mRNA mutations of type I protein kinase A regulatory subunit {alpha} in T lymphocytes of a subject with systemic lupus erythematosus

Dama Laxminarayana and Gary M. Kammer

Section on Rheumatology and Clinical Immunology, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA

Correspondence to: G. M. Kammer (E-mail: gmkammer{at}wfubmc.edu) or D. Laxminarayana (E-mail: dlaxmina{at}wfubmc.edu)


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by multiple T lymphocyte immune effector dysfunctions. Protein kinase A (PKA) isozymes contribute to the regulation of T cell immune effector functions. In SLE T cells, there is a profound deficiency of PKA-I isozyme activity characterized by both reduced RI{alpha} transcript and RI{alpha} protein levels. To identify a molecular mechanism(s) for this isozyme deficiency, we utilized single-strand conformation polymorphism (SSCP) analysis to detect structural changes in the cDNA. Of 10 SLE subjects, cDNAs from a single subject revealed a shifted band. Sequence analyses demonstrated that a shifted SSCP band from SLE T cells carried heterogeneous transcript mutations, including deletions, transitions and transversions. Most of these transcript mutations are clustered adjacent to GAGAG motifs and CT repeats—regions that are susceptible to transcript editing and/or molecular misreading. By contrast, no genomic mutations were identified. These results suggest the occurrence of mRNA editing and/or defective function of RNA polymerase in a subject with SLE. Mutant RI{alpha} transcripts are pathophysiolgically significant, for they can encode diverse, aberrant RI{alpha} isoforms, including truncated, dominant-negative subunits, resulting in deficient PKA-I activity. We propose that deficient PKA-I isozyme activity contributes to the pathogenesis of SLE by hindering effective signal transduction and impairing T cell effector functions.

Keywords: autoimmunity, immune dysfunction, mutations in gene transcripts


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is an idiopathic autoimmune disorder characterized by cellular immune dysfunctions (1). T lymphocytes exhibit enhanced helper function, deficient cytotoxic/suppressor activities, and an imbalance in cytokine production during both active and inactive disease. These diverse T cell dysfunctions suggest the existence of a disorder primary to the T cell in SLE (1).

The adenylyl cyclase/cAMP/protein kinase A (AC/cAMP/PKA) pathway is an integral signal transduction system in T lymphocytes (2). In tissues, the regulatory subunits of PKA are the principal intracellular receptors for cAMP (3). PKA is comprised of two isozymes, PKA-I and PKA-II (3), which are activated by the binding of cAMP to type I regulatory (RI) and type II regulatory (RII) subunits (4,5). Upon PKA activation, the released catalytic (C) subunits can phosphorylate membrane, cytosolic and/or nuclear substrates (68). Importantly, absence of the RI{alpha} subunit is a lethal event in an animal model (9).

This laboratory identified the first defect of signal transduction in SLE T lymphocytes (10). This signaling disorder is characterized by impaired PKA-catalyzed protein phosphorylation due to deficient PKA-I isozyme phosphotransferase activity (1113). Recent analysis has revealed that there is diminished content of both RI{alpha} transcripts and RI{alpha} protein (14). Because PKA isozymes phosphorylate multiple intracellular substrates, including plasma membrane proteins (8), intracellular receptors (15) and transcription factors (1618), the potential pathophysiologic significance of deficient PKA-I activity in SLE T cells may be impaired phosphorylation of these substrates. Under-phosphorylation of proteins may hinder their capacity to function efficiently in cellular homeostasis and, ultimately, may impede programmed, physiologic T cell immune effector functions. These events may culminate in the observed T cell dysfunctions in SLE (1).

To understand the molecular mechanism(s) underlying this functional impairment of PKA-I isozyme activity in SLE T lymphocytes, we asked whether there is a structural alteration(s) in the coding region of the RI{alpha} subunit. Here we show that T cells from a subject with severe SLE possessed heterogeneous transcript mutations, including deletions, transitions and transversions. Most of these transcript mutations are clustered adjacent to GAGAG motifs and CT repeats, regions that are susceptible to transcript editing and/or molecular misreading (19). This is the first identification of mutant transcripts of a gene in SLE. These results suggest the occurrence of mRNA editing and/or defective function of RNA polymerase in RI{alpha} transcripts that are predicted to produce mutated RI{alpha} proteins. The co-existence of mutant RI{alpha} and wild-type RI{alpha} proteins may result in random formation of heterodimers between mutant RI{alpha} monomers and/or wild-type RI{alpha} monomers (20). Because PKA-I holoenzymes may be composed of tetramers containing both mutant and wild-type RI{alpha} monomers, cAMP-inducible catalysis may be significantly compromised (20) and contribute to the observed PKA-I deficiency in SLE T cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Patient and control populations
The diagnosis of SLE, recruitment of SLE subjects and controls, gauging of SLE disease activity, and procurement of T cell specimens for analysis have been previously detailed (1214). SLE and healthy control subjects were age, gender and racially matched. SLE subjects had not been treated with an immunosuppressive agent for >1 year. To confirm the presence of mRNA mutations in a single SLE subject, three additional blood samples taken at different times were used to synthesize cDNAs.

