A MutSß-Dependent Contribution of MutSα to Repeat Expansions in Fragile X Premutation Mice?

The fragile X-related disorders result from expansion of a CGG/CCG microsatellite in the 5' UTR of the FMR1 gene. We have previously demonstrated that the MSH2/MSH3 complex, MutSß, that is important for mismatch repair, is essential for almost all expansions in a mouse model of these disorders. Here we show that the MSH2/MSH6 complex, MutSα also contributes to the production of both germ line and somatic expansions as evidenced by the reduction in the number of expansions observed in Msh6-/- mice. This effect is not mediated via an indirect effect of the loss of MSH6 on the level of MSH3. However, since MutSß is required for 98% of germ line expansions and almost all somatic ones, MutSα is apparently not able to efficiently substitute for MutSß in the expansion process. Using purified human proteins we demonstrate that MutSα, like MutSß, binds to substrates with loop-outs of the repeats and increases the thermal stability of the structures that they form. We also show that MutSα facilitates binding of MutSß to these loop-outs. These data suggest possible models for the contribution of MutSα to repeat expansion. In addition, we show that unlike MutSß, MutSα may also act to protect against repeat contractions in the Fmr1 gene.

Mutsß generates both expansions and contractions in a mouse model of the Fragile X-associated disorders.

Fragile X-associated disorders are Repeat Expansion Diseases that result from expansion of a CGG/CCG-repeat in the FMR1 gene. Contractions of the repeat tract also occur, albeit at lower frequency. However, these contractions can potentially modulate disease symptoms or generate an allele with repeat numbers in the normal range. Little is known about the expansion mechanism and even less about contractions. We have previously demonstrated that the mismatch repair (MMR) protein MSH2 is required for expansions in a mouse model of these disorders. Here, we show that MSH3, the MSH2-binding partner in the MutSß complex, is required for 98% of germ line expansions and all somatic expansions in this model. In addition, we provide evidence for two different contraction mechanisms that operate in the mouse model, a MutSß-independent one that generates small contractions and a MutSß-dependent one that generates larger ones. We also show that MutSß complexes formed with the repeats have altered kinetics of ATP hydrolysis relative to complexes with bona fide MMR substrates and that MutSß increases the stability of the CCG-hairpins at physiological temperatures. These data may have important implications for our understanding of the mechanism(s) of repeat instability and for the role of MMR proteins in this process.

X inactivation plays a major role in the gender bias in somatic expansion in a mouse model of the fragile X-related disorders: implications for the mechanism of repeat expansion.

The Fragile X-related disorders are X-linked disorders resulting from the inheritance of FMR1 alleles with >54 CGG/CCG repeats in their 5' UTR. The repeats expand both somatically and on intergenerational transmission and increased repeat numbers are associated with increased risk of disease and increased risk of further expansion. The mechanism responsible for expansion is unknown. Here, we show in a knockin mouse model of these disorders that somatic expansion is much less common in females than in males. We show that this is due in large part to the fact that expansions occur only when the repeat is on the active X chromosome. However, even when this is taken into account, expansions in females are still less common than expected. This additional gender effect is not due to a protective effect of estrogen, a deleterious effect of testosterone or to differences in the expression of the Fmr1 gene or a variety of X-linked and autosomal DNA repair genes. However, our data do suggest that a higher level of expression of genes that protect against oxidative damage in females may contribute to their lower levels of expansion. Whatever the basis, our data suggest that the risk for somatic expansion may be lower in women than it is in men. This could help explain the reduced penetrance of some aspects of disease pathology in women. The fact that expansion only occurs when the Fmr1 allele is on the active X chromosome has important implications for the mechanism of repeat expansion.

The transcription-coupled repair protein ERCC6/CSB also protects against repeat expansion in a mouse model of the fragile X premutation.

