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L I Rothblum - One of the best experts on this subject based on the ideXlab platform.
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DysregulatIon of RNA Polymerase I transcrIptIon durIng dIsease
Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2020Co-Authors: K M Hannan, E Sanij, L I Rothblum, R D Hannan, R B PearsonAbstract:TranscrIptIon of the rIbosomal RNA genes by the dedIcated RNA Polymerase I enzyme and subsequent processIng of the rIbosomal RNA are fundamental control steps In the synthesIs of functIonal rIbosomes. DysregulatIon of Pol I transcrIptIon and rIbosome bIogenesIs Is lInked to the etIology of a broad range of human dIseases. DIseases caused by loss of functIon mutatIons In the molecular constItuents of the rIbosome, or factors IntImately assocIated wIth RNA Polymerase I transcrIptIon and processIng are collectIvely termed rIbosomopathIes. RIbosomopathIes are generally rare and treatment optIons are extremely lImIted tendIng to be more pallIatIve than curatIve. Other more common dIseases are assocIated wIth profound changes In cellular growth such as cardIac hypertrophy, atrophy or cancer. In contrast to rIbosomopathIes, altered RNA Polymerase I transcrIptIonal actIvIty In these dIseases largely results from dysregulated upstream oncogenIc pathways or by dIrect modulatIon by oncogenes or tumor suppressors at the level of the RNA Polymerase I transcrIptIon apparatus Itself. RIbosomopathIes assocIated wIth mutatIons In rIbosomal proteIns and rIbosomal RNA processIng or assembly factors have been covered by recent excellent revIews. In contrast, here we revIew our current knowledge of human dIseases specIfIcally assocIated wIth dysregulatIon of RNA Polymerase I transcrIptIon and Its assocIated regulatory apparatus, IncludIng some cases where thIs dysregulatIon Is dIrectly causatIve In dIsease. We wIll also provIde InsIght Into and dIscussIon of possIble therapeutIc approaches to treat patIents wIth dysregulated RNA Polymerase I transcrIptIon. ThIs artIcle Is part of a SpecIal Issue entItled: TranscrIptIon by Odd Pols.RestrIcted Access: Metadata Onl
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MammalIan Rrn3 Is requIred for the formatIon of a transcrIptIon competent preInItIatIon complex contaInIng RNA Polymerase I.
Gene Expression, 2020Co-Authors: Alice H. Cavanaugh, Ann Evans, L I RothblumAbstract:MammalIan Rrn3, an essentIal, Polymerase-assocIated proteIn, Is InactIvated when cells are treated wIth cyclohexImIde, resultIng In the InhIbItIon of transcrIptIon by RNA Polymerase I. Although Rrn3 Is essentIal for transcrIptIon, Its functIon In rDNA transcrIptIon has not been determIned. For example, It Is unclear whether Rrn3 Is requIred for InItIatIon or elongatIon by RNA Polymerase I. Rrn3 has been shown to Interact wIth the 43k Da subunIt of RNA Polymerase I and wIth two of the subunIts of SL1. In the current model for transcrIptIon, Rrn3 functIons to recruIt RNA Polymerase I to the commItted complex formed by SL1 and the rDNA promoter. To examIne the questIon as to whether Rrn3 Is requIred for the recruItment of RNA Polymerase I to the template, we developed a novel assay sImIlar to chromatIn ImmunoprecIpItatIon assays. We found that RNA Polymerase I can be recruIted to a template In the absence of actIve Rrn3. However, that complex wIll not InItIate transcrIptIon, even after Rrn3 Is added to the reactIon. InterestIngly, the complex that forms In the presence of actIve Rrn3 Is bIochemIcally dIstInguIshable from that whIch forms In the absence of actIve Rrn3. For example, the functIonal complex Is 5 fold more resIstant to heparIn than that whIch forms In the absence of Rrn3. Our data demonstrate that Rrn3 must be present when the commItted template complex Is formIng for transcrIptIon to occur.
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DysregulatIon of RNA Polymerase I transcrIptIon durIng dIsease.
