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1.  Ebola Virus RNA Editing Depends on the Primary Editing Site Sequence and an Upstream Secondary Structure 
PLoS Pathogens  2013;9(10):e1003677.
Ebolavirus (EBOV), the causative agent of a severe hemorrhagic fever and a biosafety level 4 pathogen, increases its genome coding capacity by producing multiple transcripts encoding for structural and nonstructural glycoproteins from a single gene. This is achieved through RNA editing, during which non-template adenosine residues are incorporated into the EBOV mRNAs at an editing site encoding for 7 adenosine residues. However, the mechanism of EBOV RNA editing is currently not understood. In this study, we report for the first time that minigenomes containing the glycoprotein gene editing site can undergo RNA editing, thereby eliminating the requirement for a biosafety level 4 laboratory to study EBOV RNA editing. Using a newly developed dual-reporter minigenome, we have characterized the mechanism of EBOV RNA editing, and have identified cis-acting sequences that are required for editing, located between 9 nt upstream and 9 nt downstream of the editing site. Moreover, we show that a secondary structure in the upstream cis-acting sequence plays an important role in RNA editing. EBOV RNA editing is glycoprotein gene-specific, as a stretch encoding for 7 adenosine residues located in the viral polymerase gene did not serve as an editing site, most likely due to an absence of the necessary cis-acting sequences. Finally, the EBOV protein VP30 was identified as a trans-acting factor for RNA editing, constituting a novel function for this protein. Overall, our results provide novel insights into the RNA editing mechanism of EBOV, further understanding of which might result in novel intervention strategies against this viral pathogen.
Author Summary
Ebola virus (EBOV) causes severe hemorrhagic fever with case fatality rates of up to 90% and no therapy or vaccine currently available. A better understanding of the EBOV life cycle is important to develop new countermeasures against this virus; however, research with live EBOV is restricted to high containment laboratories. One unique feature of the EBOV life cycle is that its surface glycoprotein is expressed only after editing of the glycoprotein mRNA by the viral polymerase, leading to an insertion of a non-templated nucleotide into the mRNA. While this phenomenon has been long known, the mechanism of mRNA editing for EBOV is not understood. We have developed a unique minigenome system that allows the study of EBOV mRNA editing outside of a high containment laboratory. Using this system we have characterized EBOV mRNA editing and defined the sequence requirements for this process. Interestingly, we could show that signals both up- and downstream of the editing site are important, and that a secondary structure in the RNA upstream of the editing site as well as the viral protein VP30 contribute to editing. These findings provide new detailed molecular information about an essential process in the EBOV life cycle, which might be a potential novel target for antivirals.
doi:10.1371/journal.ppat.1003677
PMCID: PMC3798607  PMID: 24146620
2.  Hypoxia-inducible C-to-U coding RNA editing downregulates SDHB in monocytes 
PeerJ  2013;1:e152.
Background. RNA editing is a post-transcriptional regulatory mechanism that can alter the coding sequences of certain genes in response to physiological demands. We previously identified C-to-U RNA editing (C136U, R46X) which inactivates a small fraction of succinate dehydrogenase (SDH; mitochondrial complex II) subunit B gene (SDHB) mRNAs in normal steady-state peripheral blood mononuclear cells (PBMCs). SDH is a heterotetrameric tumor suppressor complex which when mutated causes paraganglioma tumors that are characterized by constitutive activation of hypoxia inducible pathways. Here, we studied regulation, extent and cell type origin of SDHB RNA editing.
Methods. We used short-term cultured PBMCs obtained from random healthy platelet donors, performed monocyte enrichment by cold aggregation, employed a novel allele-specific quantitative PCR method, flow cytometry, immunologic cell separation, gene expression microarray, database analysis and high-throughput RNA sequencing.
Results. While the editing rate is low in uncultured monocyte-enriched PBMCs (average rate 2.0%, range 0.4%–6.3%, n = 42), it is markedly upregulated upon exposure to 1% oxygen tension (average rate 18.2%, range 2.8%–49.4%, n = 14) and during normoxic macrophage differentiation in the presence of serum (average rate 10.1%, range 2.7%–18.8%, n = 17). The normoxic induction of SDHB RNA editing was associated with the development of dense adherent aggregates of monocytes in culture. CD14-positive monocyte isolation increased the percentages of C136U transcripts by 1.25-fold in normoxic cultures (n = 5) and 1.68-fold in hypoxic cultures (n = 4). CD14-negative lymphocytes showed no evidence of SDHB editing. The SDHB genomic DNA remained wild-type during increased RNA editing. Microarray analysis showed expression changes in wound healing and immune response pathway genes as the editing rate increased in normoxic cultures. High-throughput sequencing of SDHB and SDHD transcripts confirmed the induction of C136U RNA editing in normoxic cultures but showed no additional verifiable coding edits. Analysis of SDHB RNA sequence data from 16 normal human tissues from the Illumina Body Map and from 45 samples representing 23 different cell types from the ENCODE projects confirmed the occurrence of site-specific C136U editing in whole blood (1.7%) and two primary CD14+ monocyte samples (1.9% and 2.6%). In contrast, the other cell types showed an average of 0.2% and 0.1% C136U editing rates in the two databases, respectively.
Conclusions. These findings demonstrate that C-to-U coding RNA editing of certain genes is dynamically induced by physiologically relevant environmental factors and suggest that epigenetic downregulation of SDHB by site-specific RNA editing plays a role in hypoxia adaptation in monocytes.
doi:10.7717/peerj.152
PMCID: PMC3775634  PMID: 24058882
Epigenetic; Environment; RNA editing; Cytidine deaminase; Monocyte; Macrophage; Mitochondrion; Hypoxia; Complex II
3.  Mutations in Complement Regulatory Proteins Predispose to Preeclampsia: A Genetic Analysis of the PROMISSE Cohort 
PLoS Medicine  2011;8(3):e1001013.
Jane Salmon and colleagues studied 250 pregnant patients with SLE and/or antiphospholipid antibodies and found an association of risk variants in complement regulatory proteins in patients who developed preeclampsia, as well as in preeclampsia patients lacking autoimmune disease.
Background
Pregnancy in women with systemic lupus erythematosus (SLE) or antiphospholipid antibodies (APL Ab)—autoimmune conditions characterized by complement-mediated injury—is associated with increased risk of preeclampsia and miscarriage. Our previous studies in mice indicate that complement activation targeted to the placenta drives angiogenic imbalance and placental insufficiency.
Methods and Findings
We use PROMISSE, a prospective study of 250 pregnant patients with SLE and/or APL Ab, to test the hypothesis in humans that impaired capacity to limit complement activation predisposes to preeclampsia. We sequenced genes encoding three complement regulatory proteins—membrane cofactor protein (MCP), complement factor I (CFI), and complement factor H (CFH)—in 40 patients who had preeclampsia and found heterozygous mutations in seven (18%). Five of these patients had risk variants in MCP or CFI that were previously identified in atypical hemolytic uremic syndrome, a disease characterized by endothelial damage. One had a novel mutation in MCP that impairs regulation of C4b. These findings constitute, to our knowledge, the first genetic defects associated with preeclampsia in SLE and/or APL Ab. We confirmed the association of hypomorphic variants of MCP and CFI in a cohort of non-autoimmune preeclampsia patients in which five of 59 were heterozygous for mutations.
Conclusion
The presence of risk variants in complement regulatory proteins in patients with SLE and/or APL Ab who develop preeclampsia, as well as in preeclampsia patients lacking autoimmune disease, links complement activation to disease pathogenesis and suggests new targets for treatment of this important public health problem.
Study Registration
ClinicalTrials.gov NCT00198068
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Most pregnancies culminate in the birth of a healthy baby but, sadly, about a quarter of women lose their babies during pregnancy. A common pregnancy-related medical problem that threatens the life of both baby and mother is preeclampsia. Mild and severe preeclampsia affects up to 10% and 1%–2% of pregnancies, respectively. Preeclampsia occurs because of a problem with the function of the placenta, the organ that transfers nutrients and oxygen from mother to baby and removes waste products from the baby. Although preeclampsia begins early in pregnancy, it is diagnosed by the onset of high blood pressure (hypertension) and the appearance of protein in the urine (proteinuria) after 20 weeks of pregnancy. Other warning signs include headaches and swelling of the hands and face. The only cure for preeclampsia is delivery, and labor is usually induced early to prevent eclampsia (seizures), stroke, liver and kidney failure, and breathing and blood vessel problems developing in the mother. Although delivery before 37 weeks of pregnancy is not generally recommended, in cases of preeclampsia it may be too dangerous for both the baby and the mother to allow the pregnancy to continue. Unfortunately when severe preeclampsia occurs in the second trimester, babies weighing only 500 grams may be delivered and they may not survive.
Why Was This Study Done?