The fragile X-related disorders (FXDs) are members of the group of diseases known as the repeat expansion diseases. The FXDs result from expansion of an unstable CGG/CCG repeat tract in the 5' UTR of the FMR1 gene. Contractions are also seen, albeit at lower frequency. We have previously shown that ERCC6/CSB plays an auxiliary role in promoting germ line and somatic expansions in a mouse model of the FXDs. However, work in model systems of other repeat expansion diseases has suggested that CSB may protect against expansions by promoting contractions. Since FXD mice normally have such a high expansion frequency, it is possible that such a protective effect would have been masked. We thus examined the effect of the loss of CSB in an Msh2(+/-) background where the germ line expansion frequency is reduced and in an Msh2(-/-) background where expansions do not occur, but contractions do. Our data show that in addition to promoting repeat expansion, CSB does in fact protect the genome from germ line expansions in the FXD mouse model. However, it likely does so not by promoting contractions but by promoting an error-free process that preserves the parental allele.

Gender and cell-type-specific effects of the transcription-coupled repair protein, ERCC6/CSB, on repeat expansion in a mouse model of the fragile X-related disorders.

The repeat expansion diseases are human genetic disorders that arise from the expansion of a tandem-repeat tract. The Fragile X-related disorders are members of this disease group in which the repeat unit is CGG/CCG and is located in the 5' untranslated region of the FMR1 gene. Affected individuals often show mosaicism with respect to repeat number resulting from both expansion and contraction of the repeat tract; however, the mechanism responsible for these changes in repeat number is unknown. The work from a variety of model systems suggests that transcription-coupled repair (TCR) may contribute to repeat instability in diseases resulting from CAG/CTG-repeat expansion. To test whether TCR could contribute to repeat instability in the Fragile X-related disorders, we tested the effect of mutations in Csb (Cockayne syndrome group B), a gene essential for TCR, in a knock-in mouse model of these disorders. We found that the loss of CSB affects expansions in a gender and cell-type-specific manner. Our data also show an unanticipated gender difference in instability even in Csb+/+ animals that may have implications for our understanding of the mechanism of repeat expansion in the FX mouse model and perhaps for humans as well.

The mismatch repair protein MSH2 is rate limiting for repeat expansion in a fragile X premutation mouse model.

Fragile X-associated tremor and ataxia syndrome, Fragile X-associated primary ovarian insufficiency, and Fragile X syndrome are Repeat Expansion Diseases caused by expansion of a CGG•CCG-repeat microsatellite in the 5 UTR of the FMR1 gene. To help understand the expansion mechanism responsible for these disorders, we have crossed mice containing∼147 CGG•CCG repeats in the endogenous murine Fmr1 gene with mice containing a null mutation in the gene encoding the mismatch repair protein MSH2. MSH2 mutations are associated with elevated levels of generalized microsatellite instability. However, we show here for the first time that in the FX mouse model, all maternally and paternally transmitted expansions require Msh2. Even the loss of one Msh2 allele reduced the intergenerational expansion frequency significantly. Msh2 is also required for all somatic expansions and loss of even one functional Msh2 allele reduced the extent of somatic expansion in some organs. Tissues with lower levels of MSH2 were more sensitive to the loss of a single Msh2 allele. This suggests that MSH2 is rate limiting for expansion in this mouse model and that MSH2 levels may be a key factor that accounts for tissue-specific differences in expansion risk.

Intrathecal baclofen, a GABAB receptor agonist, inhibits the expression of p-CREB and NR2B in the spinal dorsal horn in rats with diabetic neuropathic pain.

This study aimed to investigate the effect of baclofen, a γ-aminobutyric acid B (GABAB) receptor agonist, on the expression of p-CREB and NR2B in the spinal dorsal horn of rats with diabetic neuropathic pain (DNP). The DNP rats, which were successfully induced with streptozocin, were distributed among 3 groups that were treated with saline (D1 group), baclofen (D2 group), or CGP55845 + baclofen (D3 group) continuously for 4 days. The rats induced with saline and subsequently treated with saline were used as controls (C group). The times for the paw withdrawal threshold and thermal withdrawal latency of the D1 group were lower than those for the C group, and were significantly increased after baclofen treatment, but not when GABA receptor was pre-blocked with CGP55845 (D3 group). Increased protein expression levels of NR2B and p-CREB and mRNA levels of NR2B were found in the D1 group when compared with the controls. Baclofen treatment significantly suppressed their expression, bringing it close to the levels of controls. However, in the D3 group, the expression of p-CREB and NR2B were still significantly higher than that of the controls. Activation of GABAB receptor by baclofen attenuates diabetic neuropathic pain, which may partly be accomplished via down-regulating the expression of p-CREB and NR2B.

