Nonsense-mediated RNA Decay Research Articles

Abstract 4964: Nonsense MED12 mutation in a patient with T-cell acute lymphoblastic leukemia

Abstract Mediator complex subunit 12 (MED12) is part of a Mediator, which is a large, highly conserved protein complex that participates in the regulation of general as well as gene-specific transcription. First somatic MED12 mutations were identified when exome sequencing revealed highly specific mutations in exon 2 in 70% of uterine leiomyomas studied. Additional mutations have subsequently been observed in exon 1, which further highlight the role of MED12 in these tumors. Functional analyses have indicated that exon 1 and exon 2 mutations lead to a diminished interaction between MED12 and other components of the Mediator kinase module, reduced Mediator-associated CDK kinase activity, and a unique global gene expression pattern. Recently, we observed similar mutations in chronic lymphocytic leukemia (CLL). Mutations were observed in 5% of the patients and the positive mutation status was associated with well-known poor prognosis markers in CLL. All the observed mutations both in uterine leiomyomas and CLL have been missense mutations or small insertions or deletions leading to an in-frame transcript. Interestingly, the first MED12 mutation in a patient with T-cell acute lymphoblastic leukemia (T-ALL) was recently identified through exome sequencing. This is a nonsense mutation affecting the mutation hotspot observed in CLL. It is also the first nonsense mutation identified in the MED12 N-terminal mutation hotspot area in any tumor type. The patient has received bone marrow transplantation and is now in complete remission, but small amounts of cancer cells were collected before the treatment. RNA was extracted from the cells, and cDNA sequencing revealed that only the mutant allele is expressed. We have now generated a construct with the MED12 specific mutation and transfected it into HEK293 Flp-In cells. Wild-type MED12 is being used as a control. Western blotting with N- and C-terminal MED12 antibodies indicates that the mutant protein is being produced but that the N-terminal part is lacking. We now aim to further characterize the function of this highly unusual mutation. Mutant and wild type protein will be produced for proteome-wide mass spectrometry analyses to detect possible effects on protein-protein interactions and also to gain more information on the translation start site. Also transcriptome-wide expression data will be produced to study the effect of the mutation on global gene expression profile. Overall, a mutation affecting the 5′ terminus of MED12 has now been identified in yet another cancer type, T-ALL. Our preliminary analyses indicate that the observed nonsense mutation escapes nonsense-mediated RNA decay and leads to a truncated protein product. Besides providing additional knowledge on the role of MED12 mutations in cancer, this exceptional mutation has potential to produce novel information on the normal MED12 function. Citation Format: Tuomas Heikkinen, Kati Kämpjärvi, Salla Keskitalo, Mikko Turunen, Heikki Kuusanmäki, Netta Mäkinen, Satu Mustjoki, Caroline Heckman, Markku Varjosalo, Pia Vahteristo. Nonsense MED12 mutation in a patient with T-cell acute lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4964. doi:10.1158/1538-7445.AM2015-4964

RNA degradation drives stem cell differentiation.

The mechanisms by which multi‐potent stem cells switch their program to become functional differentiated cells have intrigued biologists for decades. Most focus has been on transcriptional pathways, but whether they have sufficient dynamic range to cause discrete shifts in cell state is not clear. Because the steady‐state level of RNAs is also dictated by their decay rate, an attractive possibility is that specific RNA decay mechanisms also have a role in promoting differentiation mechanisms. In this issue of The EMBO Journal , Li et al (2015) obtained evidence that a highly conserved RNA degradation pathway called nonsense‐mediated RNA decay (NMD) is critical for the differentiation of embryonic stem (ES) cells.

Nonsense-mediated RNA decay--a switch and dial for regulating gene expression.

Nonsense-mediated RNA decay (NMD) represents an established quality control checkpoint for gene expression that protects cells from consequences of gene mutations and errors during RNA biogenesis that lead to premature termination during translation. Characterization of NMD-sensitive transcriptomes has revealed, however, that NMD targets not only aberrant transcripts but also a broad array of mRNA isoforms expressed from many endogenous genes. NMD is thus emerging as a master regulator that drives both fine and coarse adjustments in steady-state RNA levels in the cell. Importantly, while NMD activity is subject to autoregulation as a means to maintain homeostasis, modulation of the pathway by external cues provides a means to reprogram gene expression and drive important biological processes. Finally, the unanticipated observation that transcripts predicted to lack protein-coding capacity are also sensitive to this translation-dependent surveillance mechanism implicates NMD in regulating RNA function in new and diverse ways.