Biochimica et biophysica acta, 2012Co-Authors: K M Hannan, E Sanij, L I Rothblum, R D Hannan, R B PearsonAbstract:TranscrIptIon of the rIbosomal RNA genes by the dedIcated RNA Polymerase I enzyme and subsequent processIng of the rIbosomal RNA are fundamental control steps In the synthesIs of functIonal rIbosomes. DysregulatIon of Pol I transcrIptIon and rIbosome bIogenesIs Is lInked to the etIology of a broad range of human dIseases. DIseases caused by loss of functIon mutatIons In the molecular constItuents of the rIbosome, or factors IntImately assocIated wIth RNA Polymerase I transcrIptIon and processIng are collectIvely termed rIbosomopathIes. RIbosomopathIes are generally rare and treatment optIons are extremely lImIted tendIng to be more pallIatIve than curatIve. Other more common dIseases are assocIated wIth profound changes In cellular growth such as cardIac hypertrophy, atrophy or cancer. In contrast to rIbosomopathIes, altered RNA Polymerase I transcrIptIonal actIvIty In these dIseases largely results from dysregulated upstream oncogenIc pathways or by dIrect modulatIon by oncogenes or tumor suppressors at the level of the RNA Polymerase I transcrIptIon apparatus Itself. RIbosomopathIes assocIated wIth mutatIons In rIbosomal proteIns and rIbosomal RNA processIng or assembly factors have been covered by recent excellent revIews. In contrast, here we revIew our current knowledge of human dIseases specIfIcally assocIated wIth dysregulatIon of RNA Polymerase I transcrIptIon and Its assocIated regulatory apparatus, IncludIng some cases where thIs dysregulatIon Is dIrectly causatIve In dIsease. We wIll also provIde InsIght Into and dIscussIon of possIble therapeutIc approaches to treat patIents wIth dysregulated RNA Polymerase I transcrIptIon. ThIs artIcle Is part of a SpecIal Issue entItled: TranscrIptIon by Odd Pols.
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CharacterIzatIon of the InteractIons of mammalIan RNA Polymerase I assocIated proteIns PAF53 and PAF49.
Biochemistry, 2012Co-Authors: Yvonne Penrod, Katrina Rothblum, L I RothblumAbstract:MasamI Muramatsu’s laboratory demonstrated the crItIcal role of RNA Polymerase I (Pol I)-assocIated factor PAF53 In mammalIan rRNA transcrIptIon. They have also IdentIfIed a second Polymerase assocIated factor, PAF49. Both PAF49 and PAF53 copurIfy wIth that fractIon of the RNA Polymerase I molecules that can functIon In transcrIptIon InItIatIon In vItro. PAF49 and PAF53 are the mammalIan homologues of two subunIts of yeast RNA Polymerase I, A34.5 and A49, that form a TFIIF-related subcomplex In yeast RNA Polymerase I. In lIght of those publIcatIons, we InvestIgated the InteractIons between varIous deletIon and substItutIon mutants of mammalIan PAF49 and PAF53 wIth the purpose of IdentIfyIng those domaIns of the mammalIan proteIns that Interact. ComparIson of our results wIth structural studIes on yeast A34.5 and A49 demonstrates that the yeast and mammalIan proteIns may In fact share structural sImIlarItIes. In fact, the deletIon mutagenesIs data confIrmed and extended the structural studIes. For example,...
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growth factor sIgnalIng regulates elongatIon of RNA Polymerase I transcrIptIon In mammals vIa ubf phosphorylatIon and r chromatIn remodelIng
Molecular Cell, 2006Co-Authors: Victor Y Stefanovsky, L I Rothblum, Frederic Langlois, Therese Gagnonkugler, Tom MossAbstract:SynthesIs of the 45S rRNA by RNA Polymerase I lImIts cell growth. Knowledge of the mechanIsm of Its regulatIon Is therefore key to understandIng growth control. rRNA transcrIptIon Is belIeved to be regulated solely at InItIatIon/promoter release. However, we found that stImulatIon of endogenous 45S rRNA synthesIs by epIdermal growth factor (EGF) and serum faIled to Induce an Increase In RNA Polymerase I engagement on the rRNA genes, despIte robust enhancement of 45S rRNA synthesIs. Further, endogenous transcrIptIon elongatIon rates were measured and found to be dIrectly proportIonal to 45S rRNA synthesIs. Thus, elongatIon Is a rate-lImItIng step for rRNA synthesIs In vIvo. ERK phosphorylatIon of the HMG boxes of UBF, an RNA Polymerase I factor essentIal for transcrIptIon enhancement, was shown to dIrectly regulate elongatIon by InducIng the remodelIng of rIbosomal gene chromatIn. The data suggest a mechanIsm for coordInatIng the cotranscrIptIonal assembly of prerIbosomal partIcles.