Chromatin changes in the development and pathology of the Fragile X-associated disorders and Friedreich ataxia.

The Fragile X-associated disorders (FXDs) and Friedreich ataxia (FRDA) are genetic conditions resulting from expansion of a trinucleotide repeat in a region of the affected gene that is transcribed but not translated. In the case of the FXDs, pathology results from expansion of CGG•CCG-repeat tract in the 5' UTR of the FMR1 gene, while pathology in FRDA results from expansion of a GAA•TTC-repeat in intron 1 of the FXN gene. Expansion occurs during gametogenesis or early embryogenesis by a mechanism that is not well understood. Associated Expansion then produces disease pathology in various ways that are not completely understood either. In the case of the FXDs, alleles with 55-200 repeats express higher than normal levels of a transcript that is thought to be toxic, while alleles with >200 repeats are silenced. In addition, alleles with >200 repeats are associated with a cytogenetic abnormality known as a fragile site, which is apparent as a constriction or gap in the chromatin that is seen when cells are grown in presence of inhibitors of thymidylate synthase. FRDA alleles show a deficit of the FXN transcript. This review will address the role of repeat-mediated chromatin changes in these aspects of FXD and FRDA disease pathology. This article is part of a Special Issue entitled: Chromatin in time and space.

The relationship between processing speed and remodeling spatial patterns of intrinsic brain activity in the elderly with different sleep duration.

Objective: Brain neuroplasticity in which sleep affects the speed of information processing in the elderly population has not been reported. Therefore, this study was conducted to explore the effects of sleep on information processing speed and its central plasticity mechanism in the elderly. Methods: A total of 50 individuals aged 60 and older were enrolled in this case control study. All subjects were divided into two groups according to the sleep time: short sleep duration (< 360 min) (6 men and 19 women; mean age: 66.96 ± 4.28 years old), and non-short sleep duration (> 360 min) (13 men and 12 women). Resting-state functional magnetic resonance imaging (rs-fMRI) data were collected, and the amplitude of low frequency fluctuation (ALFF), regional homogeneity (ReHo), and degree centrality (DC) were calculated for each participant. Two-sample t-tests were performed to compare the ALFF, ReHo, and DC maps between the two groups. Then, the relationships among clinical features, fMRI and cognitive function were analyzed using general linear model. Results: Short sleep duration group showed significantly increased ALFF value in the bilateral middle frontal gyrus and right insula; significantly increased ReHo value in the left superior parietal gyrus, and decreased ReHo value in the right crebellum; significantly decreased DC value in the left inferior occipital gyrus, left superior parietal gyrus and right cerebellum (p < 0.05, AlphaSim correction). The ALFF value of right insula is significantly associated with symbol digit modalities test (SDMT) score (ß = -0.363, p = 0.033). Conclusion: Short sleep duration and processing speed are significantly associated with remodeling spatial patterns of intrinsic brain activity in the elderly.

[Concentration, Source, and Health Risk Assessment of PM1 Heavy Metals in Typical Pollution Processes in Zhengzhou].

Heavy metal elements in particulate matter can cause adverse effects on human health, and the smaller the particle size, the greater the harm. A total of 16 heavy metal elements (Al, Si, K, Ca, V, Cr, Mn, Fe, Ni, Cu, Zn, As, Se, Ba, Pb, and Cd) in PM1 were continuously determined by an online heavy metal observation instrument in Zhengzhou city from January 7 to 25, 2021. The results showed that ρ(K) concentration was the highest during the observation period (0.62 µg·m-3). According to pollutant concentration and meteorological characteristics, the observation period was divided into clean days, dust days, and haze days. The contribution of heavy metal pollution characteristics and health risk assessment in atmospheric PM1 was different under different pollution processes. The US EPA health risk assessment method was used to assess the health risks of heavy metals, and the enrichment factor method and positive matrix factorization (PMF) were used to analyze the sources of heavy metals. The influence of the transmission was evaluated by using the concentration-weighted trajectory (CWT) method and the backward trajectory method. The results show that the enrichment factors of Zn, As, Se, Pb, and Cd were more than 100 under different pollution processes, which were greatly affected by human activities. During the sampling period, the main sources of heavy metals were industrial sources, coal/biomass sources, motor vehicle sources, and dust sources. The results of the health risk assessment were substituted into PMF analysis, and it was found that industrial sources were the main contributing sources of carcinogenic and non-carcinogenic health risks during cleaning days, dust days, and haze days, and the carcinogenic risk of heavy metal elements in PM1 in this region for adults exceeded that for children. CWT and backward trajectory methods revealed that regional transmission was one of the main factors affecting local health risks.