The unfolded protein response is shaped by the NMD pathway.

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD's ability to regulate normal mRNAs.

RNA metabolism: putting the brake on the UPR.

The unfolded protein response (UPR) is a major signaling cascade that determines cell fate under conditions of endoplasmic reticulum (ER) stress. The kinetics and amplitude of UPR responses are tightly controlled by several feedback loops and the expression of positive and negative regulators. In this issue of EMBO Reports, the Wilkinson lab uncovers a novel function of nonsense-mediated RNA decay (NMD) in fine-tuning the UPR. NMD is an mRNA quality control mechanism known to destabilize aberrant mRNAs that contain premature termination codons. In this work, NMD was shown to determine the threshold of stress necessary to activate the UPR, in addition to adjusting the amplitude of downstream responses and the termination phase. These effects were mapped to the control of the mRNA stability of IRE1, a major ER stress transducer. This study highlights the dynamic crosstalk between mRNA metabolism and the proteostasis network demonstrating the physiological relevance of normal mRNA regulation by the NMD pathway.

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Objectives Proper regulation of human leukocyte antigen-C (HLA-C) expression on trophoblast cells is essential for the immunological privilege of these cells. Endoplasmic reticulum aminopeptidase 2 (ERAP2) is known to trim antigenic peptide precursors for loading onto HLA. Altered expression of the ERAP2 could play a critical role in shaping HLA-C expression and antigen presentation, but their relationships have not been defined. Methods We examined the genotype and expression levels of ERAP2 and HLA-C in several choriocarcinoma cell lines (BeWo, JAr, and JEG-3) by allelic discrimination qPCR, Western blotting and immunocytochemistry. Additionally, expression profiling of untreated versus gamma-interferon (IFN- γ , 20 ng/ml)-treated cells was performed. Lastly, to determine if there is a relationship between ERAP2 and HLA-C expression levels, transfection of major and minor allele ERAP2 variants was performed. Results JAr cells were homozygous for the rs2248374 minor A allele, and express ERAP2 protein. BeWo and JEG-3 cells are homozygous for ERAP2 genetic variant (major G allele of SNP rs2248374) that causes non-sense mediated RNA decay, and thus lack ERAP2 protein expression. Interestingly, BeWo and JEG-3 cells express HLA-C, whereas, JAr cells do not. IFN- γ treatment of BeWo and JEG-3 cells increased HLA-C expression by 2-fold ( p = 0.0037) and 3-fold ( p = 0.0446) above control levels, respectively, but ERAP2 remained undetectable. In contrast, JAr cells treated with IFN- γ had increased ERAP2 protein expression by 2-fold ( p = 0.003), but had no effect on HLA-C expression. Transfection of JEG-3 cells (ERAP2 null) with ERAP2 plasmids with the SNP rs2549782, encoding an amino acid substitution (N392K) that alters substrate specificities and antigen presentation, revealed that the different ERAP2 isoforms do not affect the expression level of HLA-C. Conclusions Our observations suggest that the expression patterns of ERAP2 and HLA-C allow choriocarcinoma cells to escape immune surveillance. Disclosures E.D. Lee: None. S. Brockett: None. D. Hilliard: None. M.E. Teves: None. R. Ramus: None. J.F. Strauss: None.

Two Novel Mutations (p.I454T and p.Y472X) and a Homozygous p.A109T Mutation Associated with Factor XI Deficiency in a Turkish Family