Because the exact cause of preeclampsia is unknown, it is difficult to develop treatments for the condition or to find ways to prevent it. Many experts think that immune system problems—in particular, perturbations in complement activation—may be involved in preeclampsia. The complement system is a set of blood proteins that attacks invading bacteria and viruses. The activation of complement proteins is usually tightly regulated (overactivation of the complement system causes tissue damage) and, because preeclampsia may run in families, one hypothesis is that mutations (genetic changes) in complement regulatory proteins might predispose women to preeclampsia. In this study, the researchers test this hypothesis by sequencing genes encoding complement regulatory proteins in pregnant women with the autoimmune diseases systemic lupus erythematosus (SLE) and/or antiphospholipid antibodies (APL Ab) who developed preeclampsia. In autoimmune diseases, the immune system attacks healthy human cells instead of harmful invaders. Both SLE and APL Ab are characterized by complement-mediated tissue injury and are associated with an increased risk of preeclampsia and miscarriage.
What Did the Researchers Do and Find?
Two hundred fifty women with SLE and/or APL Ab were enrolled into the PROMISSE study (a multi-center observational study to identify predictors of pregnancy outcome in women with SLE and/or APL Ab) when they were 12 weeks pregnant and followed through pregnancy. Thirty patients developed preeclampsia during the study and ten more had had preeclampsia during a previous pregnancy. The researchers sequenced the genes for complement regulatory proteins: membrane cofactor protein (MCP), factor I, and factor H in these 40 patients. Seven women (18%) had mutations in one copy of one of these genes (there are two copies of most genes in human cells). Five mutations were alterations in MCP or factor I that are gene variants that increase the risk of hemolytic uremic syndrome, a disease characterized by blood vessel damage. The sixth mutation was a new MCP mutation that impaired MCP's ability to regulate complement component C4b. The final mutation was a factor H mutation that did not have any obvious functional effect. No mutations in complement regulatory proteins were found in 34 matched participants in PROMISSE without preeclampsia but, among a group of non-autoimmune women who developed preeclampsia during pregnancy, 10% had mutations in MCP or factor I.
What Do These Findings Mean?
These findings identify MCP and factor I mutations as genetic defects associated with preeclampsia in pregnant women with SLE and/or APL Ab. Importantly, they also reveal an association between similar mutations and preeclampsia in women without any underlying autoimmune disease. Taken together with evidence from previous animal experiments, these findings suggest that dysregulation of complement activation is involved in the development of preeclampsia. Although further studies are needed to confirm and extend these findings, these results suggest that proteins involved in the regulation of complement activation could be new targets for the treatment of preeclampsia and raise the possibility that tests could be developed to identify women at risk of developing preeclampsia.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001013.
Tommy's, a UK charity that funds scientific research into the causes and prevention of miscarriage, premature birth, and stillbirth, has information on preeclampsia
The March of Dimes Foundation, a nonprofit organization for pregnancy and baby health, has information on preeclampsia
The UK National Health Services Choices website also has information about preeclampsia
Wikipedia has pages on the complement system, on autoimmune disease, and on preeclampsia (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
More information on the PROMISSE study is available
doi:10.1371/journal.pmed.1001013
PMCID: PMC3062534  PMID: 21445332
4.  Comprehensive High-Resolution Analysis of the Role of an Arabidopsis Gene Family in RNA Editing 
PLoS Genetics  2013;9(6):e1003584.
In flowering plants, mitochondrial and chloroplast mRNAs are edited by C-to-U base modification. In plant organelles, RNA editing appears to be generally a correcting mechanism that restores the proper function of the encoded product. Members of the Arabidopsis RNA editing-Interacting Protein (RIP) family have been recently shown to be essential components of the plant editing machinery. We report the use of a strand- and transcript-specific RNA-seq method (STS-PCRseq) to explore the effect of mutation or silencing of every RIP gene on plant organelle editing. We confirm RIP1 to be a major editing factor that controls the editing extent of 75% of the mitochondrial sites and 20% of the plastid C targets of editing. The quantitative nature of RNA sequencing allows the precise determination of overlapping effects of RIP factors on RNA editing. Over 85% of the sites under the influence of RIP3 and RIP8, two moderately important mitochondrial factors, are also controlled by RIP1. Previously uncharacterized RIP family members were found to have only a slight effect on RNA editing. The preferential location of editing sites controlled by RIP7 on some transcripts suggests an RNA metabolism function for this factor other than editing. In addition to a complete characterization of the RIP factors for their effect on RNA editing, our study highlights the potential of RNA-seq for studying plant organelle editing. Unlike previous attempts to use RNA-seq to analyze RNA editing extent, our methodology focuses on sequencing of organelle cDNAs corresponding to known transcripts. As a result, the depth of coverage of each editing site reaches unprecedented values, assuring a reliable measurement of editing extent and the detection of numerous new sites. This strategy can be applied to the study of RNA editing in any organism.
Author Summary
RNA editing is a co- or post-transcriptional RNA processing reaction that changes the nucleotide sequence of the RNA substrate. In flowering plants, mRNA editing is confined to organelle transcripts, altering cytidine to uridine. Recently, some members of a small Arabidopsis gene family were found to be important for editing of chloroplast and mitochondrial transcripts. Several methods have been developed to measure the amount of edited transcripts at specific Cs, but most of these methods either lack sensitivity or are unable to determine the number and location of edited Cs in a particular transcript. While sensitive assays have been previously developed, they are costly and labor-intensive precluding their use on a large-scale. In order to characterize the role of an entire gene family in RNA editing, we have successfully adapted RNA sequencing technology to characterize the effect of mutation and silencing of family members on organelle RNA editing. Our method to measure editing extent is sensitive, reliable, and cost-effective. As well as detecting additional family members that play a role in RNA editing, we have detected numerous new editing sites. Our strategy should benefit the investigation of RNA editing in any organism.
doi:10.1371/journal.pgen.1003584
PMCID: PMC3688494  PMID: 23818871
5.  Characterization of a germline Vk gene encoding cationic anti-DNA antibody and role of receptor editing for development of the autoantibody in patients with systemic lupus erythematosus. 
Journal of Clinical Investigation  1996;98(8):1843-1850.
We found previously that cationic anti-DNA autoantibodies (autoAbs) have nephritogenic potential and usage of a specific germline Vk gene, A30, has major influences on cationic charge of the autoAb in human lupus nephritis. In the present study, we have characterized A30 germline Vk gene using cosmid cloning technique in patients with SLE. A30 gene locus locates in less than 250 kb from the Ck region, and the cationic anti-DNA mRNA used the upstream Jk2 gene, indicating that cationic anti-DNA mRNA is a product of primary gene rearrangement. By using PCR technique, we found that A30 gene locus in the genome was defective in eight out of nine SLE patients without nephritis. In contrast, all nine patients with lupus nephritis had intact A30 gene. The presence and absence of A30 gene was associated with the development of lupus nephritis or not (P < 0.01, by Fisher's exact test, two-sided). It was thus suggested that absence of functional A30 gene may rescue from developing lupus nephritis in the patients. A30 is reported to be a potentially functional but rarely expressed Vk gene in humans. It is possible that normal B cells edit primarily rearranged A30 gene with autoreactive potentials by receptor editing mechanism for changing the affinity of the B cell Ag receptor to avoid self-reactivity, whereas SLE B cells may have a defect in this mechanism. Indeed, we found that normal B cells edit A30-Jk2 gene in their genome possibly by inversion mechanism, whereas SLE B cells contain rearranged A30-Jk2-Ck gene in the genome and express A30-associated mRNA, suggesting that receptor editing mechanism is also defective in patients with SLE. Our study suggests that polymorphism of Ig Vk locus, and failure of receptor editing may contribute to the development of pathogenic anti-DNA responses in humans.
PMCID: PMC507624  PMID: 8878436
6.  Identification of Widespread Ultra-Edited Human RNAs 
PLoS Genetics  2011;7(10):e1002317.
Adenosine-to-inosine modification of RNA molecules (A-to-I RNA editing) is an important mechanism that increases transciptome diversity. It occurs when a genomically encoded adenosine (A) is converted to an inosine (I) by ADAR proteins. Sequencing reactions read inosine as guanosine (G); therefore, current methods to detect A-to-I editing sites align RNA sequences to their corresponding DNA regions and identify A-to-G mismatches. However, such methods perform poorly on RNAs that underwent extensive editing (“ultra”-editing), as the large number of mismatches obscures the genomic origin of these RNAs. Therefore, only a few anecdotal ultra-edited RNAs have been discovered so far. Here we introduce and apply a novel computational method to identify ultra-edited RNAs. We detected 760 ESTs containing 15,646 editing sites (more than 20 sites per EST, on average), of which 13,668 are novel. Ultra-edited RNAs exhibit the known sequence motif of ADARs and tend to localize in sense strand Alu elements. Compared to sites of mild editing, ultra-editing occurs primarily in Alu-rich regions, where potential base pairing with neighboring, inverted Alus creates particularly long double-stranded RNA structures. Ultra-editing sites are underrepresented in old Alu subfamilies, tend to be non-conserved, and avoid exons, suggesting that ultra-editing is usually deleterious. A possible biological function of ultra-editing could be mediated by non-canonical splicing and cleavage of the RNA near the editing sites.