[Characteristics and Sources of PM2.5 Pollution in Typical Cities of the Central Plains Urban Agglomeration in Autumn and Winter].

To study the characteristics of PM2.5 pollution and the potential sources of its main components in the central plain urban agglomeration in autumn and winter, PM2.5 samples were collected continually in the four typical cities of Zhengzhou, Luoyang, Anyang, and Xinxiang from October 2018 to January 2019. X-ray fluorescence spectrometry, carbon analysis methods, and ion chromatography were used to determine 18 kinds of inorganic elements, organic carbon (OC)/elemental carbon (EC), and 9 kinds of water-soluble inorganic ions. According to the daily PM2.5 concentration, three pollution levels were divided, and the comparative analysis for the spatial and temporal variation of PM2.5 and its main components, i.e., NO3-, OC, and 18 kinds of inorganic elements, were studied via the calculation of the nitrogen oxidation rate (NOR), secondary organic carbon (SOC), and enrichment factor. The emission sources and their contribution rates of PM2.5 pollution level in the four cities were calculated by a chemical mass balance (CMB) model; the potential pollution sources of PM2.5 and its main components, NO3- and OC, in the four cities were analyzed by a backward trajectory model (HYSPLIT) and potential source contribution factor method (PSCF). The results showed that the means of PM2.5 in Zhengzhou, Luoyang, Anyang, and Xinxiang were (82.1±45.5), (84.7±39.8), (96.8±46.1), and (81.1±36.6) µg·m-3, respectively, during the sampling period, and the maximum daily mean values were 3.3, 2.6, 3.0, and 2.3 times, respectively, of the Chinese national secondary standard; the main components of PM2.5 in the four cities were NO3- and SOC, and the concentration of NO3-, the ratio of NO3-/EC, and NOR all increased significantly with the rising of pollution levels, generally showing that the mean values of NO3-/EC and NOR of Zhengzhou and Luoyang were a little higher than those of Anyang and Xinxiang; the concentration of SOC, the proportion of SOC in OC, and the ratio of SOC/EC all increased with the rising of pollution levels. From the concentration and enrichment degree of inorganic elements, As was the highest in Zhengzhou; Mn and Fe were the highest in Luoyang; Zn, Ni, and Cr were the highest in Anyang; and Cu and Pb were the highest in Xinxiang. Secondary nitrate, secondary sulfate, organic matter, coal combustion, motor vehicle, dust, biomass burning, and industrial processes were the main PM2.5 pollution sources in the four cities, with the highest contribution rate of secondary nitrate in Zhengzhou (37.7%), the highest contribution rate of vehicle sources in Xinxiang (14.1%), and a relatively high contribution rate of industrial process source in Luoyang (7.0%) and Anyang (6.8%). The northwest direction of airflow contributed 51.6%, 49.2%, 49.6%, and 46.3% of the total airflow in Zhengzhou, Luoyang, Anyang, and Xinxiang, respectively. From the potential pollution area of each city, the Zhengzhou area was mainly concentrated in Henan province, the Luoyang area was mainly concentrated in the south of Henan province and Fen-wei plain, and the Anyang and Xinxiang areas were mainly concentrated in Henan province and the Beijing-Tianjin-Hebei transport belt. The pollution levels of OC in Anyang and Xinxiang were also affected by the northwest Anhui, southwest Shandong, southeast Shanxi, and north Shaanxi.

[Chemical Components and Sources of PM2.5 and Their Evolutive Characteristics in Zhengzhou].

