Filamin A pre-mRNA editing modulates vascularization and tumor growth.

Adenosine to inosine (A to I) editing is mediated by adenosine deaminases acting on RNA (ADAR) enzymes. Inosines are interpreted as guanosines by the translational machinery. Consequently, A to I editing in mRNAs can lead to their recoding and the formation of proteins not encoded in the genome. Filamin A is an actin-crosslinking protein. A to I editing in the filamin pre-mRNA leads to the exchange of a glutamine to an arginine in a highly interactive domain of the protein. However, the consequences of this editing event are still poorly understood. Here we show, using transgenic mice expressing either constitutively edited or constitutively uneditable filamin A that filamin A editing critically controls angiogenesis in tumors but also in a mouse ischemia model. Hyper-editing reduces angiogenesis, while hypoediting leads to increased angiogenesis, possibly by altering vascular endothelial growth factor receptor 2 (VEGFR2) turnover. Further, FLNA editing of the tumor itself seemingly affects its metastatic potential by changing its interaction with the extracellular matrix. We therefore identify filamin A editing as a critical component for angiogenesis, tumor growth, and metastasis formation.

ATM controls meiotic DNA double-strand break formation and recombination and affects synaptonemal complex organization in plants.

Meiosis is a specialized cell division that gives rise to genetically distinct gametic cells. Meiosis relies on the tightly controlled formation of DNA double-strand breaks (DSBs) and their repair via homologous recombination for correct chromosome segregation. Like all forms of DNA damage, meiotic DSBs are potentially harmful and their formation activates an elaborate response to inhibit excessive DNA break formation and ensure successful repair. Previous studies established the protein kinase ATM as a DSB sensor and meiotic regulator in several organisms. Here we show that Arabidopsis ATM acts at multiple steps during DSB formation and processing, as well as crossover (CO) formation and synaptonemal complex (SC) organization, all vital for the successful completion of meiosis. We developed a single-molecule approach to quantify meiotic breaks and determined that ATM is essential to limit the number of meiotic DSBs. Local and genome-wide recombination screens showed that ATM restricts the number of interference-insensitive COs, while super-resolution STED nanoscopy of meiotic chromosomes revealed that the kinase affects chromatin loop size and SC length and width. Our study extends our understanding of how ATM functions during plant meiosis and establishes it as an integral factor of the meiotic program.

An internal deletion of ADAR rescued by MAVS deficiency leads to a minute phenotype.

The RNA-editing protein ADAR is essential for early development in the mouse. Genetic evidence suggests that A to I editing marks endogenous RNAs as 'self'. Today, different Adar knockout alleles have been generated that show a common phenotype of apoptosis, liver disintegration, elevated immune response and lethality at E12.5. All the Adar knockout alleles can be rescued by a concomitant deletion of the innate immunity genes Mavs or Ifih1 (MDA5), albeit to different extents. This suggests multiple functions of ADAR. We analyze AdarΔ7-9 mice that show a unique growth defect phenotype when rescued by Mavs. We show that AdarΔ7-9 can form a truncated, unstable, editing deficient protein that is mislocalized. Histological and hematologic analysis of these mice indicate multiple tissue- and hematopoietic defects. Gene expression profiling shows dysregulation of Rps3a1 and Rps3a3 in rescued AdarΔ7-9. Consistently, a distortion in 40S and 60S ribosome ratios is observed in liver cells. This dysregulation is also seen in AdarΔ2-13; Mavs-/- but not in AdarE861A/E861A; Ifih1-/- mice, suggesting editing-independent functions of ADAR in regulating expression levels of Rps3a1 and Rps3a3. In conclusion, our study demonstrates the importance of ADAR in post-natal development which cannot be compensated by ADARB1.

Inosine induces context-dependent recoding and translational stalling.

RNA modifications are present in all classes of RNAs. They control the fate of mRNAs by affecting their processing, translation, or stability. Inosine is a particularly widespread modification in metazoan mRNA arising from deamination of adenosine catalyzed by the RNA-targeting adenosine deaminases ADAR1 or ADAR2. Inosine is commonly thought to be interpreted as guanosine by cellular machines and during translation. Here, we systematically test ribosomal decoding using mass spectrometry. We show that while inosine is primarily interpreted as guanosine it can also be decoded as adenosine, and rarely even as uracil. Decoding of inosine as adenosine and uracil is context-dependent. In addition, mass spectrometry analysis indicates that inosine causes ribosome stalling especially when multiple inosines are present in the codon. Indeed, ribosome profiling data from human tissues confirm inosine-dependent ribosome stalling in vivo. To our knowledge this is the first study where decoding of inosine is tested in a comprehensive and unbiased way. Thus, our study shows novel, unanticipated functions for inosines in mRNAs, further expanding coding potential and affecting translational efficiency.