Author Summary
The traditional view of mRNA as a pure intermediate between DNA and protein has changed in the last decades since the discovery of numerous RNA processing pathways. A frequent RNA modification is A-to-I editing, or the conversion of adenosine (A) to inosine (I). Since inosine is read as a guanosine (G), A-to-I editing leads to changes in the RNA sequence that can alter the function of its encoded protein. In recent years, tens of thousands of human A-to-I editing sites were discovered by computationally comparing RNA sequences to the human genome and searching for A-to-G mismatches. However, previous screens usually ignored RNA sequences that were edited to extreme, because the large number of A-to-G mismatches carried by these RNAs obscured their genomic origin. We developed a new computational framework to detect extreme A-to-I editing, or ultra-editing, based on masking potential editing sites before the alignment to the genome. Our method detected about 14,000 editing sites, with each edited molecule affected, on average, in more than 20 nucleotides. We demonstrated that the likely reason for the ultra-editing of those sequences is their potential to fold back into a particularly long double-stranded structure, which is the preferred target of the editing enzymes.
doi:10.1371/journal.pgen.1002317
PMCID: PMC3197674  PMID: 22028664
7.  Apolipoprotein B RNA sequence 3' of the mooring sequence and cellular sources of auxiliary factors determine the location and extent of promiscuous editing. 
Nucleic Acids Research  1998;26(7):1644-1652.
Apolipoprotein B (apoB) RNA editing involves a cytidine to uridine transition at nucleotide 6666 (C6666) 5' of an essential cis -acting 11 nucleotide motif known as the mooring sequence. APOBEC-1 (apoB editing catalytic sub-unit 1) serves as the site-specific cytidine deaminase in the context of a multiprotein assembly, the editosome. Experimental over-expression of APOBEC-1 resulted in an increased proportion of apoB mRNAs edited at C6666, as well as editing of sites that would otherwise not be recognized (promiscuous editing). In the rat hepatoma McArdle cell line, these sites occurred predominantly 5' of the mooring sequence on either rat or human apoB mRNA expressed from transfected cDNA. In comparison, over-expression of APOBEC-1 in HepG2 (HepG2-APOBEC) human hepatoma cells, induced promiscuous editing primarily 5' of the mooring sequence, but sites 3' of the C6666 were also used more efficiently. The capacity for promiscuous editing was common to rat, rabbit and human sources of APOBEC-1. The data suggested that differences in the distribution of promiscuous editing sites and in the efficiency of their utilization may reflect cell-type-specific differences in auxiliary proteins. Deletion of the mooring sequence abolished editing at the wild type site and markedly reduced, but did not eliminate, promiscuous editing. In contrast, deletion of a pair of tandem UGAU motifs 3' of the mooring sequence in human apoB mRNA selectively reduced promiscuous editing, leaving the efficiency of editing at the wild type site essentially unaffected. ApoB RNA constructs and naturally occurring mRNAs such as NAT-1 (novel APOBEC-1 target-1) that lack this downstream element were not promiscuously edited in McArdle or HepG2 cells. These findings underscore the importance of RNA sequences and the cellular context of auxiliary factors in regulating editing site utilization.
PMCID: PMC147457  PMID: 9512534
8.  Induction of RNA editing at heterologous sites by sequences in apolipoprotein B mRNA. 
Molecular and Cellular Biology  1993;13(12):7288-7294.
An RNA editing mechanism modifies apolipoprotein B (apo-B) mRNA in the intestine by converting cytosine at nucleotide (nt) 6666 to uracil. To define the sequence requirements for editing, mutant apo-B RNAs were analyzed for the ability to be edited in vitro by enterocyte extracts. Editing was detected by a sensitive and linear primer extension assay. An upstream region (nt 6648 to 6661) which affected the efficiency of editing was identified. RNAs with mutations in this efficiency sequence were edited at 22 to 160% of wild-type levels. Point mutations in a downstream 11-nt mooring sequence (nt 6671 to 6681) abolished editing, confirming previous studies (R. R. Shah, T. J. Knott, J. E. Legros, N. Navaratnam, J. C. Greeve, and J. Scott, J. Biol. Chem. 266:16301-16304, 1991). The optimal distance between the editing site and the mooring sequence is 5 nt, but a C positioned 8 nt upstream is edited even when nt 6666 contains U. The efficiency and mooring sequences were inserted individually and together adjacent to a heterologous C in apo-B mRNA. The mooring sequence alone induced editing of the C at nt 6597 both in vitro and in transfected rat hepatoma cells. Editing at nt 6597 was specific, was independent of editing at nt 6666, and was stimulated to wild-type levels when the efficiency sequence was also inserted. Introduction of the mooring sequence into a heterologous mRNA, luciferase mRNA, induced editing of an upstream cytidine. Although UV cross-linking studies have previously shown that proteins of 60 to 66 kDa cross-link to apo-B mRNA, these proteins did not cross-link to the luciferase translocation mutants.
Images
PMCID: PMC364799  PMID: 8246950
9.  The Zinc-Fingers of KREPA3 Are Essential for the Complete Editing of Mitochondrial mRNAs in Trypanosoma brucei 
PLoS ONE  2010;5(1):e8913.
Most mitochondrial mRNAs in trypanosomes undergo uridine insertion/deletion editing that is catalyzed by ∼20S editosomes. The editosome component KREPA3 is essential for editosome structural integrity and its two zinc finger (ZF) motifs are essential for editing in vivo but not in vitro. KREPA3 function was further explored by examining the consequence of mutation of its N- and C- terminal ZFs (ZF1 and ZF2, respectively). Exclusively expressed myc-tagged KREPA3 with ZF2 mutation resulted in lower KREPA3 abundance and a relative increase in KREPA2 and KREL1 proteins. Detailed analysis of edited RNA products revealed the accumulation of partially edited mRNAs with less insertion editing compared to the partially edited mRNAs found in the cells with wild type KREPA3 expression. Mutation of ZF1 in TAP-tagged KREPA3 also resulted in accumulation of partially edited mRNAs that were shorter and only edited in the 3′-terminal editing region. Mutation of both ZFs essentially eliminated partially edited mRNA. The mutations did not affect gRNA abundance. These data indicate that both ZFs are essential for the progression of editing and perhaps its accuracy, which suggests that KREPA3 plays roles in the editing process via its ZFs interaction with editosome proteins and/or RNA substrates.
doi:10.1371/journal.pone.0008913
PMCID: PMC2811742  PMID: 20111718
10.  Molecular Cloning of Apobec-1 Complementation Factor, a Novel RNA-Binding Protein Involved in the Editing of Apolipoprotein B mRNA 
Molecular and Cellular Biology  2000;20(5):1846-1854.
The C-to-U editing of apolipoprotein B (apo-B) mRNA is catalyzed by a multiprotein complex that recognizes an 11-nucleotide mooring sequence downstream of the editing site. The catalytic subunit of the editing enzyme, apobec-1, has cytidine deaminase activity but requires additional unidentified proteins to edit apo-B mRNA. We purified a 65-kDa protein that functionally complements apobec-1 and obtained peptide sequence information which was used in molecular cloning experiments. The apobec-1 complementation factor (ACF) cDNA encodes a novel 64.3-kDa protein that contains three nonidentical RNA recognition motifs. ACF and apobec-1 comprise the minimal protein requirements for apo-B mRNA editing in vitro. By UV cross-linking and immunoprecipitation, we show that ACF binds to apo-B mRNA in vitro and in vivo. Cross-linking of ACF is not competed by RNAs with mutations in the mooring sequence. Coimmunoprecipitation experiments identified an ACF-apobec-1 complex in transfected cells. Immunodepletion of ACF from rat liver extracts abolished editing activity. The immunoprecipitated complexes contained a functional holoenzyme. Our results support a model of the editing enzyme in which ACF binds to the mooring sequence in apo-B mRNA and docks apobec-1 to deaminate its target cytidine. The fact that ACF is widely expressed in human tissues that lack apobec-1 and apo-B mRNA suggests that ACF may be involved in other RNA editing or RNA processing events.
PMCID: PMC85365  PMID: 10669759
11.  A Novel Tumor-Promoting Function Residing in the 5′ Non-coding Region of vascular endothelial growth factor mRNA 
PLoS Medicine  2008;5(5):e94.
Background
Vascular endothelial growth factor-A (VEGF) is one of the key regulators of tumor development, hence it is considered to be an important therapeutic target for cancer treatment. However, clinical trials have suggested that anti-VEGF monotherapy was less effective than standard chemotherapy. On the basis of the evidence, we hypothesized that vegf mRNA may have unrecognized function(s) in cancer cells.