To explore the main sources of PM2.5 and the characteristics of seasonal differences in Zhengzhou, PM2.5 sampling was conducted in 2019 and the concentrations of inorganic water-soluble ions, carbon components, and various elements were analyzed. Results showed that the average mass concentration of PM2.5 in 2019 was (67.0±37.2) µg ·m-3 with the highest concentration in winter and the lowest in summer. The main components of PM2.5 were nitrate, ammonium, sulfate, organic matter, crustal matter, and elemental carbon. In spring and autumn, PM2.5 was greatly affected by crustal matter and elemental carbon, and In summer, concentrations were mainly affected by sulfate. In winter, the concentrations of organic matter and nitrate increased significantly, produced by photochemical reactions in summer and aqueous-phase reactions under high humidity in winter. Carbonaceous aerosols were greatly influenced by automobile exhaust emission, coal combustion, and biomass combustion. Source apportionment showed that secondary sources were the greatest contributors in all four seasons, particularly in in winter (56.5%). Among the primary sources, the proportion of dust in spring (15.2%) and autumn (11.4%) was slightly higher, and the contribution of motor vehicle pollution was the largest (12.3%) in summer. In winter, PM2.5was greatly affected by coal combustion (13.2%). From 2014 to 2019, PM2.5 in Zhengzhou increased annually under the influence of secondary sources. The contribution of industrial sources, biomass combustion sources, and coal combustion sources exhibited a downward trend over this period.

M1 Macrophage-Derived Exosomal MicroRNA-326 Suppresses Hepatocellular Carcinoma Cell Progression Via Mediating NF-κB Signaling Pathway.

Accumulating evidence has shown that microRNA (miR) derived from M1 macrophage-derived exosomes can regulate the progression of hepatocellular carcinoma (HCC). However, the effect of miR-326 derived from M1 macrophage-derived exosomes on HCC has not been reported. Therefore, the objective of the present study was to explore the mechanism of exosomal miR-326 from M1 macrophages in regulating HCC cell progression. RT-qPCR detected miR-326 expression in HCC cell lines. miR-326 expression in HCC was altered by transfection, and the effect of miR-326 on CD206 and NF-κB expression, cell proliferation, colony formation, migration, apoptosis and invasion was detected. Subsequently, exosomes were isolated from M1 macrophages. RT-qPCR identified miR-326 expression in M1 macrophage-derived exosomes. miR-326 expression in M1 macrophage-derived exosomes was changed by transfection. M1 macrophage-derived exosomes were co-cultured with HCC cells to figure out their effects on the biological progress of HCC cells. Finally, in vivo experiments were performed to verify the in vitro results. MiR-326 was decreased in HCC cells and enriched in M1 macrophage-derived exosomes. Up-regulating miR-326 would inhibit HCC cell proliferation, colony formation, migration, invasion, and CD206 and NF-κB expression and promoted apoptosis, and inhibited the growth of HCC tumors in vivo, while down-regulating miR-326 showed opposite effects. M1 macrophage-derived exosomes inhibited HCC cell proliferation, colony formation, migration, invasion, and CD206 and NF-κB expression and enhanced apoptosis, while overexpression of miR-326 enhanced the effect of M1 macrophage-derived exosomes on HCC cells. It is revealed that M1 macrophages-derived exosomal miR-326 suppresses proliferation, migration and invasion as well as advances apoptosis of HCC through down-regulating NF-κB expression.

The NK-1R Antagonist Aprepitant Prevents LPS-Induced Oxidative Stress and Inflammation in RAW264.7 Macrophages.

BACKGROUND: The macrophage is one of the most important types of immune cells that protect against harmful stimuli. Macrophage activation plays a pivotal role in the progression and development of various inflammatory diseases. The neurokinin 1 receptor (NK-1R) is a G protein-coupled receptor that plays an important role in inflammatory diseases. Aprepitant is a kind of NK-1R antagonist. The purpose of this study is to determine the protective effect of aprepitant in lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. METHODS: We examined the anti-inflammatory and anti-oxidant effects of aprepitant in LPS-treated RAW264.7 macrophages by using real-time PCR, ELISA, and Western blot analysis. We also assessed cellular oxidative stress signaling by measuring the levels of cellular MDA, total ROS, and NADPH oxidase expression. Cellular NO production was measured by DAF-FM DA staining. The inhibitory effect of aprepitant against NF-κB signaling was evaluated by luciferase assay and Western blot analysis. RESULTS: The expression of NK-1R is increased in LPS-induced macrophages, suggesting a potential role of the receptor in the inflammatory response. We show that aprepitant protects macrophages against oxidative stress by reducing the generation of ROS and the expression of NOX-4. Furthermore, aprepitant inhibits the secretion of pro-inflammatory cytokines and chemotactic factors by mediating the NF-κB signaling pathway. CONCLUSION: The NK-1R receptor antagonist aprepitant acts as an anti-inflammatory agent, indicating that the blockage of the NK-1R pathway in macrophages has the potential to suppress inflammation.