Introduction: Factor XI (FXI) is a homodimeric serine protease enzyme, which is produced in the liver and circulates in the plasma complexed with high molecular weight kininogen. Approximately 1% of FXI is presented in platelets. After the activation of the extrinsic pathway of coagulation, thrombin activates FXI, and this reaction has an important role in the amplification of coagulation reaction. Congenital FXI deficiency is characterized by decreased levels or activity of FXI in the plasma, and may cause an inherited bleeding disorder. Congenital FXI deficiency is very rare (less than 1/100.000) in the general population. The factor XI gene is located on the long arm of chromosome 4 (4q34-35), consists of 15 exons and 14 introns, and encodes a 607 amino acid protein. FXIa has two chains: the light chain contains the catalytic site whereas the heavy chain contains the four tandem repeat sequences (Apple domains, A1 to A4). Mutations of the FXI gene (F11 gene) leading to FXI deficiency can occur in the Apple domains or the catalytic site. One of the most common mutations is a missense mutation in the Apple 4 domain, which is important for FXI dimerization, resulting in low serum levels of FXI. Some mutations can cause a moderate to severe FXI deficiency by producing aberrant proteins. Here we present a consanguineous family with FXI deficiency.Patients and methods: In this study a family with FXI deficiency was evaluated. The index patient was a 10 years old boy with bleeding diathesis (excessive bleeding after tooth extraction). His aPTT level was 84,3 seconds (N: 32-39 seconds), FXI activity was 0%, and he was diagnosed with FXI deficiency in 2010. His parents were related. The father had a mild bleeding tendency with prolonged aPTT (48.2 seconds). FXI activity was found to be 4%. The mother and second child had no bleeding history with mildly decreased FXI activities (40% and 60%, respectively). We performed a mutational analysis for whole family, including his grandparents. Genomic DNA was extracted from whole blood. All exons and approximately 30bp exon-intron boundaries of F11 gene were amplified using sets of designed primers. PCR reactions were performed using 2X PCR master mix (HibriGen Biotech R&D, Istanbul, Turkey), primers and 25-30 ng genomic DNA. Amplified fragments were sequenced.Results: We obtained the results shown in Figure 1. [Display omitted] The patient and his father had a A109T homozygous mutation for F11; the index patient also had heterozygous I454T and Y472X mutations. The presence of a homozygous A109T mutation in father and index patient caused severe FXI deficiency. The mother and second child had heterozygous I454T and Y472X mutations. As shown in the pedigree, I454T and Y472X compound heterozygosity moderately decreases the activity of FXI.Conclusions: Approximately 220 F11 mutations have been reported to date, affecting both the catalytic and Apple domains. In this family, we found two novel mutations, I454T and Y472X, associated with a homozygous A109T mutation. This is the first case reported with a homozygous A109T mutation in the literature. Previously Guella et al. reported a heterozygous A109T mutation in an Italian family with FXI deficiency (Thromb Haemost, 2008). They showed that exon-skipping had occured due to a heterozygous A109T mutation. They explained that the unchanged enzyme activity was due to a non-sense mediated RNA decay mechanism. Our cases confirmed their results, such that a heterozygous A109T mutation did not affect enzyme activity; the enzyme activity of a person who has two heterozygous mutations (Y472X and I454T) is the same as who has three heterozygous mutations (A109T, Y472X and I454T). However, when the A109T was homozygous like in our patient and his father, the enzyme activity decreased by approximately 96% as shown in the pedigree. Another interesting point was the presence of a homozygous A109T mutation in the patient while the mother had no A109T mutation. This can be explained by somatic recombination as seen in variable diversity junction (VDJ) recombination in the immune system. Further expression studies evaluating the effects of these mutations will improve our understanding of the functional and structural features of the FXI enzyme. DisclosuresNo relevant conflicts of interest to declare.

Stress-induced inhibition of nonsense-mediated RNA decay regulates intracellular cystine transport and intracellular glutathione through regulation of the cystine/glutamate exchanger SLC7A11.

SLC7A11 encodes a subunit of the xCT cystine/glutamate amino acid transport system and plays a critical role in the generation of glutathione and the protection of cells from oxidative stress. Expression of SLC7A11 promotes tumorigenesis and chemotherapy resistance, but while SLC7A11 has been previously noted to be upregulated in hypoxic cells its regulation has not been fully delineated. We have recently shown that nonsense mediated RNA decay (NMD) is inhibited by cellular stresses generated by the tumor microenvironment, including hypoxia, and augments tumorigenesis. Here we demonstrate that the inhibition of NMD by various cellular stresses leads to the stabilization and upregulation of SLC7A11 mRNA and protein. The inhibition of NMD and upregulation of SLC7A11 augments intracellular cystine transport, and increases intracellular levels of cysteine and glutathione. Accordinglyy, the inhibition of NMD protects cells against oxidative stress via SLC7A11 upregulation. Together our studies identify a mechanism for the dynamic regulation of SLC7A11, through the stress-inhibited regulation of NMD, and add to the growing evidence that the inhibition of NMD is an adaptive response.

Nonsense-mediated RNA decay: at the 'cutting edge' of regulated snoRNA production.

Production of multiple functional RNAs from a single primary transcript is an extremely efficient use of genetic information, although it complicates the ability of the cell to independently regulate the production of each RNA. For the case of small nucleolar RNAs (snoRNAs) encoded within introns of mRNA genes, Lykke-Andersen and colleagues (pp. 2498-2517) demonstrated that alternative splicing and the SMG6 endonuclease of the nonsense-mediated RNA decay pathway are key regulators that control which RNAs accumulate.

