ three semi!synthetic antibodies and one patient!derived one\ use the VH2Ð29 gene "VH2Ð 29  DP!38#[ This gene is used in about 7) of the cases in the IgG repertoire in healthy individuals "De Wildt et al[\ unpublished#[ In addition\ one patient!derived anti! body "G7# used a gene most homologous to a poly! morphism of the VH2Ð29 related gene "P0#\ described previously "Olee et al[\ 0880#[ All VH2Ð29 encoded anti! bodies recognize the same domain "aa 29Ð59# or a con! formational epitope\ which also could be contained within this domain[ Therefore\ a correlation between epi! tope region and VH gene usage seems to be present[ A similar possible VH!gene restriction was found for K00 and K04[ Both clones use the 2Ð96 "DP!43# VH gene\ K04 uses the unmutated VH gene while K00 has two somatic mutations in its framework 2[ Both clones use a DPK07 light chain with identical CDR2s "Table 1# and both antibodies cross!react with proteins U0C\ Sm!B:B? and U0A\ although a _ne!tuned di}erence in speci_city is noted "see immunoblot Fig[ 1A and Table 2#[ This latter di}erence could be explained by the variation in CDR2 of the VH gene and:or mutations in VH gene of K00[

also accessible in the U0snRNP particle because anti! bodies to both of these parts of U0C are able to immuno! precipitate the U0snRNP complex[ The region con! taining aa 87Ð015 contains residues PAPGMRPP "aa 007Ð014\ see Table 2\ Fig[ 2B#[ A similar region is repeated twice in Sm!B and three times in Sm!B?:N "resi! dues 080Ð087 and 105Ð112 of Sm!B:B?:N and 120Ð127 of Sm!B?:N#[ This proline!rich sequence was previously shown to contain a dominant B!cell epitope "Habets et al[\ 0878\ Williams et al[\0889\ James et al[\ 0884#[ In U0A\ a similar sequence\ PPPGMIPP "aa 054Ð061# can be found although this appeared not to be an immu! nodominant epitope "Barakat et al[\ 0880\ James et al[\ 0885#[ Our cross!reactive antibodies K00 and K04\ react with this Pro!rich region con_rming that this region is targeted by anti!Sm:RNP autoantibodies in SLE[ Inter! estingly\ the speci_city of the patient!derived monoclonal antibody K25 which does not recognize the U0A or the Sm!B:B? protein\ indicates that there are also U0C!spec! i_c patient antibodies recognizing this region of the U0C protein[ Recently Satoh et al[ "0886# reported a new group of human anti!U0C autoantibodies which appeared in vitro to stabilize the association between U0C and the Sm core particle[ Our patient antibodies K00 and K04 bind both to the Sm:B:B? and U0C in the U0snRNP particle "Fig[ 3# and therefore could crosslink and sta! bilize U0C to the Sm core particle[ 3[1[ V!gene restriction Not much is known about the V!gene usage of autoan! tibodies directed to the U0snRNP complex[ Therefore the question was asked whether particular V!genes are

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R[M[A[ Hoet et al[:Molecular Immunology 24 "0887# 0934Ð0944

Although it is not clear whether the heavy and light chain pairing in the recombinant antibodies corresponds to the in vivo situation it is interesting to note the restriction in VH:VL pairing between K00 and K04[ The possibility that VH gene usage might correlate with epitope recognition has previously been suggested by us "De Wildt et al[\ 0885# in our studies of human anti!U0A monoclonal antibody fragments[ A VH!gene:epitope relationship is probably due to the natural _t of a certain heavy chain product and is not typical for the autoan! tibody repertoire since similar VH!genes are also selected from a semi!synthetic repertoire\ which is more or less comparable with the IgM "naive# response[ 3[2[ Immunolocalization of the U0C protein Until now\ no monoclonal antibodies directed to the U0C protein have been described[ Therefore\ a precise idea about the subcellular localization of this protein could not be obtained[ Using patient polyclonal anti! bodies and in situ hybridization the presence of U0snRNPs in the coiled bodies and {speckled| structures was established "Carmo!Fonseca et al[\ 0881#[ However\ U0snRNP localization was found more widespread in the nucleoplasm as compared to the other snRNPs "U1\ U3!U5# "Carmo!Fonseca et al[\ 0881\ Andrade et al[\ 0880#[ The immunolocalization studies with our anti! U0C antibodies "Fig[ 4# show that high concentrations of U0C can be found in coiled bodies\ an organelle which has been proposed to be involved in some aspect of snRNP maturation\ transport\ or recycling\ with only a relatively minor fraction being present in the nucleo! plasmic speckles[ Our results thus underline the hypoth! esis that the coiled body structure is used as a storage:assembly place for snRNP proteins such as U0C[ The anti!U0C antibodies described in this paper might be very useful for further studies of the function of U0C in splicing\ and to evaluate the possible pathological role which these autoantibodies "Tsikaris et al[ 0885\ James et al\ 0886# may have in autoimmunity[ Acknowledgements The authors would like to thank Dr J[ Klein Gun! newiek and Ing[ Y[ van Aarssen for supplying recom! binant U0C protein and W[ Roeterdink for performing some of the epitope mapping experiments[ We are grate! ful to Dr E[ Tan "Scripps Research Institute\ La Jolla\ U[S[A[# for supplying the anti!p79 coilin antibodies and to Dr W[H[ Ouwehand "University of Cambridge\ Expt[ Hematology\ Cambridge\ U[K[# for providing DNA pri! mers and the pHENIX vector[ We also like to thank Dr G[ Winter "MRC\ Cambridge\ U[K[# for kindly providing the semi!synthetic Fab and scFv libraries and Dr G[ Pruijn for critical reading of the manuscript[ This work

