Splice Forms Research Articles

Tissue specific splicing of pre-mRNA porcine mitochondrial transcription factor A

Mitochondrial transcription factor A plays, due to its role in replication and transcription of the mitochondrial DNA, an important role in mitochondrial biogenesis and regulation of its activity. In different mammalian species, mitochondrial transcription factor A is present in two different splicing forms. The aim of the study was to verify the presence of both splicing forms and to estimate their ratio in different tissues. The expression levels of both splicing forms in two skeletal muscles (m. semispinalis capitis and m. semimembranosus), heart muscle, brain, spleen, and liver in commercial hybrids and in the autochthonous Krsko polje pig breed are reported. In all analyzed tissues the expression of the long form of the mitochondrial transcription factor A was about one order of magnitude higher compared to the short form, lacking almost the entire exon 4 region. Taking into account the expression ratio of both splicing forms and biological function of the mitochondrial transcription factor A, the possible explanation for the persistence of both forms in different mammalian species is provided.

Exploring the relationship between intron retention and chromatin accessibility in plants

BackgroundIntron retention (IR) is the most prevalent form of alternative splicing in plants. IR, like other forms of alternative splicing, has an important role in increasing gene product diversity and regulating transcript functionality. Splicing is known to occur co-transcriptionally and is influenced by the speed of transcription which in turn, is affected by chromatin structure. It follows that chromatin structure may have an important role in the regulation of splicing, and there is preliminary evidence in metazoans to suggest that this is indeed the case; however, nothing is known about the role of chromatin structure in regulating IR in plants. DNase I-seq is a useful experimental tool for genome-wide interrogation of chromatin accessibility, providing information on regions of chromatin with very high likelihood of cleavage by the enzyme DNase I, known as DNase I Hypersensitive Sites (DHSs). While it is well-established that promoter regions are highly accessible and are over-represented with DHSs, not much is known about DHSs in the bodies of genes, and their relationship to splicing in general, and IR in particular.ResultsIn this study we use publicly available DNase I-seq data in arabidopsis and rice to investigate the relationship between IR and chromatin structure. We find that IR events are highly enriched in DHSs in both species. This implies that chromatin is more open in retained introns, which is consistent with a kinetic model of the process whereby higher speeds of transcription in those regions give less time for the spliceosomal machinery to recognize and splice out those introns co-transcriptionally. The more open chromatin in IR can also be the result of regulation mediated by DNA-binding proteins. To test this, we performed an exhaustive search for footprints left by DNA-binding proteins that are associated with IR. We identified several hundred short sequence elements that exhibit footprints in their DNase I-seq coverage, the telltale sign for binding events of a regulatory protein, protecting its binding site from cleavage by DNase I. A highly significant fraction of those sequence elements are conserved between arabidopsis and rice, a strong indication of their functional importance.ConclusionsIn this study we have established an association between IR and chromatin accessibility, and presented a mechanistic hypothesis that explains the observed association from the perspective of the co-transcriptional nature of splicing. Furthermore, we identified conserved sequence elements for DNA-binding proteins that affect splicing.

Loss of an Androgen-Inactivating and Isoform-Specific HSD17B4 Splice Form Enables Emergence of Castration-Resistant Prostate Cancer

SUMMARYCastration-resistant prostate cancer (CRPC) requires tumors to engage metabolic mechanisms that allow sustained testosterone and/or dihydrotestosterone to stimulate progression. 17β-Hydroxyste-roid dehydrogenase type 4 (17βHSD4), encoded by HSD17B4, is thought to inactivate testosterone and dihydrotestosterone by converting them to their respective inert 17-keto steroids. Counterintuitively, HSD17B4 expression increases in CRPC and predicts poor prognosis. Here, we show that, of five alternative splice forms, only isoform 2 encodes an enzyme capable of testosterone and dihydrotestosterone inactivation. In contrast with other transcripts, functional expression of isoform 2 is specifically suppressed in development of CRPC in patients. Genetically silencing isoform 2 shifts the metabolic balance toward 17β-OH androgens (testosterone and dihydrotestosterone), stimulating androgen receptor (AR) and CRPC development. Our studies specifically implicate HSD17B4 isoform 2 loss in lethal prostate cancer.