T lymphocyte isolation and phenotypic characterization
T cells were purified from peripheral blood mononuclear cells as previously described (13,14). Fresh, quiescent T cells, which had not been exposed to any activators or inhibitors, were utilized herein.

Isolation of DNA, RNA and cDNA synthesis
Total cellular DNA and RNA was extracted from 10x106 T cells of normal and SLE patients as described (14). Single-stranded cDNA was synthesized from 1–2 µg of total RNA by using random primer (Pharmacia, Piscataway, NJ), M-MLV H reverse transcriptase, according to the manufacturer's instructions (Gibco/BRL, Gaithersburg, MD).

Oligonucleotide primers for PCR amplification
Overlapping oligonucleotide primer sets for the RI{alpha} coding region and exon-specific primers for genomic DNA sequence were designed based on published sequences (22) using the Oligomer version 5.0 program (National Biosciences, Plymouth, MN) and synthesized by National Biosciences. The upstream and downstream primers of each overlapping segment of cDNA and specific exons of genomic DNA are shown in Tables 1 and 2GoGo.


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Table 1. Synthesized overlapping oligonucleotide primers used for PCR amplification of PKA RI{alpha} subunit coding region from cDNA.
 

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Table 2. Synthesized oligonucleotide primers used for PCR amplification of PKA RI{alpha} subunit exons from genomic DNA
 
Amplification of PKA RI{alpha} cDNA and genomic DNA from normal and SLE T cells
Each reaction mixture consisted of 10% of a single-stranded cDNA reaction or 100 ng of genomic DNA, 25 pmol of each primer (listed in Tables 1 and 2GoGo), 1xPCR buffer [10 mM Tris–HCl (pH 8.3), 50 mM KCl], 2.5 mM MgCl2, 200 µM of each dNTP, 1.25 U Taq polymerase (Perkin-Elmer Cetus, Emeryville; CA), specific primer sets and templates, and double-distilled H2O to a final volume of 50 µl. Overlapping primer pairs from Table 1Go for cDNA and exon-specific primer pairs described in Table 2Go for genomic DNA amplifications were selectively used in the PCR reactions to amplify each overlapping segment of cDNA and each specific exon of genomic DNA. The reaction mixture was subjected to 30 cycles of denaturation (94°C, 1 min), primer annealing (temperature dependent on primer pair; cf. Table 1 and 2GoGo) for 2 min and extension for 3 min at 72°C plus 2 s added for each cycle utilizing a DNA thermal cycler (Perkin-Elmer Cetus). Then 5 µl of reaction mixture was analyzed on a 2% agarose gel in Tris–HCl/acetate/EDTA (TAE) buffer; 1 µg of HaeIII-digested {Phi}X174 DNA (Gibco/BRL) was utilized as mol. wt markers: 1353, 1078, 872, 603, 310, 281, 234,194, 118 and 72 bp. PCR products were purified using the Wizard Plus purification system (Promega, Madison, WI). Specific cDNA and genomic DNA amplifications were confirmed by sequencing of PCR products by an automatic DNA sequencer (ABI Prism 377; Applied Biosystems, Foster City. CA)