was supported by the Netherlands Foundation for Chemical Research "SON# and the Technology Foun! dation "STW# with _nancial aid from the Netherlands Organization for Scienti_c Research "Grant no[ 238!2915# and by a grant from EEC "contract no[ ERBCHRXCT 829 065# to W[J[v[V[ and S[M[

References Andrade\ L[E[\ Chan\ E[K[\ Raska\ I[\ Peebles\ C[L[\ Roos\ G[\ Tan\ E[M[\ 0880[ Human autoantibody to a novel protein of the nuclear coiled body] immunological characterization and cDNA cloning of p79!coilin[ J[ Exp[ Med[ 062\ 0396Ð0308[ Barakat\ S[\ Briand\ J[P[\ Abuaf\ N[\ Van Regenmortel\ M[H[V[\ Muller\ S[\ 0880[ Mapping of epitopes on U0 snRNP polypeptide A with synthetic peptides and autoimmune sera[ Clin[ Exp[ Immu! nol[ 75\ 60Ð67[ Brahms\ H[\ Raker\ V[A[\ van Venrooij\ W[J[\ Luhrmann\ R[\ 0886[ A major\ novel systemic lupus erythematosus autoantibody class recognizes the E\ F\ and G Sm snRNP proteins as an E!F!G complex but not in their denatured states[ Arthritis Rheum[ 39\ 561Ð571[ Carmo!Fonseca\ M[\ Pepperkok\ R[\ Carvalho\ M[T[\ Lamond\ A[I[\ 0881[ Transcription!dependent colocalization of the U0\ U1\ U3:U5\ and U4 snRNPs in coiled bodies[ J[ Cell Biol[ 006\ 0Ð03[ De Wildt\ R[M[\ Finnern\ R[\ Ouwehand\ W[H[\ Gri.ths\ A[D[\ van Venrooij\ W[J[\ Hoet\ R[M[\ 0885[ Characterization of human vari! able domain antibody fragments against the U0 RNA!associated A protein\ selected from a synthetic and patient!derived combinatorial V gene library[ Eur[ J[ Immunol[ 15\ 518Ð528[ Dumortier\ H[\ Klein Gunnewiek\ J[\ Roussel\ J!P[\ van Aarssen\ Y[\ Briand\ J!P[\ van Venrooij\ W[J[\ Muller\ S[\ 0887[ At least three linear regions but not the zinc _nger domain of U0C protein are exposed at the surface of the protein in solution and on the human spliceosomal U0 snRNP particle[ Nucleic Acids Res[\ in press[ Finnern\ R[\ Pedrollo\ E[\ Fisch\ I[\ Wieslander\ J[\ Marks\ J[D[\ Lock! wood\ C[M[\ Ouwehand\ W[H[\ 0886[ Human autoimmune anti! proteinase 2 scFv from a phage display library[ Clin[ Exp[ Immunol[ 096\ 158Ð170[ Gri.ths\ A[D[\ Williams\ S[C[\ Hartley\ O[\ Tomlinson\ I[M[\ Water! house\ P[\ Crosby\ W[L[\ Kontermann\ R[E[\ Jones\ P[T[\ Low\ N[M[\ Allison\ T[J[ et al[\ 0883[ Isolation of high a.nity human antibodies directly from large synthetic repertoires[ EMBO J[ 02\ 2134Ð2159[ Habets\ W[J[\ Hoet\ M[\ Bringmann\ P[\ Luhrmann\ R[\ van Venrooij\ W[J[ 0874[ Autoantibodies to ribonucleoprotein particles containing U1 small nuclear RNA[ EMBO J[ 3\ 0434Ð0449[ Habets\ W[J[\ Sillekens\ P[T[\ Hoet\ M[H[\ McAllister\ G[\ Lerner\ M[R[\ van Venrooij\ W[J[\ 0878[ Small nuclear RNA!associated proteins are immunologically related as revealed by mapping of autoimmune reactive B!cell epitopes[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 75\ 3563Ð3567[ Halimi\ H[\ Dumortier\ H[\ Briand\ J[P[\ Muller\ S[\ 0885[ Comparison of two di}erent methods using overlapping synthetic peptides for localizing linear B cell epitopes in the U0 snRNP!C autoantigen[ J[ Immunol[ Methods 088\ 66Ð74[ Heinrichs\ V[\ Bach M[\ Winkelmann\ G[\ Luhrmann\ R[\ 0889[ U0! speci_c protein C needed for e.cient complex formation of U0snRNP with a 4? splice site[ Science 136\ 58Ð61[ Hoet\ R[M[\ De Weerd\ P[\ Gunnewiek\ J[K[\ Koornneef\ I[\ Van Ven! rooij\ W[J[\ 0881[ Epitope regions on U0 small nuclear RNA recog! nized by anti!U0RNA!speci_c autoantibodies[ J[ Clin[ Invest[ 89\ 0642Ð0651[ James\ J[A[\ Harley\ J[B[\ 0884[ Peptide autoantigenicity of the small nuclear ribonucleoprotein C[ Clin[ Exp[ Rheumatol[ 02\ 188Ð294[