Whole-genome sequencing reveals origin and evolution of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018.

The continuous variation of the seasonal influenza viruses, particularly A(H1N1)pdm09, persistently threatens human life and health around the world. In local areas of southwest china, the large time-scale genomic research on A(H1N1)pdm09 is still insufficient. Here, we sequenced 45 whole-genome sequences of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018, by next-generation sequencing technology to characterize molecular mechanisms of their origin and evolution. Our phylogenetic analyses suggest that the A(H1N1)pdm09 strains circulating in Lincang belong to clade 6B and the subclade 6B.1A predominates in 2018. Further, the strains in 2018 possess elevated evolutionary rate as compared to strains in other years. Several newly emerged mutations for HA (hemagglutinin) in 2018 are revealed (i.e., S183P and R221K). Intriguingly, the substitution R221K falls into the RBS (receptor binding site) of HA protein, which could affect antigenic properties of influenza A(H1N1)pdm09 viruses, and another substitution S183P near to RBS with a high covering frequency (11/14 strains) in 2018 is exactly located at the epitope B. Notably, the NA (neuraminidase) protein harbors a new mutation I23T, potentially involved in N-glycosylation. Based on the background with a higher evolutionary rate in 2018 strains, we deeply evaluate the potential vaccine efficacy against Lincang strains and discover a substantive decline of the vaccine efficacy in 2018. Our analyses reaffirm that the real-time molecular surveillance and timely updated vaccine strains for prevention and control of influenza A(H1N1)pdm09 are crucial in the future.

[Identification, characterization and utilization of simple sequence repeat markers derived from Salvia miltiorrhiza expressed sequence tags].

Despite Salvia miltiorrhiza being one of the most important medicine plants in China, there is a limited availability of genomic resources, especially of the expressed sequence tag-based markers. In this study, we selected and characterized functional markers in S. miltiorrhiza, which consisted of 4,192 non-redundant expressed sequence tags (ESTs) from 10,288 identified S. miltiorrhiza ESTs in dbEST data bank. Among them, 159 simple sequence repeats (SSR) were detected, which amounted to 3.79% of the non-redundant starting sequence population. This incidence was equivalent to one EST-SSR in every 12.74 kb of S. miltiorrhiza ESTs. Among the different motifs ranging from 1 bp to 6 bp, di-nucleotide repeat motif was the most abundant (77, 48.43%), followed by tri-nucleotide (41, 25.79%), hexa-nucleotide (23, 14.47%), penta-nucleotide (12, 7.55%) and tetra-nucleotide (6, 3.77%). In 47 identified motif types, the detected frequency above 5% were GA/CT (16.35%), AG/TC (15.09%), TCA/AGT (10.69%), AT/TA (6.29%), GAAAAG/CAAAAC (6.29%) and TA/AT (5.03%). Based on flank sequence of detected SSR, a total of 83 EST-SSR primer pairs were designed and tested for the amplification efficiency, polymorphism and transferability in thirteen S. mihiorrhiza samples and other ten species from the genus Salvia. The results showed that 72 primer pairs were successfully amplified in S. miltiorrhiza samples to yield and 279 loci with an average of 3.88 loci per primer pair. The cross-transferability of S. miltiorrhiza EST-SSR markers to other ten Salvia plants was very high, ranging from 60% to 100% with an average of 85%. Further analysis of the genetic similarity based on the polymorphic bands showed the EST-SSR could detect the genetic diversity on different levels among the whole test samples and distinguish the S. miltiorrhiza from other Salvia plants effectively. It is expected that the potential markers described here would add to the repertoire of DNA markers needed for genetic analysis, linkage mapping and comparative genomics studies in S. miltiorrhiza and related Salvia genus plants.