Single-strand conformation polymorphism (SSCP) analysis, cloning and sequencing of PKA RI{alpha} cDNA
SSCP is based on altered electrophoretic mobility of mutated DNA/cDNA single strands upon electrophoresis in a neutral polyacrylamide gel, and is extensively used to detect point mutations, insertions and deletions in genomic DNA or cDNA (23,24). Therefore, overlapping oligonucleotide primer sets to amplify RI{alpha} cDNA products have been designed to cover the entire coding region and portions of 5' and 3' untranslated regions using Oligomer version 5.0 (National Biosciences) that were synthesized by National Biosciences. The cDNA samples from control and SLE groups were amplified using overlapping primer sets and PCR, as mentioned earlier. PCR-amplified products were extracted with chloroform and 100 ng of amplified DNA in 10 µl volume was denatured at 45°C for 5 min by adding 1 µl of denaturation solution (0.5 M NaOH/10 mM EDTA). After denaturation, samples were cooled on ice. Then 1 µl of 90% formamide containing 0.05% bromophenol blue and 0.5% xylene cyanol was added to the samples and electrophoresed on a non-denaturing 10% polyacrylamide gel containing 5% glycerol. Electrophoresis was carried out in 0.5% TBE at 4°C and gels were stained with ethidium bromide at a concentration of 100 ng/ml of 0.5% TBE for 20 min. Gels were photographed under UV-transillumination using 667 Polaroid film.

The cDNA strands from SSCP bands with normal and differential mobilities were excised, re-amplified, subcloned into TA cloning vectors (pCR2.1) (Invitrogen, Carlsbad, CA) and sequenced using an automated DNA sequencer (ABI Prism 377). This method of SSCP analysis followed by cloning and sequencing of SSCP bands with differential mobilities has an advantage over conventional cloning and sequencing of mRNA transcripts, and helped in isolating the mutant transcripts and minimizing the cloning and sequencing of large numbers of transcripts to characterize editing-induced changes.

Cloning and sequencing of PKA RI{alpha} genomic DNA
Genomic DNA samples from T cells of control and a single SLE patient were amplified using exon-specific primer sets (see Table 2Go), as described earlier. Amplified products of exons 3, 4 and 6 were sequenced directly from purified PCR products. Because exon 5 is only 58 bp in length, we subcloned the PCR products of this exon into TA cloning vectors (pCR2.1-TOPO) (Invitrogen) and sequenced using an automated DNA sequencer (ABI Prism 377).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
SSCP analysis of PKA RI{alpha} subunit cDNAs from normal and SLE T cells
To assess the entire coding region of PKA RI{alpha} to identify a potential polymorphism(s) or mutation(s), cDNAs from T cells of 10 SLE patients and 10 age-, gender- and racially-matched normal individuals were analyzed by SSCP. RNA was extracted from SLE and normal control T cells, and reverse transcribed into cDNA. To amplify RI{alpha} cDNA products, six pairs of overlapping oligonucleotide primer sets were designed to cover the entire coding region and portions of the 5' and 3' untranslated regions (Table 1Go). Primer sets were designed by emphasizing (i) the size of the product suitable for SSCP analysis, (ii) the priming specificity to avoid non-specific amplification and (iii) the amplification efficiency to produce enough product for SSCP analysis. The coding regions of the PKA RI{alpha} gene from control and SLE groups were amplified using these overlapping primer sets and PCR, and subjected to SSCP analysis as detailed in Methods.

Using SSCP, the PCR products amplified by oligonucleotide pairs 1, 2 and 4–6 (Table 1Go) covering the coding region of RI{alpha} from 10 to 228, 140 to 346, 558 to 821, 778 to 1101 and 1054 to 1386 nucleotides respectively yielded variable combinations of stronger and lighter single-stranded cDNA bands (Fig. 1aGo, lanes 2, 4, 6, 8, 10 and 12). In these segments, neither additional abnormal bands nor band shifts in mobility were observed in either controls or SLE patients. However, the third oligonucleotide pair, covering the region from 285 to 679 nucleotides (Fig. 1aGo, lane 6), revealed a prominent band shift in mobility in a single patient (Fig. 1bGo, lane 2). This band shift in mobility was verified using cDNAs obtained from three separate bleedings from this SLE subject.