Arabidopsis thaliana FANCD2 Promotes Meiotic Crossover Formation.

Fanconi anemia (FA) is a human autosomal recessive disorder characterized by chromosomal instability, developmental pathologies, predisposition to cancer, and reduced fertility. So far, 19 genes have been implicated in FA, most of them involved in DNA repair. Some are conserved across higher eukaryotes, including plants. The Arabidopsis thaliana genome encodes a homolog of the Fanconi anemia D2 gene (FANCD2) whose function in DNA repair is not yet fully understood. Here, we provide evidence that AtFANCD2 is required for meiotic homologous recombination. Meiosis is a specialized cell division that ensures reduction of genomic content by half and DNA exchange between homologous chromosomes via crossovers (COs) prior to gamete formation. In plants, a mutation in AtFANCD2 results in a 14% reduction of CO numbers. Genetic analysis demonstrated that AtFANCD2 acts in parallel to both MUTS HOMOLOG4 (AtMSH4), known for its role in promoting interfering COs and MMS AND UV SENSITIVE81 (AtMUS81), known for its role in the formation of noninterfering COs. AtFANCD2 promotes noninterfering COs in a MUS81-independent manner and is therefore part of an uncharted meiotic CO-promoting mechanism, in addition to those described previously.

Transcriptome-wide effects of inverted SINEs on gene expression and their impact on RNA polymerase II activity.

BACKGROUND: Short interspersed elements (SINEs) represent the most abundant group of non-long-terminal repeat transposable elements in mammalian genomes. In primates, Alu elements are the most prominent and homogenous representatives of SINEs. Due to their frequent insertion within or close to coding regions, SINEs have been suggested to play a crucial role during genome evolution. Moreover, Alu elements within mRNAs have also been reported to control gene expression at different levels. RESULTS: Here, we undertake a genome-wide analysis of insertion patterns of human Alus within transcribed portions of the genome. Multiple, nearby insertions of SINEs within one transcript are more abundant in tandem orientation than in inverted orientation. Indeed, analysis of transcriptome-wide expression levels of 15 ENCODE cell lines suggests a cis-repressive effect of inverted Alu elements on gene expression. Using reporter assays, we show that the negative effect of inverted SINEs on gene expression is independent of known sensors of double-stranded RNAs. Instead, transcriptional elongation seems impaired, leading to reduced mRNA levels. CONCLUSIONS: Our study suggests that there is a bias against multiple SINE insertions that can promote intramolecular base pairing within a transcript. Moreover, at a genome-wide level, mRNAs harboring inverted SINEs are less expressed than mRNAs harboring single or tandemly arranged SINEs. Finally, we demonstrate a novel mechanism by which inverted SINEs can impact on gene expression by interfering with RNA polymerase II.

Polymorphisms in inflammatory genes and the risk of ischemic stroke and transient ischemic attack: results of a multilocus genotyping assay.

BACKGROUND: Single-nucleotide polymorphisms (SNPs) in inflammation-related genes have been linked to an increased risk of ischemic stroke. Most of these SNP results have not been replicated, however, and metaanalyses of the effects of inflammation-related genes are rare. We investigated 49 SNPs in 34 genes previously reported to be related to inflammation in our study. We tested 459 patients with acute ischemic stroke or transient ischemic attack and 459 controls individually matched by sex and age. METHODS: We studied genetic variation by PCR analysis and subsequent hybridization to linear arrays of sequence-specific oligonucleotides. We used univariate conditional logistic regression analysis to test for associations of conventional vascular risk factors and the SNPs with stroke. Variables showing significant differences (P < 0.05) between cases and controls were included in a multivariate model. ROC curves were plotted to assess the contribution of genetic variation to stroke risk in addition to that of conventional risk factors. RESULTS: Univariate regression analysis revealed 3 SNPs with significant allelic differences between patients and controls, which fulfilled the criteria for further analysis. Only one of these SNPs, the C5 (complement component 5) 2416A>G variant (rs17611), remained significant after the multivariate analysis (odds ratio, 0.585; P = 0.0037). ROC curve analysis revealed no contribution of this genetic variation to stroke risk. CONCLUSIONS: We found evidence for an association of the 2416A>G polymorphism in the C5 gene with the risk for ischemic stroke. Our data suggest that the C5 gene particularly influences the risk for patients with microangiopathy.