R[M[A[ Hoet et al[:Molecular Immunology 24 "0887# 0934Ð0944 James\ J[A[\ Gross\ T[\ Sco_eld\ R[H[\ Harley\ J[B[\ 0884[ Immu! noglobulin epitope spreading and autoimmune disease after peptide immunization] Sm B:B?!derived PPPGMRPP and PPPGIRGP induce spliceosome autoimmunity[ J[ Exp[ Med[ 070\ 342Ð350[ James\ J[A[\ Harley\ J[B[\ 0885[ Human lupus anti!spliceosome A pro! tein autoantibodies bind contiguous surface structures and segregate into two sequential epitope binding patterns[ J[ Immunol[ 045\ 3907Ð 3915[ James\ J[A[\ Sco_eld\ R[H[\ Harley\ J[B[\ 0886[ Lupus humoral auto! immunity after short peptide immunization[ In] B lymphocytes and autoimmunity[ Annals New York Academy Sciences 704\ 013Ð016[ Klein Gunnewiek\ J[M[\ van Aarssen\ Y[\ Wassenaar\ R[\ Legrain\ P[\ van Venrooij\ W[J[\ Nelissen\ R[L[\ 0884[ Homodimerization of the human U0 snRNP!speci_c protein C[ Nucleic Acids Res[ 12\ 3753Ð 3760[ Klein Gunnewiek\ J[M[\ van de Putte\ L[B[\ van Venrooij\ W[J[\ 0886[ The U0 snRNP complex] an autoantigen in connective tissue diseases[ An update[ Clin[ Exp[ Rheumatol[ 04\ 438Ð459[ Kreis\ T[E[\ Lodish\ H[F[\ 0875[ Oligomerization is essential for trans! port of vesicular stomatitis viral glycoprotein to the cell surface[ Cell 35\ 818Ð826[ Marks\ J[D[\ Hoogenboom\ H[R[\ Bonnert\ T[P[\ McCa}erty\ J[\ Gri.ths\ A[D[\ Winter\ G[\ 0880[ By!passing immunization[ Human antibodies from V!gene libraries displayed on phage[ J[ Mol[ Biol[ 111\ 470Ð486[ Misaki\ Y[\ Yamamoto\ K[\ Yanagi\ K[\ Miura\ H[\ Ichijo\ H[\ Kato\ T[\ Mato\ T[\ Welling!Wester\ S[\ Nishioka\ K[\ Ito\ K[\ 0882[ B cell epitope on the U0 snRNP!C autoantigen contains a sequence similar to that of the herpes simplex virus protein[ Eur[ J[ Immunol[ 12\ 0953Ð0960[ Nelissen\ R[L[\ Heinrichs\ V[\ Habets\ W[J[\ Simons\ F[\ Luhrmann\ R[\ van Venrooij\ W[J[\ 0880[ Zinc _nger!like structure in U0!speci_c protein C is essential for speci_c binding to U0 snRNP[ Nucleic Acids Res[ 08\ 338Ð343[ Nelissen\ R[L[\ Will\ C[L[\ van Venrooij\ W[J[\ Luhrmann\ R[\ 0883[ The association of the U0!speci_c 69K and C proteins with U0 snRNPs is mediated in part by common U snRNP proteins[ EMBO J[ 02\ 3002Ð3014[ Nissim\ A[\ Hoogenboom\ H[R[\ Tomlinson\ I[M[\ Flynn\ G[\ Midgley\ C[\ Lane\ D[\ Winter\ G[\ 0883[ Antibody fragments from a {single pot| phage display library as immunochemical reagents[ EMBO J[ 02\ 581Ð587[