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Fig. 1. SSCP analysis of PKA RI{alpha} cDNA obtained from normal T lymphocyte samples. (a) Lanes 1, 3, 5, 7, 9 and 11 are mol. wt markers; lanes 2, 4, 6, 8, 10 and 12 represent SSCP analyses of cDNA segments of PKA RI{alpha} coding region amplified using overlapping primer pair numbers 1–6 respectively from Table 1Go. During SSCP analysis PCR products are denatured as described in the methods section and form single strands. These single-stranded cDNA strands fold and form secondary structures which are unique to their primary sequence, and move at different mobilities on non-denaturing polyacrylamide gel and result in two or more single-stranded cDNA bands. Non-denatured double-stranded cDNA will move with same mobility on non-denaturing polyacrylamide gel and result in a single band. Arrows ({Rightarrow},{Rightarrow}) represent double- and single-stranded cDNA respectively. (b) SSCP analysis of PKA RI{alpha} cDNA segment (position 285–679 bp) corresponds to lane 6 of (a). Lane 1 is a mol. wt marker. Lane 2 is a SLE and lane 3 is a normal T cell sample. A band shift is observed in the SLE sample in lane 2 ({blacktriangleup}) (b, lane 2). Lane 4 is a mol. wt marker. The shifted band from lane 2 of (b) was excised, amplified and analyzed by SSCP in lane 5 to confirm the altered mobility of the shifted band ({blacktriangleup}). Lane 6 represents a SSCP analysis of a normal sample used as the control for normal mobility of SSCP bands.

 
During the course of this work, we tested multiple primer pairs for overlapping segments for the entire coding region. One primer pair used to amplify the segment from 283 to 602 nucleotides, 5'-GGAGGCAAAACAGATTCA-3' and 5'-ATCCCCTTCATCACCTTG -3', amplified another product of 359 bp length in eight of 10 SLE samples and in six of 10 controls along with the 337 bp RI{alpha} segment (Fig. 2Go). To identify this 359 bp band, it was separated on a non-denaturing polyacrylamide gel, excised from the gel, re-amplified and sequenced. Our results indicated that this 359 bp band is a transcript of a PKA RI{alpha} pseudogene. The human PKA RI{alpha} pseudogene is 89% homologous at the nucleotide level to the open reading frame of the human RI{alpha} gene (25). Importantly, SSCP, cloning and sequencing analyses revealed that none of the primer sets shown in Table 1Go amplified pseudogene cDNA products.



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Fig. 2. PCR amplification and SSCP analysis of PKA RI{alpha} and the PKA RI{alpha} pseudogene. The PKA RI{alpha} (position: 283 to 602 bp) and RI{alpha} pseudogene (position 401–742 bp) were amplified from cDNAs using the following primer set: 5'-GGAGGCAAAACAGATTCA-3' and 5'-ATCCCCTTCATCACCTTG-3'. Lanes 1 and 4 are mol. wt markers; lanes 2 and 5 are PCR amplifications of PKA RI{alpha} and RI{alpha} pseudogene cDNAs obtained from normal and SLE T lymphocyte samples, respectively. Arrows ({blacktriangleup}, {triangleup}) point to double-stranded cDNAs of PKA RI{alpha} and RI{alpha} pseudogene respectively. Lanes 3 and 6 represent SSCP analyses of PCR-amplified cDNA segments of PKA RI{alpha} and RI{alpha} pseudogene corresponding to lanes 2 and 5 respectively. Arrows ({Rightarrow},{Rightarrow}) represent single-stranded cDNA of PKA RI{alpha} and RI{alpha} pseudogene respectively.

 
Transcript mutations observed in SLE RI{alpha} coding region
The third overlapping segment corresponding to positions 285–679 nucleotides of the PKA RI{alpha} coding region encodes amino acids 60–195. This stretch of amino acids is encoded in exons 3–6 of genomic DNA, and contains three GAGAG motifs and one CT repeat. The transcript from this region encodes a pseudosubstrate site and part of the cAMP-binding A domain (21). Because the pseudosubstrate site is a proteolytically sensitive hinge region to which the C subunit attaches (26), this region is functionally important in PKA-I isozyme catalysis. Thus, the identification of a band shift in mobility in this region raised the possibility of a polymorphism(s) or a mutation(s) that could be functionally significant.

To identify any polymorphisms or mutations, we excised the shifted single-stranded cDNA band (Fig. 1bGo, lane 2 denoted by arrowhead) and the corresponding normal single-stranded cDNA band (Fig. 1bGo, lane 3, upper band) from control samples, and re-amplified, subcloned and sequenced the products. Because PCR and cloning are well known to induce artifacts that may produce false mutations, we calculated the frequency of these artifacts in single-stranded cDNA strands from controls with normal mobilities to be 1.2x10–4/bp. By contrast, sequence analyses of the shifted single-stranded cDNA band revealed that 66% of the single-stranded cDNA strands carried discrete transcript mutations, including deletions, transitions and transversions (Fig. 3Go). These mutations (i) are heterogeneous in nature, (ii) reveal multiple mutations in some clones and (iii) demonstrate no clonal relationship among most of the clones carrying mutations. The frequency of these transcript mutations was 2.6x10–3/bp, a 22-fold increase over control cells (Table 3Go). A -> G transitions were more frequently observed compared with other mutations (Table 4Go). The remaining mutations were T -> A, C -> A and G -> T transversions, G -> A, T -> C and C -> T transitions, and base deletions. Multiple abnormalities were observed in some clones compared with other clones (Table 4Go, clones 7, 9 and 24). Deletions in homopolymeric runs were observed in 2/20 clones with mutations (Table 4Go, clones 7 and 9, and Fig. 3dGo).



