Transfection Analysis of Expression of mRNA Isoforms Encoding the Nuclear Autoantigen La/SS-B

Abstract

Transcription of the gene encoding for the nuclear autoantigen La resulted in La mRNA isoforms. A promoter switching combined with an alternative splicing pathway replaced the exon 1 with the exon 1′. The exon 1′ contained GC-rich regions and an oligo(U) tail of 23 uridine residues. Moreover, it encoded for three open reading frames upstream of the La protein reading frame. Despite this unusual structure, when exon 1′ La mRNAs were expressed in transfected cells, both exon 1 and 1′ La mRNAs were translated to La protein, whereas the upstream open reading frames of the exon 1′ were not translated. In addition to full-length exon 1′ La mRNAs 5′-shortened exon 1′ La mRNAs were detected. The exon 1′ 5′-starts varied in dependence on the analyzed tissues. Like the full-length exon 1′ La mRNA a 5′-shortened exon 1′ construct starting downstream of the oligo(U) tail but upstream of the open reading frames 2 and 3 was also well translated when transfected in mouse cells. Thus all La mRNA forms represent functional La mRNAs. Transcription of the gene encoding for the nuclear autoantigen La resulted in La mRNA isoforms. A promoter switching combined with an alternative splicing pathway replaced the exon 1 with the exon 1′. The exon 1′ contained GC-rich regions and an oligo(U) tail of 23 uridine residues. Moreover, it encoded for three open reading frames upstream of the La protein reading frame. Despite this unusual structure, when exon 1′ La mRNAs were expressed in transfected cells, both exon 1 and 1′ La mRNAs were translated to La protein, whereas the upstream open reading frames of the exon 1′ were not translated. In addition to full-length exon 1′ La mRNAs 5′-shortened exon 1′ La mRNAs were detected. The exon 1′ 5′-starts varied in dependence on the analyzed tissues. Like the full-length exon 1′ La mRNA a 5′-shortened exon 1′ construct starting downstream of the oligo(U) tail but upstream of the open reading frames 2 and 3 was also well translated when transfected in mouse cells. Thus all La mRNA forms represent functional La mRNAs. One of the target antigens of sera suffered from autoimmune patients with rheumatoid diseases such as systemic lupus erythematosus or primary Sjögren's syndrome (pSS) 1The abbreviations used are: pSS, primary Sjögren's syndrome; PBL, peripheral blood lymphocyte; ORF, open reading frame; DMEM, Dulbecco's modified Eagle's medium; mAb, monoclonal antibody; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; UAP, universal amplification primer; FCS, fetal calf serum; PBS, phosphate-buffered saline; cLSM, confocal laser scanning microscopy; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase. is the nuclear autoantigen La (SS-B) (1Tan E.M. Adv. Immunol. 1989; 44: 93-151Google Scholar). The La protein was described to associate at least transiently with all primary RNA polymerase III transcripts including precursor molecules of ribosomal 5 S RNA, tRNAs, and some 4.5 S RNAs, as well as a portion of the uridine-rich small nuclear RNAs U1 and U6 (2Hendrick J.P. Wolin S. Rinke J. Lerner M. Steitz J. Mol. Cell. Biol. 1981; 12: 1138-1149Google Scholar, 3Madore S.J. Wieben E.D. Pederson T. J. Biol. Chem. 1984; 259: 1929-1933Google Scholar, 4Rinke J. Steitz J.A. Nucleic Acids Res. 1985; 13: 2617-2629Google Scholar, 5Stefano J.E. Cell. 1984; 36: 145-154Google Scholar). Common to all primary RNA polymerase III transcripts is their 3′-terminal oligo(U) tail, which is transcribed during the transcription termination step. These oligo(U) tails were shown to be a binding site for the La protein (6Pruijn G.J.M. Slobbe R.L. van Venrooij W.J. Mol. Biol. Rep. 1990; 14: 43-48Google Scholar). In addition to the oligouridylated RNA polymerase III transcripts, an association of the La protein with some nonoligouridylated RNAs has been reported especially for some viral RNAs including the leader RNAs of the vesicular stomatitis virus and rabies virus (7Wilusz J. Kurilla M.G. Keene J.D. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5827-5831Google Scholar, 8Kurilla M.G. Cabradilla C.D. Holloway B.P. Keene J.D. J. Virol. 1984; 50: 773-778Google Scholar). The La protein is assumed to be involved in transcription termination of RNA polymerase III and internal initiation of translation of at least the poliovirus mRNA (9Gottlieb E. Steitz J.A. EMBO J. 1989; 8: 841-850Google Scholar, 10Gottlieb E. Steitz J.A. EMBO J. 1989; 8: 851-861Google Scholar, 11Meerovitch K. Svitkin Y.V. Lee H.S. Leibkowicz F. Kenan D. Chan E.K.L. Agol V.I. Keene J.D. Sonenberg N.J. J. Virol. 1993; 67: 3798-3807Google Scholar, 12Bachmann M. Pfeifer K. Schröder H.C. Müller W.E.G. Cell. 1990; 60: 85-93Google Scholar). Most recently five La cDNAs were isolated when a cDNA library made from peripheral blood lymphocytes (PBLs) of a patient with pSS was screened with her own anti-La serum (13Tröster H. Metzger T.E. Semsei I. Schwemmle M. Winterpacht A. Zabel B. Bachmann M. J. Exp. Med. 1994; 180: 2059-2067Google Scholar). In two of these five La cDNAs the exon 1 was replaced with an alternative 5′-end. Genomic analysis revealed that these La cDNAs represented alternatively spliced transcripts of the La gene. An additional promoter site was identified in the intron between exons 1 and 2, which served as initiation site for transcription of the alternative exon 1′. The exon 1′ La mRNA form had an unusual 5′ terminus. It contained GC-rich regions and an oligo(U) tail of 23 uridine residues and encoded for three upstream open reading frames (ORF1 to 3). The ORF1 encoded for a putative peptide of 5.4 kDa. It was interrupted from the La protein reading frame by two stop codons. The ORF2 and ORF3 were not in the reading frame of the La protein. Qualitative and quantitative analysis of expression of the exon 1 and exon 1′ La mRNAs showed that both La mRNA forms represented finally processed abundant cytoplasmic mRNAs. Exon 1 to exon 1′ La mRNAs were expressed at ratios between 1:1 and 1:5 (15Hilker M. Tröster H. Grölz D. Hake U. Bachmann M. Cell Tissue Res. 1996; 284: 383-389Google Scholar, 16Bachmann M. Hilker M. Grölz D. Tellmann G. Hake U. Kater L. deWilde P. Tröster H. J. Autoimmunity. 1996; 9: 757-766Google Scholar). Due to the unusual structure of the exon 1′ La mRNA it still remained unclear whether the exon 1′ La mRNA is a translatable mRNA and if so, which of the reading frames is used for translation. This was of special interest because one of the exon 1′ La cDNAs contained a frameshift mutation in a recently detected hot spot region in the exon 7 of the La gene (14Bachmann M. Bartsch H. Tröster H. Grölz D. J. Autoimmunity. 1996; 9: 747-756Google Scholar). The frameshift mutation caused a premature stop codon in the La protein reading frame. Thus the mutant exon 1′ La mRNA could encode for a C-terminally truncated mutant La peptide of 29 kDa. Here we present evidence that exon 1′ La mRNAs can be translated to La protein, whereas the upstream ORFs are not used for translation. The following materials were obtained:BstEII, EcoO109, EcoRI,KpnI, pGEX-2T, and T7-sequencing kit from Pharmacia Biotech Inc. (Freiburg, Germany); SalI, NheI, andNcoI from MBI Fermentas (St. Leon-Rot, Germany); QIAprep-spin kit and QIAEX were from Qiagen (Hilden, Germany); DMEM, Opti-MEM medium, LipofectAMINE, and Taq polymerase from Life Technologies, Inc. (Eggenstein, Germany); BglII,Pfu polymerase, pBluescript SK(−) from Stratagene GmbH (Heidelberg, Germany); CDP-Star Tropix, pCI-neo, pCI, and pGEM-T vector systems from Promega (Serva, Heidelberg, Germany); shrimp alkaline phosphatase, BstXI, HindIII, Taqbuffer (10 ×), DNA molecular from Germany); and from Germany); with in with serum and with alkaline phosphatase, and from the from Germany); and from Germany); and from U. S. The anti-La which was to be to the of La protein G.J.M. W.J. J. was described by J. Immunol. 