Human nonsense-mediated RNA decay initiates widely by endonucleolysis and targets snoRNA host genes.

Eukaryotic RNAs with premature termination codons (PTCs) are eliminated by nonsense-mediated decay (NMD). While human nonsense RNA degradation can be initiated either by an endonucleolytic cleavage event near the PTC or through decapping, the individual contribution of these activities on endogenous substrates has remained unresolved. Here we used concurrent transcriptome-wide identification of NMD substrates and their 5'-3' decay intermediates to establish that SMG6-catalyzed endonucleolysis widely initiates the degradation of human nonsense RNAs, whereas decapping is used to a lesser extent. We also show that a large proportion of genes hosting snoRNAs in their introns produce considerable amounts of NMD-sensitive splice variants, indicating that these RNAs are merely by-products of a primary snoRNA production process. Additionally, transcripts from genes encoding multiple snoRNAs often yield alternative transcript isoforms that allow for differential expression of individual coencoded snoRNAs. Based on our findings, we hypothesize that snoRNA host genes need to be highly transcribed to accommodate high levels of snoRNA production and that the expression of individual snoRNAs and their cognate spliced RNA can be uncoupled via alternative splicing and NMD.

Suppressing nonsense--a surprising function for 5-azacytidine.

In this issue of EMBO Molecular Medicine, Bhuvanagiri et al report on a chemical means to convert molecular junk into gold. They identify a chemical inhibitor of a quality control pathway that is best known for its ability to clear cells of rubbish, but that in certain cases can be detrimental because it eliminates “useful” garbage. The chemical inhibitor identified by Bhuvanagiri et al perturbs Nonsense-Mediated RNA Decay (NMD), a RNA surveillance pathway that targets mRNAs harboring premature termination codons (PTCs) for degradation (Kervestin & Jacobson, 2012).

5-azacytidine inhibits nonsense-mediated decay in a MYC-dependent fashion.

Nonsense-mediated RNA decay (NMD) is an RNA-based quality control mechanism that eliminates transcripts bearing premature translation termination codons (PTC). Approximately, one-third of all inherited disorders and some forms of cancer are caused by nonsense or frame shift mutations that introduce PTCs, and NMD can modulate the clinical phenotype of these diseases. 5-azacytidine is an analogue of the naturally occurring pyrimidine nucleoside cytidine, which is approved for the treatment of myelodysplastic syndrome and myeloid leukemia. Here, we reveal that 5-azacytidine inhibits NMD in a dose-dependent fashion specifically upregulating the expression of both PTC-containing mutant and cellular NMD targets. Moreover, this activity of 5-azacytidine depends on the induction of MYC expression, thus providing a link between the effect of this drug and one of the key cellular pathways that are known to affect NMD activity. Furthermore, the effective concentration of 5-azacytidine in cells corresponds to drug levels used in patients, qualifying 5-azacytidine as a candidate drug that could potentially be repurposed for the treatment of Mendelian and acquired genetic diseases that are caused by PTC mutations.

Human cancer xenografts in outbred nude mice can be confounded by polymorphisms in a modifier of tumorigenesis.

Tumorigenicity studies often employ outbred nude mice, in the absence of direct evidence that this mixed genetic background will negatively affect experimental outcome. Here we show that outbred nude mice carry two different alleles of Pla2g2a, a genetic modifier of intestinal tumorigenesis in mice. Here, we identify previous unreported linked polymorphisms in the promoter, noncoding and coding sequences of Pla2g2a and show that outbred nude mice from different commercial providers are heterogeneous for this polymorphic Pla2g2a allele. This heterogeneity even extends to mice obtained from a single commercial provider, which display mixed Pla2g2a genotypes. Notably, we demonstrated that the polymorphic Pla2g2a allele affects orthotopic xenograft establishment of human colon cancer cells in outbred nude mice. This finding establishes a non-cell-autonomous role for Pla2g2a in suppressing intestinal tumorigenesis. Using in vitro reporter assays and pharmacological inhibitors, we show promoter polymorphisms and nonsense-mediated RNA decay (NMD) as underlying mechanisms that lead to low Pla2g2a mRNA levels in tumor-sensitive mice. Together, this study provides mechanistic insight regarding Pla2g2a polymorphisms and demonstrates a non-cell-autonomous role for Pla2g2a in suppressing tumors. Moreover, our direct demonstration that mixed genetic backgrounds of outbred nude mice can significantly affect baseline tumorigenicity cautions against future use of outbred mice for tumor xenograft studies.