Methods and Findings
Knockdown of VEGF with vegf-targeting small-interfering (si) RNAs increased susceptibility of human colon cancer cell line (HCT116) to apoptosis caused with 5-fluorouracil, etoposide, or doxorubicin. Recombinant human VEGF165 did not completely inhibit this apoptosis. Conversely, overexpression of VEGF165 increased resistance to anti-cancer drug-induced apoptosis, while an anti-VEGF165-neutralizing antibody did not completely block the resistance. We prepared plasmids encoding full-length vegf mRNA with mutation of signal sequence, vegf mRNAs lacking untranslated regions (UTRs), or mutated 5′UTRs. Using these plasmids, we revealed that the 5′UTR of vegf mRNA possessed anti-apoptotic activity. The 5′UTR-mediated activity was not affected by a protein synthesis inhibitor, cycloheximide. We established HCT116 clones stably expressing either the vegf 5′UTR or the mutated 5′UTR. The clones expressing the 5′UTR, but not the mutated one, showed increased anchorage-independent growth in vitro and formed progressive tumors when implanted in athymic nude mice. Microarray and quantitative real-time PCR analyses indicated that the vegf 5′UTR-expressing tumors had up-regulated anti-apoptotic genes, multidrug-resistant genes, and growth-promoting genes, while pro-apoptotic genes were down-regulated. Notably, expression of signal transducers and activators of transcription 1 (STAT1) was markedly repressed in the 5′UTR-expressing tumors, resulting in down-regulation of a STAT1-responsive cluster of genes (43 genes). As a result, the tumors did not respond to interferon (IFN)α therapy at all. We showed that stable silencing of endogenous vegf mRNA in HCT116 cells enhanced both STAT1 expression and IFNα responses.
Conclusions
These findings suggest that cancer cells have a survival system that is regulated by vegf mRNA and imply that both vegf mRNA and its protein may synergistically promote the malignancy of tumor cells. Therefore, combination of anti-vegf transcript strategies, such as siRNA-based gene silencing, with anti-VEGF antibody treatment may improve anti-cancer therapies that target VEGF.
Shigetada Teshima-Kondo and colleagues find that cancer cells have a survival system that is regulated by vegf mRNA and that vegf mRNA and its protein may synergistically promote the malignancy of tumor cells.
Editors' Summary
Background
Normally, throughout life, cell division (which produces new cells) and cell death are carefully balanced to keep the body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—disorganized masses of cells. When a cancer is small, it uses the body's existing blood supply to get the oxygen and nutrients it needs for its growth and survival. But, when it gets bigger, it has to develop its own blood supply. This process is called angiogenesis. It involves the release by the cancer cells of proteins called growth factors that bind to other proteins (receptors) on the surface of endothelial cells (the cells lining blood vessels). The receptors then send signals into the endothelial cells that tell them to make new blood vessels. One important angiogenic growth factor is “vascular endothelial growth factor” (VEGF). Tumors that make large amounts of VEGF tend to be more abnormal and more aggressive than those that make less VEGF. In addition, high levels of VEGF in the blood are often associated with poor responses to chemotherapy, drug regimens designed to kill cancer cells.
Why Was This Study Done?
Because VEGF is a key regulator of tumor development, several anti-VEGF therapies—drugs that target VEGF and its receptors—have been developed. These therapies strongly suppress the growth of tumor cells in the laboratory and in animals but, when used alone, are no better at increasing the survival times of patients with cancer than standard chemotherapy. Scientists are now looking for an explanation for this disappointing result. Like all proteins, cells make VEGF by “transcribing” its DNA blueprint into an mRNA copy (vegf mRNA), the coding region of which is “translated” into the VEGF protein. Other, “noncoding” regions of vegf mRNA control when and where VEGF is made. Scientists have recently discovered that the noncoding regions of some mRNAs suppress tumor development. In this study, therefore, the researchers investigate whether vegf mRNA has an unrecognized function in tumor cells that could explain the disappointing clinical results of anti-VEGF therapeutics.
What Did the Researchers Do and Find?
The researchers first used a technique called small interfering (si) RNA knockdown to stop VEGF expression in human colon cancer cells growing in dishes. siRNAs are short RNAs that bind to and destroy specific mRNAs in cells, thereby preventing the translation of those mRNAs into proteins. The treatment of human colon cancer cells with vegf-targeting siRNAs made the cells more sensitive to chemotherapy-induced apoptosis (a type of cell death). This sensitivity was only partly reversed by adding VEGF to the cells. By contrast, cancer cells engineered to make more vegf mRNA had increased resistance to chemotherapy-induced apoptosis. Treatment of these cells with an antibody that inhibited VEGF function did not completely block this resistance. Together, these results suggest that both vegf mRNA and VEGF protein have anti-apoptotic effects. The researchers show that the anti-apoptotic activity of vegf mRNA requires a noncoding part of the mRNA called the 5′ UTR, and that whereas human colon cancer cells expressing this 5′ UTR form tumors in mice, cells expressing a mutated 5′ UTR do not. Finally, they report that the expression of several pro-apoptotic genes and of an anti-tumor pathway known as the interferon/STAT1 tumor suppression pathway is down-regulated in tumors that express the vegf 5′ UTR.
What Do These Findings Mean?
These findings suggest that some cancer cells have a survival system that is regulated by vegf mRNA and are the first to show that a 5′UTR of mRNA can promote tumor growth. They indicate that VEGF and its mRNA work together to promote their development and to increase their resistance to chemotherapy drugs. They suggest that combining therapies that prevent the production of vegf mRNA (for example, siRNA-based gene silencing) with therapies that block the function of VEGF might improve survival times for patients whose tumors overexpress VEGF.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050094.
This study is discussed further in a PLoS Medicine Perspective by Hughes and Jones
The US National Cancer Institute provides information about all aspects of cancer, including information on angiogenesis, and on bevacizumab, an anti-VEGF therapeutic (in English and Spanish)
CancerQuest, from Emory University, provides information on all aspects of cancer, including angiogenesis (in several languages)
Cancer Research UK also provides basic information about what causes cancers and how they develop, grow, and spread, including information about angiogenesis
Wikipedia has pages on VEGF and on siRNA (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
doi:10.1371/journal.pmed.0050094
PMCID: PMC2386836  PMID: 18494554
12.  MicroRNA-3148 Modulates Allelic Expression of Toll-Like Receptor 7 Variant Associated with Systemic Lupus Erythematosus 
PLoS Genetics  2013;9(2):e1003336.
We previously reported that the G allele of rs3853839 at 3′untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10−10, odds ratio (OR) (95%CI) = 1.27 (1.17–1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10−11, OR = 1.24 [1.18–1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3′UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R2 = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3′UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta = 2.0×10−19, OR = 1.25 [1.20–1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.
Author Summary
Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease contributed to by excessive innate immune activation involving toll-like receptors (TLRs, particularly TLR7/8/9) and type I interferon (IFN) signaling pathways. TLR7 responds against RNA–containing nuclear antigens and activates IFN-α pathway, playing a pivotal role in the development of SLE. While a genomic duplication of Tlr7 promotes lupus-like disease in the Y-linked autoimmune accelerator (Yaa) murine model, the lack of common copy number variations at TLR7 in humans led us to identify a functional single nucleotide polymorphism (SNP), rs3853839 at 3′ UTR of the TLR7 gene, associated with SLE susceptibility in Eastern Asians. In this study, we fine-mapped the TLR7-TLR8 region and confirmed rs3853839 exhibiting the strongest association with SLE in European Americans, African Americans, and Amerindian/Hispanics. Individuals carrying the risk G allele of rs3853839 exhibited increased TLR7 expression at the both mRNA and protein level and decreased transcript degradation. MicroRNA-3148 (miR-3148) downregulated the expression of non-risk allele (C) containing transcripts preferentially, suggesting a likely mechanism for increased TLR7 levels in risk-allele carriers. This trans-ancestral mapping provides evidence for the global association with SLE risk at rs3853839, which resides in a microRNA–gene regulatory site affecting TLR7 expression.
doi:10.1371/journal.pgen.1003336
PMCID: PMC3585142  PMID: 23468661
13.  An Enhancer Element Harboring Variants Associated with Systemic Lupus Erythematosus Engages the TNFAIP3 Promoter to Influence A20 Expression 
PLoS Genetics  2013;9(9):e1003750.