1985; Scholar). The was by of The anti-La serum we used the serum of the patient M. Pfeifer K. Schröder H.C. Müller W.E.G. Cell. 1990; 60: 85-93Google Scholar). was a The in 1 Taq contained 2 of and 1 of was by for 3 to of at at and 1 at the was for at and to The were analyzed by gel were by were or pGEM-T (13Tröster H. Metzger T.E. Semsei I. Schwemmle M. Winterpacht A. Zabel B. Bachmann M. J. Exp. Med. 1994; 180: 2059-2067Google Scholar). DNA was the QIAprep-spin kit and by to a of 1 in (10 1 DNA was as described (13Tröster H. Metzger T.E. Semsei I. Schwemmle M. Winterpacht A. Zabel B. Bachmann M. J. Exp. Med. 1994; 180: 2059-2067Google Scholar). an analysis of the of the La mRNAs we the amplification of cDNA by Life Technologies, Inc. It a cDNA and a amplification step. transcription of were to RNA The RNA were isolated from of the fetal of the patient with pSS, and of a and mouse cells, which were or transfected with the La gene or La exon 1 La cDNA Kenan D. Keene J.D. J. Biol. Chem. Scholar). the of the patient not the an abundant expression of the not The RNA was isolated as described (13Tröster H. Metzger T.E. Semsei I. Schwemmle M. Winterpacht A. Zabel B. Bachmann M. J. Exp. Med. 1994; 180: 2059-2067Google Scholar). The and the following were to the of the the the the exon was used This was with and in The amplification was the of the as upstream and the in the exon 3 as downstream The cDNA and the amplification is in The cDNAs were The of the downstream exon 2 in with the exon 1 upstream or the exon 1′ upstream also 2 the cDNAs were a of the universal amplification of the with the exon 1′ downstream or the exon 1 downstream as in and 3 exon 1 and 1′ were in the two and to the following from the La cDNA in pBluescript The La in at the with an tail and represented a 5′-shortened exon 1′ La mRNA in a an exon 1′ full-length La cDNA had to be In an exon 1 full-length La cDNA was at the 5′ with this tail was to 5 residues to the 5′-shortened exon 1′ we that contained a frameshift mutation the region M. Bartsch H. Tröster H. Grölz D. J. Autoimmunity. 1996; 9: 747-756Google also ORF1 all were of in a the reading frame was replaced with the reading which was from the La cDNA the of the 1′ and 1 were and by the In the of the exon 1′ was DNA of a as The was from the Kenan D. Keene J.D. J. Biol. Chem. which was a by J. D. Keene of DNA with resulted in a and a The was isolated and pBluescript This contained the exons 1 and 2 the intron between exons 1 and 2 including the exon 1′. was as upstream the site is and as downstream The exon 1′ at the of exon 1′ (13Tröster H. Metzger T.E. Semsei I. Schwemmle M. Winterpacht A. Zabel B. Bachmann M. J. Exp. Med. 1994; 180: 2059-2067Google and downstream of an which downstream of the in the La The exon 1′ were with and and the of this was with and the isolated DNA was with Despite a of had to be because the of had an of residues in the tail from to residues. a was isolated with the of 23 residues. The 1 was as we used the La cDNA which was described by Chan E.K.L. E.M. Nucleic Acids Res. 1989; and which was a of E. K. L. Chan La exon 1 served the the site is The downstream in the exon downstream of the site in the La The exon 1 were with and and the of this was with and the isolated DNA was with The exon 1 and 1′ La were isolated from the pBluescript SK(−) by with and and and pCI, which were with The exon 1′ construct in was as ORF1 also In the of the 5′-shortened exon 1′ a was as upstream the site is in with as downstream and as The was with and and in the of the reading frame of the exon 1 and 1′ was as cDNA the La was with which in the exon of the La and which in the exon 5 of the La The exon 1 and 1′ and were with and of the DNA with of was in the of the exon 1 and 1′ or The were The and were in in a and and were in in a in for of or on for The mouse were transfected transiently to the following was in were to of in of to the was and the were for with 2 of and In of DNA was in of Opti-MEM and combined with of and of of the 2 of DMEM, of