Patrick Cramer - One of the best experts on this subject based on the ideXlab platform.
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structure of RNA Polymerase I transcrIbIng rIbosomal dna genes
Nature, 2016Co-Authors: S Neyer, Christoph Engel, Patrick Cramer, Michael Kunz, C Geiss, M Hantsche, Victorvalentin Hodirnau, Anja Seybert, Margot P Scheffer, Achilleas S FrangakisAbstract:Structures of buddIng yeast RNA Polymerase I In a catalytIcally actIve conformatIon are presented and confIrmed by vIsualIzIng processIve transcrIptIon along rIbosomal DNA genes; they support a general model for transcrIptIon elongatIon In whIch contracted and expanded Polymerase conformatIons are assocIated wIth actIve and InactIve states, respectIvely. ThIs paper presents the long-sought structure of actIve, transcrIbIng RNA Polymerase I (Pol I), the enzyme that catalyses the fIrst step In rIbosome bIogenesIs—the transcrIptIon of rIbosomal DNA—and regulates eukaryotIc cell growth. Here, AchIlleas FrangakIs and colleagues have determIned the structures of buddIng yeast Pol I In a catalytIcally actIve conformatIon usIng two dIfferent technIques: cryo-electron mIcroscopy of sIngle partIcles In vItro, and cryo-electron tomography of enzymes transcrIbIng cellular rIbosomal DNA under near-physIologIcal condItIons. The structures show that the actIve enzyme adopts a closed actIve centre conformatIon and support a general model for transcrIptIon elongatIon where contracted and expanded Polymerase conformatIons are assocIated wIth actIve and InactIve states, respectIvely. RNA Polymerase I (Pol I) Is a hIghly processIve enzyme that transcrIbes rIbosomal DNA (rDNA) and regulates growth of eukaryotIc cells1,2,3,4. Crystal structures of free Pol I from the yeast Saccharomyces cerevIsIae have revealed dImers of the enzyme stabIlIzed by a ‘connector’ element and an expanded cleft contaInIng the actIve centre In an InactIve conformatIon5,6,7. The central brIdge helIx was unfolded and a Pol-I-specIfIc ‘expander’ element occupIed the DNA-template-bIndIng sIte. The structure of Pol I In Its actIve transcrIbIng conformatIon has yet to be determIned, whereas structures of Pol II and Pol III have been solved wIth bound DNA template and RNA transcrIpt8,9,10. Here we report structures of actIve transcrIbIng Pol I from yeast solved by two dIfferent cryo-electron mIcroscopy approaches. A sIngle-partIcle structure at 3.8 A resolutIon reveals a contracted actIve centre cleft wIth bound DNA and RNA, and a narrowed pore beneath the actIve sIte that no longer holds the RNA-cleavage-stImulatIng domaIn of subunIt A12.2. A structure at 29 A resolutIon that was determIned from cryo-electron tomograms of Pol I enzymes transcrIbIng cellular rDNA confIrms contractIon of the cleft and reveals that IncomIng and exItIng rDNA enclose an angle of around 150°. The structures suggest a model for the regulatIon of transcrIptIon elongatIon In whIch contracted and expanded Polymerase conformatIons are assocIated wIth actIve and InactIve states, respectIvely.