Functional characterization of causal variants present on risk haplotypes identified through genome-wide association studies (GWAS) is a primary objective of human genetics. In this report, we evaluate the function of a pair of tandem polymorphic dinucleotides, 42 kb downstream of the promoter of TNFAIP3, (rs148314165, rs200820567, collectively referred to as TT>A) recently nominated as causal variants responsible for genetic association of systemic lupus erythematosus (SLE) with tumor necrosis factor alpha inducible protein 3 (TNFAIP3). TNFAIP3 encodes the ubiquitin-editing enzyme, A20, a key negative regulator of NF-κB signaling. A20 expression is reduced in subjects carrying the TT>A risk alleles; however, the underlying functional mechanism by which this occurs is unclear. We used a combination of electrophoretic mobility shift assays (EMSA), mass spectrometry (MS), reporter assays, chromatin immunoprecipitation-PCR (ChIP-PCR) and chromosome conformation capture (3C) EBV transformed lymphoblastoid cell lines (LCL) from individuals carrying risk and non-risk TNFAIP3 haplotypes to characterize the effect of TT>A on A20 expression. Our results demonstrate that the TT>A variants reside in an enhancer element that binds NF-κB and SATB1 enabling physical interaction of the enhancer with the TNFAIP3 promoter through long-range DNA looping. Impaired binding of NF-κB to the TT>A risk alleles or knockdown of SATB1 expression by shRNA, inhibits the looping interaction resulting in reduced A20 expression. Together, these data reveal a novel mechanism of TNFAIP3 transcriptional regulation and establish the functional basis by which the TT>A risk variants attenuate A20 expression through inefficient delivery of NF-κB to the TNFAIP3 promoter. These results provide critical functional evidence supporting a direct causal role for TT>A in the genetic predisposition to SLE.
Author Summary
A key objective of human genetics is the identification and characterization of variants responsible for association with complex diseases. A pair of single nucleotide polymorphisms (rs148314165, rs200820567) 42 kb downstream from the promoter of TNFAIP3, have been proposed as the variants responsible for association with systemic lupus erythematosus based on comprehensive genetic and bioinformatic analyses. TNFAIP3 encodes for the ubiquitin-editing enzyme, A20, which plays a central role in maintaining immune system homeostasis through restriction of NF-κB signaling. Cells that carry this risk haplotype express low levels of TNFAIP3 compared to cells carrying the nonrisk haplotype. How the risk alleles of rs148314165 and rs200820567 might influence low TNFAIP3 expression is unknown. In this paper, we demonstrate that these variants reside in an enhancer element that binds NF-κB and SATB1 enabling the interaction of the enhancer with the TNFAIP3 promoter through long-range DNA looping. Impaired binding of NF-κB directly to the risk alleles or shRNA-mediated knockdown of SATB1 inhibits interaction of the enhancer with the TNFAIP3 promoter resulting in reduced A20 expression. These results clarify the functional mechanism by which rs148314165 and rs200820567 attenuate A20 expression and support a causal role for these variants in the predisposition to autoimmune disease.
doi:10.1371/journal.pgen.1003750
PMCID: PMC3764111  PMID: 24039598
14.  RNA Sequence and Base Pairing Effects on Insertion Editing in Trypanosoma brucei 
Molecular and Cellular Biology  2002;22(5):1567-1576.
RNA editing inserts and deletes uridylates (U's) in kinetoplastid mitochondrial pre-mRNAs by a series of enzymatic steps. Small guide RNAs (gRNAs) specify the edited sequence. Editing, though sometimes extensive, is precise. The effects of mutating pre-mRNA and gRNA sequences in, around, and upstream of the editing site on the specificity and efficiency of in vitro insertion editing were examined. U's could be added opposite guiding pyrimidines, but guiding purines, particularly A's, were required for efficient ligation. A base pair between mRNA and gRNA immediately upstream of the editing site was not required for insertion editing, although it greatly enhanced its efficiency and accuracy. In addition, a gRNA/mRNA duplex upstream of the editing site enhanced insertion editing when it was close to the editing site, but prevented cleavage, and hence editing, when immediately adjacent to the editing site. Thus, several aspects of mRNA-gRNA interaction, as well as gRNA base pairing with added U's, optimize editing efficiency, although they are not required for insertion editing.
PMCID: PMC134691  PMID: 11839822
15.  Dependencies among Editing Sites in Serotonin 2C Receptor mRNA 
PLoS Computational Biology  2012;8(9):e1002663.
The serotonin 2C receptor (5-HT2CR)–a key regulator of diverse neurological processes–exhibits functional variability derived from editing of its pre-mRNA by site-specific adenosine deamination (A-to-I pre-mRNA editing) in five distinct sites. Here we describe a statistical technique that was developed for analysis of the dependencies among the editing states of the five sites. The statistical significance of the observed correlations was estimated by comparing editing patterns in multiple individuals. For both human and rat 5-HT2CR, the editing states of the physically proximal sites A and B were found to be strongly dependent. In contrast, the editing states of sites C and D, which are also physically close, seem not to be directly dependent but instead are linked through the dependencies on sites A and B, respectively. We observed pronounced differences between the editing patterns in humans and rats: in humans site A is the key determinant of the editing state of the other sites, whereas in rats this role belongs to site B. The structure of the dependencies among the editing sites is notably simpler in rats than it is in humans implying more complex regulation of 5-HT2CR editing and, by inference, function in the human brain. Thus, exhaustive statistical analysis of the 5-HT2CR editing patterns indicates that the editing state of sites A and B is the primary determinant of the editing states of the other three sites, and hence the overall editing pattern. Taken together, these findings allow us to propose a mechanistic model of concerted action of ADAR1 and ADAR2 in 5-HT2CR editing. Statistical approach developed here can be applied to other cases of interdependencies among modification sites in RNA and proteins.
Author Summary
The serotonin receptor 2C is a key regulator of diverse neurological processes that affect feeding behavior, sleep, sexual behavior, anxiety and depression. The function of the receptor itself is regulated via so-called pre-mRNA editing, i.e. site-specific adenosine deamination in five distinct sites. The greater the number of edited sites in the serotonin receptor mRNA, the lower the activity of the receptor it encodes. Here we used the results of extensive massively parallel sequencing from human and rat brains to elucidate the dependencies among the editing states of the five sites. Despite the apparent simplicity of the problem, disambiguation of these dependencies is a difficult task that required development of a new statistical technique. We employed this method to analyse the dependencies among editing in the 5 susceptible sites of the receptor mRNA and found that the proximal, juxtaposed sites A and B are strongly interdependent, and that the editing state of these two sites is a major determinant of the editing states of the other three sites, and hence the overall editing pattern. The statistical approach we developed for the analysis of mRNA editing can be applied to other cases of multiple site modification in RNA and proteins.
doi:10.1371/journal.pcbi.1002663
PMCID: PMC3435259  PMID: 22969417
16.  Intronic Alus Influence Alternative Splicing 
PLoS Genetics  2008;4(9):e1000204.
Examination of the human transcriptome reveals higher levels of RNA editing than in any other organism tested to date. This is indicative of extensive double-stranded RNA (dsRNA) formation within the human transcriptome. Most of the editing sites are located in the primate-specific retrotransposed element called Alu. A large fraction of Alus are found in intronic sequences, implying extensive Alu-Alu dsRNA formation in mRNA precursors. Yet, the effect of these intronic Alus on splicing of the flanking exons is largely unknown. Here, we show that more Alus flank alternatively spliced exons than constitutively spliced ones; this is especially notable for those exons that have changed their mode of splicing from constitutive to alternative during human evolution. This implies that Alu insertions may change the mode of splicing of the flanking exons. Indeed, we demonstrate experimentally that two Alu elements that were inserted into an intron in opposite orientation undergo base-pairing, as evident by RNA editing, and affect the splicing patterns of a downstream exon, shifting it from constitutive to alternative. Our results indicate the importance of intronic Alus in influencing the splicing of flanking exons, further emphasizing the role of Alus in shaping of the human transcriptome.
Author Summary
The human genome is crowded with over one million copies of primate-specific retrotransposed elements, termed Alu. A large fraction of Alu elements are located within intronic sequences. The human transcriptome undergoes extensive RNA editing (A-to-I), to higher levels than any other tested organism. RNA editing requires the formation of a double-stranded RNA structure in order to occur. Over 90% of the editing sites in the human transcriptome are found within Alu sequences. Thus, the high level of RNA editing is indicative of extensive secondary structure formation in mRNA precursors driven by intronic Alu-Alu base pairing. Splicing is a molecular mechanism in which introns are removed from an mRNA precursor and exons are ligated to form a mature mRNA. Here, we show that Alu insertions into introns can affect the splicing of the flanking exons. We experimentally demonstrate that two Alu elements that were inserted into the same intron in opposite orientation undergo base-pairing, and consequently shift the splicing pattern of the downstream exon from constitutive inclusion in all mature mRNA molecules to alternative skipping. This emphasizes the impact of Alu elements on the primate-specific transcriptome evolution, as such events can generate new isoforms that might acquire novel functions.
doi:10.1371/journal.pgen.1000204
PMCID: PMC2533698  PMID: 18818740
17.  High ACSL5 Transcript Levels Associate with Systemic Lupus Erythematosus and Apoptosis in Jurkat T Lymphocytes and Peripheral Blood Cells 
PLoS ONE  2011;6(12):e28591.