and were to the cells, and the DNA an 1 of and was the of the was replaced by 2 of and the were for of or with for In we that of the the expression of the was with the In addition to transiently transfected cells, transfected were These were by K. of the of J. and T. The were transfected with the La gene or the exon 1 La cDNA Kenan D. Keene J.D. J. Biol. Chem. Scholar). The transfected were in the of the were with at for 1 of with the anti-La was by the cells, which had been for 5 with PBS, with of the anti-La for at least The were with and the anti-La was detected in The for the was The at and the were by with 5 The were laser scanning was a The were of the of the was with the of transiently transfected were by with of from or transfected were by of hot were by The were for 5 to and at for 5 at were with and used for and was as described Scholar, P. J. Biol. Chem. Scholar). and the were with of the anti-La were the the anti-La were and the was processed a the anti-La or the The were detected or with alkaline and as the a protein of and a portion of ORF1 was this an exon 1′ (6Pruijn G.J.M. Slobbe R.L. van Venrooij W.J. Mol. Biol. Rep. 1990; 14: 43-48Google was by the upstream the site is A a mutation in the and the downstream the site is pGEM-T and with site of the of the serum and to for or to an expression of the ORF1 were In a the exon 1′ was in the vector the translation initiation site of the ORF1 in the exon 1′ is not to M. J. Cell Biol. a construct was in which the translation initiation site was to the This construct was ORF1 the ORF1 was downstream of a truncated exon This was as ORF1 also The ORF1 in the vector was as The vector the full-length exon 1′ La cDNA was the DNA was and The contained the 5′ leader of the exon 1′ La mRNA the ORF1 at and at including the It at in the exon 1′. The ORF1 construct was as The full-length exon 1′ La cDNA served as for as upstream the the site is the initiation site to is in The the site is served the downstream The was in isolated by and and the of The ORF1 construct contained the portion of the which was also in the The ORF1 construct was as The exon 1′ full-length construct in was with and The La mutant encoded for of La protein. The ORF1 was from the ORF1 in pGEM-T as upstream and as downstream the site is The was and and in the site of the truncated La In a T. P. J. J. Autoimmunity. 1993; a mouse transfected with the La gene. This expressed La protein in addition to the mouse La protein. shown in 1 a between the La protein and the mouse La protein in this mouse was by two including 1 and 1 shown in A transfected with the La gene were with the anti-La whereas were not 1 and the anti-La with a protein to a molecular of from the of the transfected with the La gene The was also by the anti-La 1 The anti-La not with the of the 1 In the with a protein to a molecular of in the of both the transfected 1 and the 1 3). These the following the and the mouse La protein can be by the protein with a molecular of represented the La protein, the protein with a molecular of represented the mouse La protein, the anti-La with both the and the mouse La protein, and the anti-La with the but not with the mouse La protein. of mouse with a exon 1′ La construct the if the exon 1′ La mRNA is a translatable Moreover, a mouse transfected with an exon 1′ La construct but also the transfected with the La gene be to for translation of the upstream ORFs of the exon 1′ La One to the transfected with the La gene for such a was that the exon 1′ La mRNA was made and expressed to cells. in a we analyzed if the mouse the expression of the exon 1′ La mRNA from the La gene. this were as in The the amplification were a of the exon 2 downstream with an exon 1 2, or exon 1′ upstream or of the upstream in with an exon 1′ downstream 2, and also shown in 2 both the exon 1′ and exon 1 La mRNA forms could be detected in the mouse transfected with the La whereas were not in the 2, 3). The exon 1′ La mRNAs were also not in a