RNA secondary structures located in the interchromosomal region of human ACAT1 chimeric mRNA are required to produce the 56-kDa isoform.

We have previously reported that the human ACAT1 gene produces a chimeric mRNA through the interchromosomal processing of two discontinuous RNAs transcribed from chromosomes 1 and 7. The chimeric mRNA uses AUG(1397-1399) and GGC(1274-1276) as translation initiation codons to produce normal 50-kDa ACAT1 and a novel enzymatically active 56-kDa isoform, respectively, with the latter being authentically present in human cells, including human monocyte-derived macrophages. In this work, we report that RNA secondary structures located in the vicinity of the GGC(1274-1276) codon are required for production of the 56-kDa isoform. The effects of the three predicted stem-loops (nt 1255-1268, 1286-1342 and 1355-1384) were tested individually by transfecting expression plasmids into cells that contained the wild-type, deleted or mutant stem-loop sequences linked to a partial ACAT1 AUG open reading frame (ORF) or to the ORFs of other genes. The expression patterns were monitored by western blot analyses. We found that the upstream stem-loop(1255-1268) from chromosome 7 and downstream stem-loop(1286-1342) from chromosome 1 were needed for production of the 56-kDa isoform, whereas the last stem-loop(1355-1384) from Chromosome 1 was dispensable. The results of experiments using both monocistronic and bicistronic vectors with a stable hairpin showed that translation initiation from the GGC(1274-1276) codon was mediated by an internal ribosome entry site (IRES). Further experiments revealed that translation initiation from the GGC(1274-1276) codon requires the upstream AU-constituted RNA secondary structure and the downstream GC-rich structure. This mechanistic work provides further support for the biological significance of the chimeric nature of the human ACAT1 transcript.

FAN1 protects against repeat expansions in a Fragile X mouse model.

The Fragile X-related disorders (FXDs) are members of a large group of human neurological or neurodevelopmental conditions known as the Repeat Expansion Diseases. The mutation responsible for all of these diseases is an expansion in the size of a disease-specific tandem repeat tract. However, the underlying cause of this unusual mutation is unknown. Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) in the vicinity of the FAN1 (MIM* 613534) gene that are associated with variations in the age at onset of a number of Repeat Expansion Diseases. FAN1 is a nuclease that has both 5'-3' exonuclease and 5' flap endonuclease activities. Here we show in a model for the FXDs that Fan1-/- mice have expansions that, in some tissues including brain, are 2-3 times as extensive as they are in Fan1+/+ mice. However, no effect of the loss of FAN1 was apparent for germ line expansions. Thus, FAN1 plays an important role in protecting against somatic expansions but is either not involved in protecting against intergenerational repeat expansions or is redundant with other related enzymes. However, since loss of FAN1 results in increased expansions in brain and other somatic tissue, FAN1 polymorphisms may be important disease modifiers in those Repeat Expansion Diseases in which somatic expansion contributes to age at onset or disease severity.

Timing of Expansion of Fragile X Premutation Alleles During Intergenerational Transmission in a Mouse Model of the Fragile X-Related Disorders.

Fragile X syndrome (FXS) is caused by the maternal expansion of an unstable CGG-repeat tract located in the first exon of the FMR1 gene. Further changes in repeat number occur during embryogenesis resulting in individuals sometimes being highly mosaic. Here we show in a mouse model that, in males, expansions are already present in primary spermatocytes with no additional expansions occurring in later stages of gametogenesis. We also show that, in females, expansion occurs in the post-natal oocyte. Additional expansions and a high frequency of large contractions are seen in two-cell stage embryos. Expansion in oocytes, which are non-dividing, would be consistent with a mechanism involving aberrant DNA repair or recombination rather than a problem with chromosomal replication. Given the difficulty of replicating large CGG-repeat tracts, we speculate that very large expanded alleles may be prone to contract in the mitotically proliferating spermatagonial stem cells in men. However, expanded alleles may not be under such pressure in the non-dividing oocyte. The high degree of both expansions and contractions seen in early embryos may contribute to the high frequency of somatic mosaicism that is observed in humans. Our data thus suggest an explanation for the fact that FXS is exclusively maternally transmitted and lend support to models for repeat expansion that are based on problems arising during DNA repair.