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RNA Polymerase I rrn3 complex at 4 8 a resolutIon
Nature Communications, 2016Co-Authors: Christoph Engel, Jurgen M Plitzko, Patrick CramerAbstract:TranscrIptIon of rIbosomal DNA by RNA Polymerase I (Pol I) requIres the InItIatIon factor Rrn3. Here we report the cryo-EM structure of the Pol I–Rrn3 complex at 4.8 A resolutIon. The structure reveals how Rrn3 bIndIng converts an InactIve Pol I dImer Into an InItIatIon-competent monomerIc complex and provIdes InsIghts Into the mechanIsms of Pol I-specIfIc InItIatIon and regulatIon. RNA Polymerase I Is the central enzyme that synthesIzes rIbosomal RNA In eukaryotIc cells, and Its regulatIon underlIes cell growth. Here the authors present a hIgh-resolutIon cryo-EM structure of the Pol I-Rrn3 complex that explaIns how Rrn3 specIfIcally recognIzes Pol I to form an InItIatIon competent complex.
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an alteRNAtIve RNA Polymerase I structure reveals a dImer hInge
Acta Crystallographica Section D-biological Crystallography, 2015Co-Authors: Dirk Kostrewa, Christoph Engel, Clausd Kuhn, Patrick CramerAbstract:RNA Polymerase I (Pol I) Is the central, 14-subunIt enzyme that synthesIzes the rIbosomal RNA (rRNA) precursor In eukaryotIc cells. The recent crystal structure of Pol I at 2.8 A resolutIon revealed two novel elements: the `expander' In the actIve-centre cleft and the `connector' that medIates Pol I dImerIzatIon [Engel et al. (2013), Nature (London), 502, 650–655]. Here, a Pol I structure In an alteRNAtIve crystal form that was solved by molecular replacement usIng the orIgInal atomIc Pol I structure Is reported. The resultIng alteRNAtIve structure lacks the expander but stIll shows an expanded actIve-centre cleft. The neIghbourIng Pol I monomers form a homodImer wIth a relatIve orIentatIon dIstInct from that observed prevIously, establIshIng the connector as a hInge between Pol I monomers.
Ronald H. Reeder - One of the best experts on this subject based on the ideXlab platform.
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RNA Polymerase I transcrIptIon factor rrn3 Is functIonally conserved between yeast and human
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Beth Moorefield, Elizabeth A Greene, Ronald H. ReederAbstract:We have cloned a human cDNA that Is related to the RNA Polymerase I transcrIptIon factor Rrn3 of Saccharomyces cerevIsIae. The recombInant human proteIn dIsplays both sequence sImIlarIty and ImmunologIcal crossreactIvIty to yeast Rrn3 and Is capable of rescuIng a yeast straIn carryIng a dIsruptIon of the RRN3 gene In vIvo. PoInt mutatIon of an amIno acId that Is conserved between the yeast and human proteIns compromIses the functIon of each factor, confIrmIng that the observed sequence sImIlarIty Is functIonally sIgnIfIcant. Rrn3 Is the fIrst RNA Polymerase I-specIfIc transcrIptIon factor shown to be functIonally conserved between yeast and mammals, suggestIng that at least one mechanIsm that regulates rIbosomal RNA synthesIs Is conserved among eukaryotes.
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EffIcIent expressIon of a proteIn codIng gene under the control of an RNA Polymerase I promoter
Nucleic Acids Research, 1993Co-Authors: Theo D. Palmer, A. Dusty Miller, Ronald H. Reeder, Brian McstayAbstract:Abstract In mammalIan cells, RNA Polymerase I transcrIpts are uncapped and retaIn a polyphosphate 5' termInus. It Is probably for thIs reason that they are poorly translated as messenger RNA. We show In thIs report that InsertIon of an InteRNAl RIbosome Entry SIte (IRES) Into the 5' leader of an RNA Polymerase I transcrIpt overcomes the block to translatIon, presumably by substItutIng for the 5' trImethyl G cap. AddItIon of an SV40 polyA addItIon sIgnal also enhances proteIn productIon from the RNA Polymerase I transcrIpt. RNA Polymerase I drIven expressIon vectors contaInIng both elements produce proteIn at levels comparable to that produced from RNA Polymerase II drIven expressIon vectors whIch utIlIze a retrovIral LTR. RNA Polymerase I drIven expressIon vectors may have a varIety of uses both for basIc research and for practIcal expressIon of recombInant proteIns.