Background
Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease in which increased apoptosis and decreased apoptotic cells removal has been described as most relevant in the pathogenesis. Long-chain acyl-coenzyme A synthetases (ACSLs) have been involved in the immunological dysfunction of mouse models of lupus-like autoimmunity and apoptosis in different in vitro cell systems. The aim of this work was to assess among the ACSL isoforms the involvement of ACSL2, ACSL4 and ACSL5 in SLE pathogenesis.
Findings
With this end, we determined the ACSL2, ACSL4 and ACSL5 transcript levels in peripheral blood mononuclear cells (PBMCs) of 45 SLE patients and 49 healthy controls by quantitative real time-PCR (q-PCR). We found that patients with SLE had higher ACSL5 transcript levels than healthy controls [median (range), healthy controls = 16.5 (12.3–18.0) vs. SLE = 26.5 (17.8–41.7), P = 3.9×10 E-5] but no differences were found for ACSL2 and ACSL4. In in vitro experiments, ACSL5 mRNA expression was greatly increased when inducing apoptosis in Jurkat T cells and PBMCs by Phorbol-Myristate-Acetate plus Ionomycin (PMA+Io). On the other hand, short interference RNA (siRNA)-mediated silencing of ACSL5 decreased induced apoptosis in Jurkat T cells up to the control levels as well as decreased mRNA expression of FAS, FASLG and TNF.
Conclusions
These findings indicate that ACSL5 may play a role in the apoptosis that takes place in SLE. Our results point to ACSL5 as a potential novel functional marker of pathogenesis and a possible therapeutic target in SLE.
doi:10.1371/journal.pone.0028591
PMCID: PMC3232234  PMID: 22163040
18.  The Versatility of Paramyxovirus RNA Polymerase Stuttering 
Journal of Virology  1999;73(7):5568-5576.
Paramyxoviruses cotranscriptionally edit their P gene mRNAs by expanding the number of Gs of a conserved AnGn run. Different viruses insert different distributions of guanylates, e.g., Sendai virus inserts a single G, whereas parainfluenza virus type 3 inserts one to six Gs. The sequences conserved at the editing site, as well as the experimental evidence, suggest that the insertions occur by a stuttering process, i.e., by pseudotemplated transcription. The number of times the polymerase “stutters” at the editing site before continuing strictly templated elongation is directed by a cis-acting sequence found upstream of the insertions. We have examined the stuttering process during natural virus infections by constructing recombinant Sendai viruses with mutations in their cis-acting sequences. We found that the template stutter site is precisely determined (C1052) and that a relatively short region (∼6 nucleotides) just upstream of the AnGn run can modulate the overall frequency of mRNA editing as well as the distribution of the nucleotide insertions. The positions more proximal to the 5′ AnGn run are the most important in this respect. We also provide evidence that the stability of the mRNA/template hybrid plays a determining role in the overall frequency and range of mRNA editing. When the template U run is extended all the way to the stutter site, adenylates rather than guanylates are added at the editing site and their distribution begins to resemble the polyadenylation associated with mRNA 3′ end formation by the viral polymerase. Our data suggest how paramyxovirus mRNA editing and polyadenylation are related mechanistically and how editing sites may have evolved from poly(A)-termination sites or vice versa.
PMCID: PMC112614  PMID: 10364305
19.  Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease. 
Journal of Clinical Investigation  1996;98(5):1107-1113.
The pathogenesis of systemic lupus erythematosus (SLE) is multifactorial and multigenetic. The apoptosis genes, fas and fas ligand (fasL), are candidate contributory genes in human SLE, as mutations of these genes result in autoimmunity in several murine models of this disease. In humans, fas mutations result in a familial autoimmune lymphoproliferative syndrome, but defects in FasL have not yet been identified. In this study, DNA from 75 patients with SLE was screened by single-stranded conformational polymorphism analysis for potential mutations of the extracellular domain of FasL. A heterozygous single-stranded conformational polymorphism for FasL, was identified in one SLE patient, who exhibited lymphadenopathy. Molecular cloning and sequencing indicated that the genomic DNA of this patient contained an 84-bp deletion within exon 4 of the fasL gene, resulting in a predicted 28 amino acid in-frame deletion. Analysis of PBMC from this patient revealed decreased FasL activity, decreased activation-induced cell death, and increased T cell proliferation after activation. This is the first report of defective FasL-mediated apoptosis related to a mutation of the human Fasl, gene in a patient with SLE and suggests that fasL mutations are an uncommon cause of the disease.
PMCID: PMC507531  PMID: 8787672
20.  Selective Involvement of the Amygdala in Systemic Lupus Erythematosus 
PLoS Medicine  2006;3(12):e499.
Background
Antibodies specifically affect the amygdala in a mouse model of systemic lupus erythematosus (SLE). The aim of our study was to investigate whether there is also specific involvement of the amygdala in human SLE.
Methods and Findings
We analyzed a group of 37 patients with neuropsychiatric SLE (NP-SLE), 21 patients with SLE, and a group of 12 healthy control participants with diffusion weighted imaging (DWI). In addition, in a subset of eight patients, plasma was available to determine their anti-NMDAR antibody status. From the structural magnetic resonance imaging data, the amygdala and the hippocampus were segmented, as well as the white and gray matter, and the apparent diffusion coefficient (ADC) was retrieved. ADC values between controls, patients with SLE, and patients with NP-SLE were tested using analysis of variance with post-hoc Bonferroni correction. No differences were found in the gray or white matter segments. The average ADC in the amygdala of patients with NP-SLE and SLE (940 × 10−6 mm2/s; p = 0.006 and 949 × 10−6 mm2/s; p = 0.019, respectively) was lower than in healthy control participants (1152 × 10−6 mm2/s). Mann-Whitney analysis revealed that the average ADC in the amygdala of patients with anti-NMDAR antibodies (n = 4; 802 × 10−6 mm2/s) was lower (p = 0.029) than the average ADC of patients without anti-NMDAR antibodies (n = 4; 979 × 10−6 mm2/s) and also lower (p = 0.001) than in healthy control participants.
Conclusions
This is the first study to our knowledge to observe damage in the amygdala in patients with SLE. Patients with SLE with anti-NMDAR antibodies had more severe damage in the amygdala compared to SLE patients without anti-NMDAR antibodies.
Patients with SLE who also had antibodies against the NMDA receptor had more severe damage in the amygdala as compared with patients with SLE without these antibodies.
Editors' Summary
Background.
The human body is continually attacked by viruses, bacteria, fungi, and parasites, but the immune system usually prevents these pathogens from causing disease. To be effective, the immune system has to respond rapidly to foreign antigens (bits of proteins that are unique to the pathogen) but ignore self-antigens. In autoimmune diseases, this ability to discriminate between self and nonself fails for unknown reasons, and the immune system begins to destroy human tissues. In the chronic autoimmune disease systemic lupus erythematosus (SLE or lupus), the immune system attacks the skin, joints, nervous system, and many other organs. Patients with SLE make numerous “autoantibodies” (antibodies are molecules made by the immune system that recognize and attack antigens; autoantibodies attack self-antigens). These autoantibodies start the attack on the body; then other parts of the immune system join in, causing inflammation and forming deposits of immune cells, both of which damage tissues. Common symptoms of SLE include skin rashes and arthritis, but some patients develop NP-SLE, a form of SLE that includes neuropsychiatric symptoms such as amnesia, dementia, mood disorders, strokes, and seizures. There is no cure for SLE, but mild cases are controlled with ibuprofen and other non-steroidal anti-inflammatory drugs; severe cases are kept in check with corticosteroids and other powerful immunosuppressants.
Why Was This Study Done?
In most of the tissues affected by SLE, the damage done by autoantibodies and immune cells can be seen when the tissues are examined with a microscope. But there is little microscopic damage visible in the brains of patients with NP-SLE. More generally, it is unclear how or even whether the immune system affects mental functions and emotion. In this study, researchers used magnetic resonance imaging (MRI) to investigate whether there are any structural changes in the brains of patients with NP-SLE that could explain their neuropsychiatric symptoms. They have also examined whether any changes in the brain can be linked to the presence of autoantibodies that recognize a protein called the NMDA receptor (anti-NMDAR antibodies) that is present on brain cells.
What Did the Researchers Do and Find?
The researchers used an MRI technique called diffusion weighted imaging to examine the brains of several patients with NP-SLE or SLE and the brains of several healthy individuals. Using this technique, it is possible to quantify the amount of structural damage in different regions of the brain. The researchers found no differences in most areas of the brain between the two groups of patients and the healthy controls. However, there were clear signs of damage in the amygdala (the part of the brain that regulates emotions and triggers responses to danger) in the patients with SLE or NP-SLE when compared to the control individuals. The researchers also found that the damage was more severe in the patients who had anti-NMDAR autoantibodies than in those that did not have these autoantibodies.