transfected with the exon 1 La cDNA 2, exon whereas the exon 1 La mRNA was in this 2, exon when the exon 1 and 1′ were and the could be as exon 1 or exon 1′ during of the of the exon 1′ La mRNA a of 5′-shortened exon 1′ La cDNAs were The of the of the exon 1′ La mRNA was made on the 1′ La cDNA by the when the for mRNAs isolated from the transfected with the La gene were on a gel 2, were were and the were this exon 1 and exon 1′ were in from mRNA to the that is in the amplification 2, the were as in 3 The amplification was the in with an exon 1 or an exon 1′ downstream The mRNA used for the were from including and of a patient with pSS A and and a A and fetal and shown in 3 the exon 1 a when on an The from the mRNAs of the were and and a of were exon 1 the of the exon 1 La mRNA as in was between the mRNA In when the exon 1′ were on an gel 3 to at least five were Moreover, the exon 1′ between the and the for the 3 and and the fetal 3 regions of the gel as by the in were The were and of were The of these were in and The exon 1′ La cDNAs upstream of the ORF1 at the of the exon 1′ the downstream of the ORF2 but upstream of the ORF3 or downstream of the ORF3 In these the and we had to for not of the exon 1′ full-length La mRNA but also of the 5′-shortened exon 1′ La mRNAs. In a full-length exon full-length exon and a 5′-shortened exon 1′ construct starting downstream of the oligo(U) tail but upstream of the ORF2 were and mouse were transiently The transfected were analyzed for expression of La protein by and shown in the anti-La the nuclear which was also for the transfected with the La gene 1 on the transiently transfected with the full-length exon full-length exon or the 5′-shortened exon 1′ La was on transfected with a a La The of the three It in the of the 5′-shortened exon 1′ by the exon 1 and the exon 1′ full-length were when of the construct the construct was In all the construct a protein expression the analysis of transiently transfected mouse cells. An of mouse transfected with a construct or a construct of the exon 1 the exon 1′ or the exon 1′ was analyzed with the anti-La 2, The with were by shown in 5 the patient anti-La with the mouse La protein in transiently transfected with the construct a La The mouse La protein was also detected in all the from the transiently transfected In addition to the mouse La protein, the anti-La also detected the La protein in the mouse transfected with full-length exon 1 full-length exon 1′ and the 5′-shortened exon 1′ was between the molecular of La protein translated from the exon 1 or the exon 1′ La mRNAs. the and the we that all La mRNA forms can be translated to the La protein. The of the of the full-length exon 1′ La mRNA the of the upstream ORFs be to La protein when translated from an exon 1′ La we analyzed whether the ORF1 of the exon 1′ is translated. this a protein was and used for of a of the serum to GST, the with the protein but still with the protein not was detected in of including or and mouse transfected with the La gene or full-length exon 1′ La mRNA not the of the ORF1 serum and to for a of the ORF1 exon 1′ La were as in The ORF1 construct represented a full-length exon 1′ La cDNA in but contained a frameshift mutation in the exon The frameshift mutation caused a premature stop Thus the mutant exon 1′ La mRNA encoded a C-terminally truncated La peptide of 29 (14Bachmann M. Bartsch H. Tröster H. Grölz D. J. Autoimmunity. 