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Fig. 3. PKA RI{alpha} cDNA sequence analysis. The shifted band from lane 2 of Fig. 1Go(b) was excised, amplified, subcloned into TA cloning vectors (pCR2.1) and sequenced using an automated DNA sequencer (ABI Prism 377). Panels (a), (c), (e), (g) and (i) represent normal sequence. Panel (b) represents a deletion of three bases (AAT). The three base deletion (AAT) corresponds to the code of amino acid 142 (asn). Panel (d) represents a T deletion. This T deletion results in a frame shift mutation and a stop codon at amino acid 171. Panel (f) represents an adenosine (A) (•) to guanosine (G) (arrow) transition. The A to G substitution results in a change of amino acid 183 from Tyr (ATG) to Val (GTG). All these transcript mutations are clustered adjacent to GAGAG motifs (cf. b, d and f). Panel (h) shows cytosine (C) (•) to adenosine (A) (arrow) transversion, which results in a change of amino acid 85 from Pro (CCC) to His (CAC). Panel (j) demonstrates a thymidine (T) (•) to cytosine (C) (arrow) transition and a change in amino acid 172 from Phe (TTC) to Ser (TCC). The transcript mutations in (h) and (j) are in the CT-rich region.

 

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Table 3. Sequence analysis of SSCP bands of PKA RI{alpha} subunit transcripts from normal and SLE T lymphocytes
 

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Table 4. Transcript mutations in PKA RI{alpha} subunit of lupus T lymphocytes
 
Comparisons of the sequence analyses from the third segment of controls and the affected SLE subject with that of the RI{alpha} pseudogene revealed information pertinent to the interpretation of our results. First, the primer set used to amplify this segment did not amplify RI{alpha} pseudogene products. Second, none of these transcript mutations matched the sequence variations of the pseudogene. Third, these mutant transcripts have normal splice junctions and exclude the possibility of abnormal splicing. Therefore, these results exclude the possibility that the identified transcript mutations in the SLE RI{alpha} cDNA were derived from the pseudogene. Lastly, we sequenced the RI{alpha} genomic sequence from the affected SLE subject's DNA corresponding to this cDNA region and identified no such mutations. The heterogeneous nature of the transcript mutations in the SLE cDNA and lack of such alterations in genomic DNA and pseudogene provides credible evidence for co- or post-transcriptional modification of mRNA. Repeated sequencing of new cDNAs obtained at different times from the affected SLE subject revealed some identical transcript mutations, thus verifying their presence.

Most of these transcript mutations are clustered near GAGAG motifs and CT repeats, which are susceptible to transcript editing and/or molecular misreading (19). Compelling evidence for this can be observed in cDNA clones obtained from the affected SLE subject at different times. For example, clones 3 and 17 have an A -> G transition in codon 142. Additionally, clone 3 carries a G -> A transition in codon 168. Finally, clone 29 carries a codon 142 (AAT) deletion (Table 4Go). Taken together, these results suggest that these three clones are products of different mRNA transcripts carrying identical and/or different mutations in the same codon located adjacent to a GAGAG motif.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Eighty percent of subjects with SLE harbor a deficiency of PKA-I isozyme phosphotransferase activity in T cells (27). Although a reduction in the amounts of RI{alpha} transcript and protein contribute to this isozyme deficiency (14), the molecular mechanism(s) underlying altered RI{alpha} expression have not yet been identified. T cell effector functions, such as helper and suppressor/cytotoxic activities, are well known to be profoundly impaired in SLE, resulting in defective cellular immunity (28). Because deficient PKA-I activity results in under-phosphorylation of its substrates (1113) and is likely to impede effective signal transduction in SLE T cells, effector T cell dysfunctions, such as cytotoxicity, may be a direct outcome of aberrant signaling. One mechanism that may account for the observed RI{alpha} disorder is a genomic polymorphism(s) or mutation(s). To determine the presence of a polymorphism, we initially utilized SSCP to walk along the coding region of the RI{alpha} cDNA to detect nucleotide changes. Of 10 SLE subjects, cDNAs from a single subject revealed a shifted band in a segment containing nucleotides 285–679 that encode amino acids 60–195.