Pure and syndromic optic atrophy explained by deep intronic OPA1 mutations and an intralocus modifier.

The genetic diagnosis in inherited optic neuropathies often remains challenging, and the emergence of complex neurological phenotypes that involve optic neuropathy is puzzling. Here we unravel two novel principles of genetic mechanisms in optic neuropathies: deep intronic OPA1 mutations, which explain the disease in several so far unsolved cases; and an intralocus OPA1 modifier, which explains the emergence of syndromic 'optic atrophy plus' phenotypes in several families. First, we unravelled a deep intronic mutation 364 base pairs 3' of exon 4b in OPA1 by in-depth investigation of a family with severe optic atrophy plus syndrome in which conventional OPA1 diagnostics including gene dosage analyses were normal. The mutation creates a new splice acceptor site resulting in aberrant OPA1 transcripts with retained intronic sequence and subsequent translational frameshift as shown by complementary DNA analysis. In patient fibroblasts we demonstrate nonsense mediated messenger RNA decay, reduced levels of OPA1 protein, and impairment of mitochondrial dynamics. Subsequent site-specific screening of >360 subjects with unexplained inherited optic neuropathy revealed three additional families carrying this deep intronic mutation and a base exchange four nucleotides upstream, respectively, thus confirming the clinical significance of this mutational mechanism. Second, in all severely affected patients of the index family, the deep intronic mutation occurred in compound heterozygous state with an exonic OPA1 missense variant (p.I382M; NM_015560.2). The variant alone did not cause a phenotype, even in homozygous state indicating that this long debated OPA1 variant is not pathogenic per se, but acts as a phenotypic modifier if it encounters in trans with an OPA1 mutation. Subsequent screening of whole exomes from >600 index patients identified a second family with severe optic atrophy plus syndrome due to compound heterozygous p.I382M, thus confirming this mechanism. In summary, we provide genetic and functional evidence that deep intronic mutations in OPA1 can cause optic atrophy and explain disease in a substantial share of families with unsolved inherited optic neuropathies. Moreover, we show that an OPA1 modifier variant explains the emergence of optic atrophy plus phenotypes if combined in trans with another OPA1 mutation. Both mutational mechanisms identified in this study-deep intronic mutations and intragenic modifiers-might represent more generalizable mechanisms that could be found also in a wide range of other neurodegenerative and optic neuropathy diseases.

Translation of Small Open Reading Frames within Unannotated RNA Transcripts in Saccharomyces cerevisiae

High-throughput gene expression analysis has revealed a plethora of previously undetected transcripts in eukaryotic cells. In this study, we investigate >1,100 unannotated transcripts in yeast predicted to lack protein-coding capacity. We show that a majority of these RNAs are enriched on polyribosomes akin to mRNAs. Ribosome profiling demonstrates that many bind translocating ribosomes within predicted open reading frames 10-96 codons in size. We validate expression of peptides encoded within a subset of these RNAs and provide evidence for conservation among yeast species. Consistent with their translation, many of these transcripts are targeted for degradation by the translation-dependent nonsense-mediated RNA decay (NMD) pathway. We identify lncRNAs that are also sensitive to NMD, indicating that translation of noncoding transcripts also occurs in mammals. These data demonstrate transcripts considered to lack coding potential are bona fide protein coding and expand the proteome of yeast and possibly other eukaryotes.

Identification and Characterization of Small Molecules That Inhibit Nonsense-Mediated RNA Decay and Suppress Nonsense p53 Mutations

Many of the gene mutations found in genetic disorders, including cancer, result in premature termination codons (PTC) and the rapid degradation of their mRNAs by nonsense-mediated RNA decay (NMD). We used virtual library screening, targeting a pocket in the SMG7 protein, a key component of the NMD mechanism, to identify compounds that disrupt the SMG7-UPF1 complex and inhibit NMD. Several of these compounds upregulated NMD-targeted mRNAs at nanomolar concentrations, with minimal toxicity in cell-based assays. As expected, pharmacologic NMD inhibition disrupted SMG7-UPF1 interactions. When used in cells with PTC-mutated p53, pharmacologic NMD inhibition combined with a PTC "read-through" drug led to restoration of full-length p53 protein, upregulation of p53 downstream transcripts, and cell death. These studies serve as proof-of-concept that pharmacologic NMD inhibitors can restore mRNA integrity in the presence of PTC and can be used as part of a strategy to restore full-length protein in a variety of genetic diseases.