Impact of alphaENaC polymorphisms on the risk of ischemic cerebrovascular events: a multicenter case-control study.

BACKGROUND: Amiloride-sensitive epithelial sodium channels (ENaCs) are important candidates in the development of hypertension, which is a major risk factor for stroke. Two single-nucleotide polymorphisms (SNPs) in the gene that encodes the ENac alpha-subunit (alphaENaC) have been identified. We evaluated those SNPs for a possible association with ischemic cerebrovascular events (ICEs). METHODS AND RESULTS: We genotyped 1399 patients with ICEs [median age, 70 years; interquartile range, 58-78 years; 745 (53%) men] and 1076 control individuals without vascular disease [47 (39-58) years; 557 (52%) men] for the SNPs Trp493Arg and Ala663Thr. The SNP frequencies at nucleotide 3977 (Trp493Arg) in the alphaENaC gene were significantly different in patients and controls. Carriers of 493Arg had a 1.78-fold increased risk (95% confidence interval, 1.02-3.12) for ICEs compared with Trp/Trp carriers. Interaction analysis revealed that the relative risk was even higher in women in the lowest age tertile [adjusted odds ratio, 3.26 (1.10-9.72)]. CONCLUSIONS: Carriers of the 493Arg allele are at increased risk for ICEs compared with Trp/Trp carriers. The effect is independent of traditional vascular risk factors and is particularly evident in younger women. The Trp493Arg variant in alphaENaC may represent an important candidate genetic susceptibility factor in the development of ICEs.

Vienna Stroke Registry. Impact of the platelet glycoprotein Ib alpha Kozak polymorphism on the risk of ischemic cerebrovascular events: a case-control study.

We performed a multicenter case-control study to evaluate the impact of the glycoprotein 1b alpha (-5)T/C Kozak polymorphism on the risk of ischemic cerebrovascular events. The genetic analysis in 1399 patients (745 men; median age, 70 years; interquartile ratio, 58-78) and 1066 control subjects (549 men; median age, 47 years; interquartile ratio, 39-59) was carried out with mutagenically separated polymerase chain reaction. Homozygous C/C genotype carriers had a 3.5-fold increased risk for ischemic cerebrovascular events (95% confidence interval, 1.5-7.9, P = 0.003) over T/T or T/C genotype carriers together. The effect was independent of well-established atherosclerotic risk factors. Our findings could be explained by the reported gene dose effect of the Kozak polymorphism on the density of the glycoprotein 1b alpha/IX/V receptors on platelets. According to our data, the (-5)C Kozak allele may represent a candidate genetic susceptibility factor for ischemic cerebrovascular events.

Postdelivery levels of anti-D IgG prophylaxis in D- mothers depend on maternal body weight.

BACKGROUND: Current recommendations for anti-D prophylaxis for women who deliver a D+ offspring vary from country to country, and the introduction of new reagents require pharmacokinetic studies that show serum levels after the injection. Serum levels of anti-D may depend on the maternal body mass index (BMI). STUDY DESIGN AND METHODS: Serum concentrations of total anti-D IgG and IgG1-4 subclasses were determined by flow cytometry in 26 D- women, who had received prophylaxis after delivery of a D+ offspring. Blood samples were drawn on Days 1, 2, 3, and 14 after injection, and the BMI was recorded. RESULTS: Anti-D levels increased continuously in all women during the first 3 days. The increase was significantly affected by the BMI if higher than 27 kg per m2 (p<0.001). The higher the BMI, the less was the increase of serum anti-D. Mean peak levels 72 hours after injection was 89 ng per mL in lean women, but estimated levels were 28 to 60 percent lower in women with a BMI of 28 to 40 kg per m2. The effect of a BMI higher than 27 kg per m2 on anti-D was not gradual but progressive. Similarly, the BMI affected serum concentrations of anti-D subclasses IgG1-4 (p<0.001). CONCLUSION: The BMI needs consideration for the adjustment of the dosage of anti-D, provided its bioavailability to suppress alloimmunization is reflected by measurable amounts in the serum.