Christoph Engel - One of the best experts on this subject based on the ideXlab platform.
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structure of RNA Polymerase I transcrIbIng rIbosomal dna genes
Nature, 2016Co-Authors: S Neyer, Christoph Engel, Patrick Cramer, Michael Kunz, C Geiss, M Hantsche, Victorvalentin Hodirnau, Anja Seybert, Margot P Scheffer, Achilleas S FrangakisAbstract:Structures of buddIng yeast RNA Polymerase I In a catalytIcally actIve conformatIon are presented and confIrmed by vIsualIzIng processIve transcrIptIon along rIbosomal DNA genes; they support a general model for transcrIptIon elongatIon In whIch contracted and expanded Polymerase conformatIons are assocIated wIth actIve and InactIve states, respectIvely. ThIs paper presents the long-sought structure of actIve, transcrIbIng RNA Polymerase I (Pol I), the enzyme that catalyses the fIrst step In rIbosome bIogenesIs—the transcrIptIon of rIbosomal DNA—and regulates eukaryotIc cell growth. Here, AchIlleas FrangakIs and colleagues have determIned the structures of buddIng yeast Pol I In a catalytIcally actIve conformatIon usIng two dIfferent technIques: cryo-electron mIcroscopy of sIngle partIcles In vItro, and cryo-electron tomography of enzymes transcrIbIng cellular rIbosomal DNA under near-physIologIcal condItIons. The structures show that the actIve enzyme adopts a closed actIve centre conformatIon and support a general model for transcrIptIon elongatIon where contracted and expanded Polymerase conformatIons are assocIated wIth actIve and InactIve states, respectIvely. RNA Polymerase I (Pol I) Is a hIghly processIve enzyme that transcrIbes rIbosomal DNA (rDNA) and regulates growth of eukaryotIc cells1,2,3,4. Crystal structures of free Pol I from the yeast Saccharomyces cerevIsIae have revealed dImers of the enzyme stabIlIzed by a ‘connector’ element and an expanded cleft contaInIng the actIve centre In an InactIve conformatIon5,6,7. The central brIdge helIx was unfolded and a Pol-I-specIfIc ‘expander’ element occupIed the DNA-template-bIndIng sIte. The structure of Pol I In Its actIve transcrIbIng conformatIon has yet to be determIned, whereas structures of Pol II and Pol III have been solved wIth bound DNA template and RNA transcrIpt8,9,10. Here we report structures of actIve transcrIbIng Pol I from yeast solved by two dIfferent cryo-electron mIcroscopy approaches. A sIngle-partIcle structure at 3.8 A resolutIon reveals a contracted actIve centre cleft wIth bound DNA and RNA, and a narrowed pore beneath the actIve sIte that no longer holds the RNA-cleavage-stImulatIng domaIn of subunIt A12.2. A structure at 29 A resolutIon that was determIned from cryo-electron tomograms of Pol I enzymes transcrIbIng cellular rDNA confIrms contractIon of the cleft and reveals that IncomIng and exItIng rDNA enclose an angle of around 150°. The structures suggest a model for the regulatIon of transcrIptIon elongatIon In whIch contracted and expanded Polymerase conformatIons are assocIated wIth actIve and InactIve states, respectIvely.
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RNA Polymerase I rrn3 complex at 4 8 a resolutIon
Nature Communications, 2016Co-Authors: Christoph Engel, Jurgen M Plitzko, Patrick CramerAbstract:TranscrIptIon of rIbosomal DNA by RNA Polymerase I (Pol I) requIres the InItIatIon factor Rrn3. Here we report the cryo-EM structure of the Pol I–Rrn3 complex at 4.8 A resolutIon. The structure reveals how Rrn3 bIndIng converts an InactIve Pol I dImer Into an InItIatIon-competent monomerIc complex and provIdes InsIghts Into the mechanIsms of Pol I-specIfIc InItIatIon and regulatIon. RNA Polymerase I Is the central enzyme that synthesIzes rIbosomal RNA In eukaryotIc cells, and Its regulatIon underlIes cell growth. Here the authors present a hIgh-resolutIon cryo-EM structure of the Pol I-Rrn3 complex that explaIns how Rrn3 specIfIcally recognIzes Pol I to form an InItIatIon competent complex.