1996; 9: 747-756Google Scholar). the by the anti-La is upstream of the frameshift it still with the truncated La protein The La region contained a site upstream of the premature stop This site was used to a ORF1 to the of the La Thus the construct ORF1 contained a ORF1 upstream of but interrupted by two stop from the La region and a ORF1 to the of the truncated La shown in the ORF1 La with the was for the ORF1 construct of whether the construct was transfected or mouse was in the and The were from the the was processed a the anti-La shown in and the anti-La with the La protein in all In the of the transfected the anti-La detected of the truncated La peptide the ORF1 and or the peptide the of the peptide was the of the truncated La peptide the These that the ORF1 to the of the La peptide was made and by the In the ORF1 encoded from the exon 1′ La mRNA but upstream of the La protein reading frame was not made at an or was This was by the with two ORF1 One construct contained the exon 1′ In the of the construct we to the of a translation of the ORF1 reading frame by of the translation initiation site of the ORF1 in both ORF1 translation was detected when these were transfected or mouse not the that were by for all the ORF1 were by and In of these was an ORF1 peptide detected not In these the following the ORF1 of an exon 1′ La mRNA is not translated to a ORF1 peptide or a ORF1 La protein and the La protein reading frame in an exon 1′ La mRNA is translated. sera of patients with pSS or systemic lupus erythematosus to nuclear One of the of is the nuclear autoantigen (1Tan E.M. Adv. Immunol. 1989; 44: 93-151Google Scholar). The La protein was described as a protein Kenan D. Keene J.D. J. Biol. Chem. Scholar). It was to be involved in of RNA polymerase III and in internal initiation of translation especially of mRNAs (9Gottlieb E. Steitz J.A. EMBO J. 1989; 8: 841-850Google Scholar, 10Gottlieb E. Steitz J.A. EMBO J. 1989; 8: 851-861Google Scholar, 11Meerovitch K. Svitkin Y.V. Lee H.S. Leibkowicz F. Kenan D. Chan E.K.L. Agol V.I. Keene J.D. Sonenberg N.J. J. Virol. 1993; 67: 3798-3807Google Scholar, 12Bachmann M. Pfeifer K. Schröder H.C. Müller W.E.G. Cell. 1990; 60: 85-93Google Scholar). In a we identified alternative La mRNAs (13Tröster H. Metzger T.E. Semsei I. Schwemmle M. Winterpacht A. Zabel B. Bachmann M. J. Exp. Med. 1994; 180: 2059-2067Google Scholar). La mRNA forms were as abundant finally processed cytoplasmic mRNAs the to the unusual 5′ of the exon 1′ La mRNA still remained The exon 1′ was contained an oligo(U) tail of 23 uridine and encoded for three ORFs upstream of the La protein reading frame. mRNAs with GC-rich upstream ORFs had also been described in the of and also in the of and involved in and M. J. Cell Biol. Scholar). In of these it was that the alternative mRNA forms a It was assumed that the mRNA are expressed in to the protein of or represent which are alternatively expressed of the functional gene in were the upstream ORFs used for translation. on this we which if of the reading frames encoded by the exon 1′ La mRNA can be translated protein. to the upstream ORF1 to ORF3 it that the ORF1 could be translated to a whereas the ORF2 and ORF3 reading frames are and not in the La protein frame. we for expression of a peptide from the ORF1 or the La protein reading frame of the exon 1′ La for an expression of the ORF1 or the La protein reading frame a and had to be that a of the La protein and an ORF1 peptide when these we to whether the mouse was to the La gene to a it that the of the exon 1′ La mRNAs had also been when mRNA that were from a of tissues. The not be the of a RNA because the exon 1 La mRNAs analyzed in from the RNA not and the 5′-starts of exon 1′ La mRNAs were in which contained the during of the mRNAs. It is also that the regions of the exon 1′ La cDNAs are the of premature during to in the exon 1′ La mRNAs because the of a mRNA could be not exon 1′ La mRNAs the ORF1 could if with the full-length exon 1′ La we a 5′-shortened exon 1′ La mRNA in expression The 5′-shortened exon 1′ La mRNA form the two exon 1′ La cDNAs isolated from the cDNA library at the oligo(U) tail in the exon 1′. The two and showed that the La protein reading frame in full-length exon 1 and exon 1′ and the 5′-shortened exon 1′ La mRNAs can be translated in transiently transfected cells, whereas the ORF1 reading was not The translation of the full-length exon 1′ to be if with the exon 1 whereas the 5′-shortened exon 1′ La mRNA to be the still two upstream ORFs 2 and gene be to the of the La mRNA