Subcloning and sequencing of 30 clones unexpectedly revealed heterogeneous transcript mutations, including deletions, transitions and transversions (Table 4Go). In particular, one T deletion mutation results in a frame shift and predicts a protein truncation at amino acid 171. Also of note is that some clones had multiple, co-existing mutations and that most of the clones carrying mutations demonstrated no clonal relationship. Moreover, the frequency of these transcript mutations was 2.6x10–3/bp, a 22-fold increase over control cells. Having excluded contamination from transcripts of the RI{alpha} pseudogene (25), we sequenced the region of genomic DNA that encodes amino acids 60–195 of the RI{alpha} gene. We identified no mutations of the genomic DNA.

We observed that most of these transcript mutations are clustered near GAGAG motifs and CT repeats, which are susceptible to transcript editing and/or molecular misreading (19). Taken together with the heterogeneity of the mutations, these data suggest the occurrence of mRNA editing and/or molecular misreading by defective RNA polymerase as possible mechanisms that produce RI{alpha} transcript mutations in SLE T cells.

Two mechanisms that have been proposed to account for transcript misreading and the resultant transcript mutations are defective RNA polymerase and/or mRNA editing. It is now well-established that RNA editing yields co- or post-transcriptional modifications of RNA that result in the insertion, deletion or substitution of nucleotides (29). However, the precise mechanisms responsible for molecular misreading by defective RNA polymerase have not yet been identified.

Although mRNA editing is a pathological event in SLE T cells that yields mutant RI{alpha} transcripts, current evidence suggests that this editing process can also operate as a physiologic process. By modifying mRNA sequences, RNA editing can correct, extend or diversify information encoded within the corresponding genomic sequence (2933). Several fundamental mechanisms leading to mRNA editing have been identified, including (i) guide RNA-directed cleavage of pre-mRNA and subsequent deletion or insertion of uridylate (U) residues; (ii) dsRNA adenosine deaminase (dsRAD)-mediated conversion of adenosine to inosine; and (iii) cytosine deaminase-mediated conversion of cytosine to uridine. mRNA editing appears to be a common mechanism in certain lower species, including viruses, Leishmania, Acanthamoeba, Drosophila and plants (29,34). In addition to these lower species, transcript editing has also been identified in mammals. For example, spontaneous dsRAD-mediated mRNA editing has been documented in several organs of rats (35). The frequency ranged between 1/17,000 and 1/150,000 bp, brain having the highest frequency and muscle the lowest frequency. Of especial interest is the demonstration of selective transcript editing in human organs, such as brain and intestine. It has been recently observed that dsRAD-mediated adenosine deamination of transcripts leads to editing of glutamate receptors (gluR) (36,37), 5-HT2c serotonin receptors (38) and {alpha}2,6-sialyltransferase (39). These editing events are responsible for functional changes in ion channels formed from the altered gluR subunits (36,37); a 10- to 15-fold reduction in the efficiency of the interaction between the 5-HT2c serotonin receptor and G proteins (38); and formation of two isoforms of the {alpha}2,6-sialyltransferase catalytic domain, creating proteins with differing catalytic activities (39). Moreover, selective cytidine deamination of apolipoprotein B mRNA in human small intestine produces a novel truncated peptide with altered functions (40).

However, the role of transcript mutations in disease pathogenesis is just beginning to be explored and is, therefore, not well understood. Selective dinucleotide deletions near GAGAG motifs in transcripts encoding the ß-amyloid precursor and ubiquitin-B proteins have been recently implicated in the pathogenesis of both Alzheimer's disease and Down's syndrome (19). More recently, heterogeneous transcript mutations have also been identified in cDNA clones of the BCL10 gene in mucosa-associated lymphoid tissue (MALT) lymphomas (4143). These events may be occurring at a low frequency. In a single MALT tumor, sampling of cells from different blocks revealed mutant transcripts from cells in only one of five samples. Thus, even in a single tumor specimen, there is considerable variability of mutant transcript expression (41). In the T cells of this SLE subject, the frequency was estimated to be 2.6x10–3/bp. Future efforts will focus on determining the frequency of T cell RI{alpha} transcript mutations in a larger cohort of SLE subjects as well as the editing mechanism(s) by which these mutations arise.