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Ohlin\ M[\ Owman\ H[\ Mach\ M[\ Borrebaeck\ C[A[\ 0885[ Light chain shu/ing of a high a.nity antibody results in a drift in epitope recognition[ Mol[ Immunol[ 22\ 36Ð45[ Olee\ T[\ Yang\ P[\ Siminovitch\ K[A[\ Olsen\ N[J[\ Hillson\ J[\ Wu J[\ Kozin\ F[\ Carson\ D[A[\ Chen\ P[P[\ 0880[ Molecular basis of an autoantibody!associated restriction fragment length polymorphism that confers susceptibility to autoimmune diseases[ J[ Clin[ Invest[ 77\ 082Ð192[ Rossi\ F[\ Forne\ T[\ Antoine\ E[\ Tazi\ J[\ Brunel\ C[\ Cathala\ G[\ 0885[ Involvement of U0 small nuclear ribonucleoproteins "snRNP# in 4? splice site!U0 snRNP interaction[ J[ Biol[ Chem[ 160\ 12874Ð 12880[ Sambrook\ J[\ Fritsch\ E[\ Maniatis\ T[\ 0878[ Molecular cloning[ Cold Spring Harbor Lab Press ISBN 9!76858!298!5[ Satoh\ M[\ Langdon\ J[J[\ Hamilton\ K[J[\ Richards\ H[B[\ Panka\ D[\ Eisenberg\ R[A[\ Reeves\ W[H[\ 0885[ Distinctive immune response patterns of human and murine autoimmune sera to U0 small nuclear ribonucleoprotein C protein[ J[ Clin[ Invest[ 86\ 1508Ð1515[ Satoh\ M[\ Richards\ H[B[\ Hamilton\ K[J[\ Reeves\ W[H[\ 0886[ Human anti!nuclear ribonucleoprotein antigen autoimmune sera contain a novel subset of autoantibodies that stabilizes the molec! ular interaction of U0RNP!C protein with the Sm core proteins[ J[ Immunol[ 047\ 4906Ð4914[ Tang\ J[\ Abovich\ N[\ Fleming\ M[L[\ Seraphin\ B[\ Rosbash\ M[\ 0886[ Identi_cation and characterization of a yeast homolog of U0snRNP!speci_c protein C[ EMBO J[ 05\ 3971Ð3980[ Tsikaris\ V[\ Vlachoyiannopoulos\ P[G[\ Panou!Pomonis\ E[\ Marraud\ M[\ Sakarellos\ C[\ Moutsopoulos\ H[M[\ Sakarellos!Daitsiotis\ M[\ 0885[ Immunoreactivity and conformation of the P!P!G!M!R!P!P repetitive epitope of the Sm autoantigen[ Int[ J[ Pept[ Protein Res[ 37\ 208Ð216[ Will\ C[L[\ Rumpler\ S[\ Klein Gunnewiek\ J[\ van Venrooij\ W[J[\ Luhrmann\ R[\ 0885[ In vitro reconstitution of mammalian U0 snRNPs active in splicing] the U0!C protein enhances the formation of early "E# spliceosomal complexes[ Nucleic Acids Res[ 13\ 3503Ð 3512[ Williams\ D[G[\ Sharpe\ N[G[\ Wallace\ G[\ Latchman\ D[S[\ 0889[ A repeated proline!rich sequence in Sm B:B? and N is a dominant epitope recognized by human and murine autoantibodies[ J[ Auto! immunity 2\ 604Ð614[