The UPF1 RNA surveillance gene is commonly mutated in pancreatic adenosquamous carcinoma

Pancreatic adenosquamous carcinoma (ASC) is an enigmatic and aggressive tumor that has a worse prognosis and higher metastatic potential than its adenocarcinoma counterpart. Here we report that ASC tumors frequently harbor somatically acquired mutations in the UPF1 gene, which encodes the core component of the nonsense-mediated RNA decay (NMD) pathway. These tumor-specific mutations alter UPF1 RNA splicing and perturb NMD, leading to upregulated levels of NMD substrate mRNAs. UPF1 mutations are the first known unique molecular signatures of ASC.

DEPDC5 mutations in families presenting as autosomal dominant nocturnal frontal lobe epilepsy.

To study the prevalence of DEPDC5 mutations in a series of 30 small European families with a phenotype compatible with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Thirty unrelated families referred with ADNFLE were recruited in France, Italy, Germany, Belgium, and Norway. Whole-exome sequencing was performed in 10 probands and direct sequencing of the DEPDC5 coding sequence in 20 probands. Testing for nonsense-mediated messenger RNA decay (NMD) was performed in lymphoblastic cells. Exome sequencing revealed a splice acceptor mutation (c.2355-2A>G) in DEPDC5 in the proband of a German family. In addition, 3 nonsense DEPDC5 mutations (p.Arg487*, p.Arg1087*, and p.Trp1369*) were detected in the probands of 2 French and one Belgian family. The nonsense mutations p.Arg487* and p.Arg1087* were targeted by NMD, leading to the degradation of the mutated transcripts. At the clinical level, 78% of the patients with DEPDC5 mutations were drug resistant. DEPDC5 loss-of-function mutations were found in 13% of the families with a presentation of ADNFLE. The rate of drug resistance was high in patients with DEPDC5 mutations. Small ADNFLE pedigrees with DEPDC5 mutations might actually represent a part of the broader familial focal epilepsy with variable foci phenotype.

Early presentation of cystic kidneys in a family with a homozygous INVS mutation

Nephronophthisis (NPHP) is an autosomal recessive cystic kidney disease that is the most frequent monogenic cause of end-stage renal disease in children. Infantile NPHP, often in combination with other features like situs inversus, are commonly caused by mutations in the INVS gene. INVS encodes the ciliary protein inversin, and mutations induce dysfunction of the primary cilia. In this article, we present a family with two severely affected fetuses that were aborted after discovery of grossly enlarged cystic kidneys by ultrasonography before 22 weeks gestation. Exome sequencing showed that the fetuses were homozygous for a previously unreported nonsense mutation, resulting in a truncation in the IQ1 domain of inversin. This mutation induces nonsense-mediated RNA decay, as suggested by a reduced RNA level in fibroblasts derived from the fetus. However, a significant amount of mutant INVS RNA was present in these fibroblasts, yielding mutant inversin protein that was mislocalized. In control fibroblasts, inversin was present in the ciliary axoneme as well as at the basal body, whereas in the fibroblasts from the fetus, inversin could only be detected at the basal body. The phenotype of both fetuses is partly characteristic of infantile NPHP and Potter sequence. We also identified that the fetuses had mild skeletal abnormalities, including shortening and bowing of long bones, which may expand the phenotypic spectrum associated with INVS mutations.

Posttranscriptional Control of the Stem Cell and Neurogenic Programs by the Nonsense-Mediated RNA Decay Pathway

The mechanisms dictating whether a cell proliferates or differentiates have undergone intense scrutiny, but they remain poorly understood. Here, we report that UPF1, a central component in the nonsense-mediated RNA decay (NMD) pathway, plays a key role in this decision by promoting the proliferative, undifferentiated cell state. UPF1 acts, in part, by destabilizing the NMD substrate encoding the TGF-β inhibitor SMAD7 and stimulating TGF-β signaling. UPF1 also promotes the decay of mRNAs encoding many other proteins that oppose the proliferative, undifferentiated cell state. Neural differentiation is triggered when NMD is downregulated byneurally expressed microRNAs (miRNAs). This UPF1-miRNA circuitry is highly conserved and harbors negative feedback loops that act as a molecular switch. Our results suggest that the NMD pathway collaborates with the TGF-β signaling pathway to lock in the stem-like state, a cellular state that is stably reversed when neural differentiation signals that induce NMD-repressive miRNAs are received.