What Do These Findings Mean?
These findings suggest that autoantibodies produced by patients with SLE specifically damage the amygdala, a discovery that helps to explain some of the neuropsychiatric symptoms of this condition. Previous work has shown that the treatment of mice with anti-NMDAR antibodies and epinephrine, a stress hormone that causes leaks in the blood-brain barrier (antibodies can't usually get into the brain because of this barrier), results in damage to the amygdala and a deficient response to dangerous stimuli. The researchers suggest that a similar series of events might happen in SLE—patients often mention that a period of major stress precedes the development of symptoms. To provide stronger evidence for such a scenario, a detailed study of how stress relates to neuropsychiatric symptoms is needed. The damage to the amygdala (and the lack of damage elsewhere in the brain) and the possible association between brain damage and anti-NMDAR antibodies seen in this small study also need to be confirmed in more patients. Nevertheless, these findings provide an intriguing glimpse into the interplay between the immune system and the brain and into how stress might lead to physical damage in the brain.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030499.
MedlinePlus encyclopedia pages on autoimmunity and on systemic lupus erythematosus
US National Institute of Arthritis and Musculoskeletal and Skin Diseases booklet for patients with SLE
American College of Rheumatology information for patients on SLE
NHS Direct Online Health Encyclopedia pages on SLE
The Lupus Foundation of America information and support for patients with SLE
doi:10.1371/journal.pmed.0030499
PMCID: PMC1702559  PMID: 17177602
21.  MicroRNAs Implicated in the Immunopathogenesis of Lupus Nephritis 
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the deposition of immune complexes due to widespread loss of immune tolerance to nuclear self-antigens. Deposition in the renal glomeruli results in the development of lupus nephritis (LN), the leading cause of morbidity and mortality in SLE. In addition to the well-recognized genetic susceptibility to SLE, disease pathogenesis is influenced by epigenetic regulators such as microRNAs (miRNAs). miRNAs are small, noncoding RNAs that bind to the 3′ untranslated region of target mRNAs resulting in posttranscriptional gene modulation. miRNAs play an important and dynamic role in the activation of innate immune cells and are critical in regulating the adaptive immune response. Immune stimulation and the resulting cytokine milieu alter miRNA expression while miRNAs themselves modify cellular responses to stimulation. Here we examine dysregulated miRNAs implicated in LN pathogenesis from human SLE patients and murine lupus models. The effects of LN-associated miRNAs in the kidney, peripheral blood mononuclear cells, macrophages, mesangial cells, dendritic cells, and splenocytes are discussed. As the role of miRNAs in immunopathogenesis becomes delineated, it is likely that specific miRNAs may serve as targets for therapeutic intervention in the treatment of LN and other pathologies.
doi:10.1155/2013/430239
PMCID: PMC3741610  PMID: 23983769
22.  IL-37 inhibits the production of inflammatory cytokines in peripheral blood mononuclear cells of patients with systemic lupus erythematosus: its correlation with disease activity 
Background
Interleukin-37 (IL-37), a new member of IL-1 family cytokine, is recently identified as a natural inhibitor of innate immunity. This study aimed to measure the peripheral blood mononuclear cells (PBMCs) and serum levels of IL-37 in patients with systemic lupus erythematosus (SLE) and to investigate its role in SLE, including its correlation with disease activity, organ disorder and the regulation of inflammatory cytokines.
Methods
The expressions of IL-37 mRNAs in PBMCs and serum IL-37 levels in 66 SLE patients were measured by real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). SLE patients PBMCs were stimulated with recombinant IL-37, levels of cytokines TNF-α, IL-1β, IL-6 and IL-10 were detected by RT-PCR and ELISA.
Results
IL-37 mRNAs and serum protein levels were higher in patients with SLE compared with healthy controls. Patients with active disease showed higher IL-37 mRNAs and serum protein levels compared with those with inactive disease as well as healthy controls. Serum IL-37 levels correlated with SLEDAI and inversely with C3 and C4. Serum IL-37 levels were higher in SLE patients with renal involvement compared with those without renal disease. In vitro, IL-37 inhibited the production of TNF-α, IL-1β and IL-6 in PBMCs of patients with SLE, whereas the production of IL-10 was unaffected.
Conclusions
IL-37 associated with SLE disease activity, especially related with SLE renal disease activity. IL-37 is an important cytokine in the control of SLE pathogenesis by suppressing the production of inflammatory cytokines. Thus, IL-37 may provide a novel research target for the pathogenesis and therapy of SLE.
doi:10.1186/1479-5876-12-69
PMCID: PMC4003851  PMID: 24629023
Interleukin-37; Systemic lupus erythematosus; Autoimmunity; Cytokines; Peripheral blood mononuclear cell
23.  Elevated Serum Levels of Interferon-Regulated Chemokines Are Biomarkers for Active Human Systemic Lupus Erythematosus 
PLoS Medicine  2006;3(12):e491.
Background
Systemic lupus erythematosus (SLE) is a serious systemic autoimmune disorder that affects multiple organ systems and is characterized by unpredictable flares of disease. Recent evidence indicates a role for type I interferon (IFN) in SLE pathogenesis; however, the downstream effects of IFN pathway activation are not well understood. Here we test the hypothesis that type I IFN-regulated proteins are present in the serum of SLE patients and correlate with disease activity.
Methods and Findings
We performed a comprehensive survey of the serologic proteome in human SLE and identified dysregulated levels of 30 cytokines, chemokines, growth factors, and soluble receptors. Particularly striking was the highly coordinated up-regulation of 12 inflammatory and/or homeostatic chemokines, molecules that direct the movement of leukocytes in the body. Most of the identified chemokines were inducible by type I IFN, and their levels correlated strongly with clinical and laboratory measures of disease activity.
Conclusions
These data suggest that severely disrupted chemokine gradients may contribute to the systemic autoimmunity observed in human SLE. Furthermore, the levels of serum chemokines may serve as convenient biomarkers for disease activity in lupus.
A comprehensive survey of the serologic proteome in human SLE suggests that severely disrupted chemokine gradients may contribute to the systemic autoimmunity observed.
Editors' Summary
Background.
The term “lupus,” meaning wolf in Latin, is often used as an abbreviation for the disease systemic lupus erythematosus (SLE). The name may have been given because some people with SLE have a rash that slightly resembles a wolf's face. The condition affects around 50 to 100 people per 100,000, and is much more common in women than men. SLE is a complicated disease that comes about when antibodies inappropriately attack the body's own connective tissues, although it is not known why this happens. Symptoms vary between different people; the disease may get better and then worse, without explanation; and can affect many different organs including the skin, joints, kidneys, blood cells, and brain and nervous system. SLE is difficult for doctors to diagnose. Although the disease cannot be cured, patients who are diagnosed with SLE can be treated for their symptoms, and the right management can slow progress of the disease. One area of SLE research focuses on finding “molecular markers” (e.g., proteins or other compounds) that could be tested for in the blood. Researchers hope this would help doctors to more accurately diagnose SLE initially, and then also help to track progress in a patient's condition.
Why Was This Study Done?
“Gene expression” is a term meaning the process by which a gene's DNA sequence is converted into the structures and functions of a cell. These investigators had found in previous studies that certain genes were more “highly expressed” in the blood cells of patients with SLE. Some of these genes were already known to be regulated by interferons (a group of proteins, produced by certain blood cells, that are important in helping to defend against viral infections). The investigators performing this study wanted to understand more clearly the role of interferon in SLE and to see whether the genes that are more highly expressed in patients with SLE go on to produce higher levels of protein, which might then provide useful markers for monitoring the condition.
What Did the Researchers Do and Find?
This research project was a “case-control” study, in which the researchers compared the levels of certain proteins in the blood of people who had SLE with the levels in people who did not have the condition. Thirty people were recruited as cases, from a group of patients with SLE who have been under evaluation at Johns Hopkins School of Medicine since 1987. Fifteen controls were recruited from a group of healthy people of similar age and sex as the patients with SLE; everyone involved in the study gave their consent to take part. Blood samples were taken from each individual, and the serum (liquid component of blood) was separated out. The serum levels of 160 different blood proteins were then measured. When comparing levels of blood proteins between the groups, the researchers found that 30 specific proteins were present at higher or lower levels in the SLE-affected patients. Many of these proteins are cytokines, which are regulated by interferons and are involved in the process of “signaling” within the immune system. A few proteins were found at lower levels. Levels of the interferon-regulated proteins were, on average, seen at higher levels in people whose condition was more severe.
What Do These Findings Mean?