RI{alpha} transcript mutations were found in the T cells of only one of 10 SLE subjects. Of significance is that this SLE subject was entering a flare of her disease at the time blood samples were obtained; the SLE disease activity index (SLEDAI) (44) was 33, indicating very active disease (27), and the patient ultimately died. The other nine SLE subjects had varying degrees of active disease, having SLEDAI values ranging from 8 (mildly active disease) to 23 (very active disease). T cell PKA-I-specific activities were determined on several occasions during the course of her illness to be consistently below 230 pmol/min/mg protein, values <15% of the mean specific activity of normal controls (13,27). Although low PKA-I activity is unassociated with the extent of disease activity (27), we do not know yet whether the occurrence of the observed heterogeneous transcript mutations is directly related to the severity of SLE. Because this subject had not yet been treated with corticosteroids or immunosuppressive agents, the mutations cannot be attributed to therapy.

The transcript mutations identified herein predict the presence of multiple mutant RI{alpha} proteins, including truncated forms. Although these mutant RI{alpha} proteins are not derived from genomic mutations, the presence of both wild-type and mutant RI{alpha} proteins in SLE T cells is reminiscent of heterozygous murine S49 T lymphoma cells that express both wild-type and mutant RI{alpha} proteins (45). The co-existence of mutant and wild-type RI{alpha} proteins would be expected to result in the random formation of heterodimers between mutant and wild-type RI{alpha} monomers, mutant RI{alpha} homodimers, and wild-type RI{alpha} homodimers. Because mutant RI{alpha} hetero- or homodimers preferentially bind C subunits and the resultant holoenzymes are relatively resistant to activation by cAMP (20,46), this could account in part for the increase in the apparent constant (Ka) for PKA-I activation and reduction in maximal velocity (Vmax) of the isozyme that we previously demonstrated (13). Thus, mutant RI{alpha} monomers may be an etiopathogenetic mechanism contributing to profound PKA-I deficiency in SLE T cells.

In conclusion, this is the first report of an association between the occurrence of T cell transcript mutations and SLE. Based on our identification of heterogeneous RI{alpha} transcript mutations that usually cluster adjacent to GAGAG motifs and CT repeats, we propose mRNA editing and/or molecular misreading by defective RNA polymerase as possible mechanisms that produce these mutations in SLE T cells. Further, we propose that these transcript base deletions and substitutions observed in SLE T cell RI{alpha} mRNA may contribute to deficient PKA-I isozyme activity in these cells. Such mRNA mutations are expected to produce amino acid deletions, changes or frame shift mutations in the A domain of the cAMP-binding site. These structural alterations of RI{alpha} transcripts may be pathophysiolgically significant, because mutant RI{alpha} transcripts could produce truncated, dominant-negative RI{alpha} subunits (47). Moreover, such mutations could yield a heterogeneous group of mutant RI{alpha} monomers that form heterodimers with wild-type RI{alpha} monomers and exhibit altered cAMP binding, relative resistance to cAMP activation and diminished cAMP activatable kinase activities. This is supported by our previous identification of markedly altered PKA-I kinetics in SLE T cells (13). Finally, these transcript mutations may impact on SLE pathogenesis by impeding effective signaling from the cell surface to the nucleus, potentially altering gene regulation. Such a mechanism would then take on wider importance and significance in the etiopathogenesis of SLE.


    Acknowledgments
 
We thank A. Herbert for a critical reading of the manuscript. This work was partially supported by grants from the National Institutes of Health (RO1-AR39501 to G. M. K.) (RO3-AR46385 to D. L), the Lupus Foundation of America (to D. L. and G. M. K.) and the General Clinical Research Center of the Wake Forest University School of Medicine (MO1 RR07122).


    Abbreviations
 
AC adenylyl cyclase
C catalytic
dsRAD dsRNA adenosine deaminase
gluR glutamate receptor
MALT mucosa-associated lymphoid tissue
PKA protein kinase A
RI type I regulatory
RII type II regulatory
SLE systemic lupus erythematosus
SLEDAI SLE disease activity index
SSCP single-strand conformation polymorphism

    Notes
 
Transmitting editor: M. Feldmann

Received 3 March 2000, accepted 13 July 2000.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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