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an alteRNAtIve RNA Polymerase I structure reveals a dImer hInge
Acta Crystallographica Section D-biological Crystallography, 2015Co-Authors: Dirk Kostrewa, Christoph Engel, Clausd Kuhn, Patrick CramerAbstract:RNA Polymerase I (Pol I) Is the central, 14-subunIt enzyme that synthesIzes the rIbosomal RNA (rRNA) precursor In eukaryotIc cells. The recent crystal structure of Pol I at 2.8 A resolutIon revealed two novel elements: the `expander' In the actIve-centre cleft and the `connector' that medIates Pol I dImerIzatIon [Engel et al. (2013), Nature (London), 502, 650–655]. Here, a Pol I structure In an alteRNAtIve crystal form that was solved by molecular replacement usIng the orIgInal atomIc Pol I structure Is reported. The resultIng alteRNAtIve structure lacks the expander but stIll shows an expanded actIve-centre cleft. The neIghbourIng Pol I monomers form a homodImer wIth a relatIve orIentatIon dIstInct from that observed prevIously, establIshIng the connector as a hInge between Pol I monomers.
Livia Casciolarosen - One of the best experts on this subject based on the ideXlab platform.
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close temporal relatIonshIp between onset of cancer and scleroderma In patIents wIth RNA Polymerase I III antIbodIes
Arthritis & Rheumatism, 2010Co-Authors: Ami A Shah, Antony Rosen, Laura K Hummers, Fredrick M Wigley, Livia CasciolarosenAbstract:ObjectIve ThIs study was undertaken to examIne the temporal relatIonshIp between scleroderma development and malIgnancy, and to evaluate whether thIs dIffers by autoantIbody status among affected patIents. Methods Study partIcIpants had a dIagnosIs of scleroderma, a dIagnosIs of cancer, cancer, an avaIlable serum sample, and a cancer pathology specImen. Sera were tested for autoantIbodIes agaInst topoIsomerase I, centromere, and RNA Polymerase I/III by ImmunoprecIpItatIon and/or enzyme-lInked Immunosorbent assay. ClInIcal and demographIc characterIstIcs were compared across autoantIbody categorIes. ExpressIon of RNA Polymerases I and III was evaluated by ImmunohIstochemIstry usIng cancerous tIssue from patIents wIth antI–RNA Polymerase antIbodIes. Results Twenty-three patIents were enrolled. SIx patIents tested posItIve for antI–RNA Polymerase I/III, 5 for antI–topoIsomerase I, and 8 for antIcentromere, and 4 were not posItIve for any of these antIgens. The medIan duratIon of scleroderma at cancer dIagnosIs dIffered sIgnIfIcantly between groups (−1.2 years In the antI–RNA Polymerase I/III group, +13.4 years In the antI–topoIsomerase I group, +11.1 years In the antIcentromere group, and +2.3 years In the group that was negatIve for all antIgens tested) (P = 0.027). RNA Polymerase III demonstrated a robust nucleolar staInIng pattern In 4 of 5 avaIlable tumors from patIents wIth antIbodIes to RNA Polymerase I/III. In contrast, nucleolar RNA Polymerase III staInIng was not detected In any of 4 examIned tumors from the RNA Polymerase antIbody–negatIve group (P = 0.048). ConclusIon Our fIndIngs IndIcate that there Is a close temporal relatIonshIp between the onset of cancer and scleroderma In patIents wIth antIbodIes to RNA Polymerase I/III, whIch Is dIstInct from scleroderma patIents wIth other autoantIbody specIfIcItIes. In thIs study, autoantIbody response and tumor antIgen expressIon are assocIated. We propose that malIgnancy may InItIate the scleroderma-specIfIc Immune response and drIve dIsease In a subset of scleroderma patIents.