These results suggest that patients with SLE are likely to have a very different pattern of regulation of certain proteins within the blood, particularly the proteins involved in signaling within the immune system. The authors propose that these proteins may be involved in the progression of the disease. There is also the possibility that some of these proteins may prove useful in diagnostic tests, or in tests for monitoring how the disease progresses. However, before any such tests could be used in clinical practice, they would need to be further developed and then thoroughly tested in clinical trials.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030491
Patient information from the UK National Health Service on systemic lupus erythematosus
Patient handout from the US National Institutes of Health
MedlinePLUS encyclopedia entry on lupus
Information on lupus from the UK Arthritis Research Campaign
doi:10.1371/journal.pmed.0030491
PMCID: PMC1702557  PMID: 17177599
24.  Interferon-lambda1 induces peripheral blood mononuclear cell-derived chemokines secretion in patients with systemic lupus erythematosus: its correlation with disease activity 
Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease involving multiple organ systems. Previous studies have suggested that interferon-lambda 1 (IFN-λ1), a type III interferon, plays an immunomodulatory role. In this study we investigated its role in SLE, including its correlation with disease activity, organ disorder and production of chemokines.
Methods
We determined levels of IFN-λ1 mRNA in peripheral blood mononuclear cells (PBMC) and serum protein levels in patients with SLE using real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunoassay (ELISA). Further, we detected the concentration of IFN-inducible protein-10 (IP-10), monokine induced by IFN-γ (MIG) and interleukin-8 (IL-8) secreted by PBMC under the stimulation of IFN-λ1 using ELISA.
Results
IFN-λ1 mRNA and serum protein levels were higher in patients with SLE compared with healthy controls. Patients with active disease showed higher IFN-λ1 mRNA and serum protein levels compared with those with inactive disease as well. Serum IFN-λ1 levels were positively correlated with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), anti-dsDNA antibody, C-reactive protein (CRP) and negatively correlated with complement 3. Serum IFN-λ1 levels were higher in SLE patients with renal involvement and arthritis compared with patients without the above-mentioned manifestations. IFN-λ1 with different concentrations displayed different effects on the secretion of the chemokines IP-10, MIG and IL-8.
Conclusions
These findings indicate that IFN-λ1 is probably involved in the renal disorder and arthritis progression of SLE and associated with disease activity. Moreover, it probably plays an important role in the pathogenesis of SLE by stimulating secretion of the chemokines IP-10, MIG and IL-8. Thus, IFN-λ1 may provide a novel research target for the pathogenesis and therapy of SLE.
doi:10.1186/ar3363
PMCID: PMC3218903  PMID: 21679442
25.  Mutations in a Novel, Cryptic Exon of the Luteinizing Hormone/Chorionic Gonadotropin Receptor Gene Cause Male Pseudohermaphroditism 
PLoS Medicine  2008;5(4):e88.
Background
Male pseudohermaphroditism, or Leydig cell hypoplasia (LCH), is an autosomal recessive disorder in individuals with a 46,XY karyotype, characterized by a predominantly female phenotype, a blind-ending vagina, absence of breast development, primary amenorrhea, and the presence of testicular structures. It is caused by mutations in the luteinizing hormone/chorionic gonadotropin receptor gene (LHCGR), which impair either LH/CG binding or signal transduction. However, molecular analysis has revealed that the LHCGR is apparently normal in about 50% of patients with the full clinical phenotype of LCH. We therefore searched the LHCGR for novel genomic elements causative for LCH.
Methods and Findings
In the present study we have identified a novel, primate-specific bona fide exon (exon 6A) within the LHCGR gene. It displays composite characteristics of an internal/terminal exon and possesses stop codons triggering nonsense-mediated mRNA decay (NMD) in LHCGR. Transcripts including exon 6A are physiologically highly expressed in human testes and granulosa cells, and result in an intracellular, truncated LHCGR protein of 209 amino acids. We sequenced exon 6A in 16 patients with unexplained LCH and detected mutations in three patients. Functional studies revealed a dramatic increase in the expression of the mutated internal exon 6A transcripts, indicating aberrant NMD. These altered ratios of LHCGR transcripts result in the generation of predominantly nonfunctional LHCGR isoforms, thereby preventing proper expression and functioning.
Conclusions
The identification and characterization of this novel exon not only identifies a new regulatory element within the genomic organization of LHCGR, but also points toward a complex network of receptor regulation, including events at the transcriptional level. These findings add to the molecular diagnostic tools for LCH and extend our understanding of the endocrine regulation of sexual differentiation.
Joerg Gromoll and colleagues describe the identification and characterization of a novel exon that appears to be a new regulatory element within the luteinizing hormone/chorionic gonadotropin receptor gene of three individuals with Leydig cell hypoplasia.
Editors' Summary
Background.
A person's sex is determined by their complement of X and Y (sex) chromosomes. Someone who has two X chromosomes is genetically female and usually has ovaries and female external sex organs. Someone who has an X and a Y chromosome is genetically male and has testes and male external sex organs. Sometimes, though, the development of the reproductive organs proceeds abnormally, resulting in a person with an “intersex” condition whose chromosomes, gonads (ovaries or testes), and external sex organs do not correspond. Leydig cell hypoplasia (LCH; also called male pseudohermaphroditism or a disorder of sex development) is an XY female intersex condition. People with this inherited condition develop testes but also have a vagina (which is not connected to a womb), and they do not develop breasts or have periods. This mixture of sexual characteristics arises because the Leydig cells in the testes are underdeveloped. Leydig cells normally secrete testosterone, the hormone that promotes the development and maintenance of male sex characteristics. Before birth, chorionic gonadotropin (CG; a hormone made by the placenta) stimulates Leydig cell development and testosterone production; after birth, luteinizing hormone (LH), which is made by the pituitary gland, stimulates testosterone production. Both hormones bind to the LH/CG receptor, a protein on the surface of Leydig cells. In LCH, this receptor either does not bind CG and LH or fails to tell the Leydig cells to make testosterone.
Why Was This Study Done?
The gene that encodes the LH/CG receptor is called LHCGR. Several mutations (genetic changes) that inactivate the LC/CG receptor have been identified in people with LCH. However, the LHCGR gene is apparently normal in 50% of people with this intersex condition. In this study, the researchers examine the LHCGR gene in detail to try to find the underlying genetic defect in these individuals.
What Did the Researchers Do and Find?
The researchers used several molecular biology techniques to identify a new exon—exon 6A—within the human LHCGR gene. (Exons are DNA sequences that contain the information for making proteins; introns are DNA sequences that interrupt the coding sequence of a gene. Both introns and exons are transcribed into messenger RNA [mRNA] and the exons are then “spliced” together to make the mature mRNA, which is translated into protein.) The researchers identify several differently spliced LHCGR mRNA transcripts that contain exon 6A—a terminal exon 6A mRNA that contains exons 1–6 and exon 6A, and two internal exon 6A mRNAs that also contain exons 7–11. The researchers report that human testes express high levels of the terminal exon 6A transcript, which is translated into a short version of LHCGR protein that remains within the cell (full-length LHCGR moves to the cell surface). By contrast, testes contain low levels of the internal exon 6A mRNAs. This is because exon 6A contains two premature stop codons (DNA sequences that mark the end of a protein), which trigger “nonsense-mediated decay” (NMD), a cellular surveillance mechanism that regulates protein synthesis by degrading mRNAs that contain internal stop codons. When the researchers screened 16 people with LCH but without known mutations in the LHCGR gene, three had mutations in exon 6A. Laboratory experiments show that these mutations greatly increased the amounts of the internal exon 6A transcripts present in cells and interfered with the cells' normal response to chorionic gonadotropin.
What Do These Findings Mean?
These findings identify a new, functional exon in the LHCGR gene and show that mutations in this exon cause some cases of LCH. This is the first time that a human disease has been associated with mutations in an exon that is a target for NMD. In addition, these findings provide important insights into how the LHCGR is regulated. The researchers speculate that a complex network that involves the exon 6A-containing transcripts and NMD normally tightly regulates the production of functional LHCGR already at the transcriptional level. When mutations are present in exon 6A, they suggest, NMD is the predominant pathway for all the exon 6A-containing transcripts, thereby drastically decreasing the amount of functional LHCGR.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0050088.
The MedlinePlus Encyclopedia has a page on intersex conditions (in English and Spanish)
Wikipedia has pages on intersexuality and on the LH/CG receptor (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Intersex Society of North America provides information and support for the parents of children with intersex conditions
The Androgen Insensitivity Syndrome Support Group also provides some general information about intersex conditions, including information about LCH and other XY female conditions (in several languages)
Sequence-Structure-Function-Analysis (SSFA), run by a group of researchers in Germany (Leibniz-Institut für Molekulare Pharmakologie; Humboldt-Universitätzu Berlin), is a database dealing the sequence, structure, and function of glycoprotein hormone receptors
Glycoprotein-hormone Receptors Information System (GRIS), from Université Libre de Bruxelles and Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, is a database giving structural information on the LHCGR
doi:10.1371/journal.pmed.0050088
PMCID: PMC2323302  PMID: 18433292

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