Abstract A129: A second-site mutation in BRAF confers resistance to RAF inhibition in a BRAF V600E-mutant brain tumor

Abstract BRAF V600E drives tumors by hyperactivating ERK signaling, is activated in a RAS-independent manner, and signals as a monomer. Selective inhibitors of RAF potently inhibit BRAF V600E and downstream ERK signaling, and have been associated with impressive clinical responses in patients whose tumors harbor V600E mutations in BRAF. Almost all patients, however, ultimately develop resistance. To date, acquired resistance mechanisms validated in patient samples have included splice forms of BRAF, mutations in NRAS, and BRAF amplification, all of which promote formation of RAF dimers, and mutations in MEK. Second-site point mutations in BRAF that promote dimer formation and are associated with acquired resistance have not previously been identified in RAF inhibitor-resistant tumors. Here, we report a complete response followed by clinical progression of a BRAF V600E-mutant pediatric brain tumor treated with the RAF inhibitor dabrafenib. We identified a second-site mutation in BRAF at the time of progression that was not present in the pretreatment tumor. Further study indicated the acquired mutation is in cis with V600E. We demonstrate that the acquired resistance mutation induces RAF dimer formation, and is sufficient to confer resistance to dabrafenib. Moreover, we showed that ERK signaling activated by the double mutation is sensitive to MEK inhibition or to the RAF plus and MEK inhibitor combination. We also find that two novel RAF dimer inhibitors may overcome resistance mediated by the novel mutation in BRAF. This is the first study, to our knowledge, that demonstrates the emergence of a confirmed second-site mutation in BRAF V600E and validates that this mutation is responsible for acquired resistance in an initially responding patient treated with dabrafenib. The mechanism of acquired resistance can be abrogated by the novel RAF dimer inhibitors. Our data confirm that a novel class of RAF dimer inhibitors are active against an acquired resistance mutation, suggesting an improved personalized treatment option for patients who harbor similar second-site mutations in BRAF. Citation Format: Jiawan Wang, Zhan Yao, Philip Jonsson, Amy Allen, Alice Can Ran Qin, David Pisapia, Neal Rosen, Barry S. Taylor, Christine A. Pratilas. A second-site mutation in BRAF confers resistance to RAF inhibition in a BRAF V600E-mutant brain tumor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A129.

Long Non-coding RNAs, Novel Culprits, or Bodyguards in Neurodegenerative Diseases.

Long non-coding RNA (lncRNA) is a kind of non-coding RNA (ncRNA), with a length of 200 nt to 100 kb, that lacks a significant open reading frame (ORF) encoding a protein. lncRNAs are widely implicated in various physiological and pathological processes, such as epigenetic regulation, cell cycle regulation, cell differentiation regulation, cancer, and neurodegenerative diseases, through their interactions with chromatin, protein, and other RNAs. Numerous studies have suggested that lncRNAs are closely linked with the occurrence and development of a variety of diseases, especially neurodegenerative diseases, of which the etiologies are complicated and the underlying mechanisms remain elusive. Determining the roles of lncRNA in the pathogenesis of neurodegenerative diseases will not only deepen understanding of the physiological and pathological processes that occur in those diseases but also provide new ideas and solutions for their diagnosis and prevention. This review aims to highlight the progress of lncRNA research in the pathological and behavioral changes of neurodegenerative diseases. Specifically, we focus on how lncRNA dysfunctions are involved in the pathogenesis of Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis.

A spliced form of CD44 expresses the unique glycan that is recognized by the prostate cancer specific antibody F77.

Prostate cancer is the most common cancer occurring in men in the United States. The monoclonal antibody F77 that was originally developed in our laboratory recognizes mainly glycolipids as well as O-linked glycosylation on proteins in prostate cancer cells. We have identified a spliced form of glycoprotein CD44 as one critical protein expressing the F77 antigen. The F77-specific glycosylation occurs on multiple potential glycosylation sites on the CD44 protein encoded by the fourteenth exon. CD44 is a tumor stem cell marker and is known to induce tumor stemness and metastasis. Knockdown of CD44 or FUT1 genes dramatically reduced F77-induced apoptosis in prostate cancer cell lines. We developed an ELISA using both a CD44 antibody and F77 to identify the special form of glycosylated CD44 from prostate cancer cells as well as from serum samples of prostate cancer patients. These results reveal a CD44-dependent mechanism for F77 to induce tumor cell apoptosis, and a new strategy for the detection of glycosylated CD44 proteins secreted by prostate cancer cells.

Regulation of cell proliferation in the retinal pigment epithelium: Differential regulation of the death-associated protein like-1 DAPL1 by alternative MITF splice forms.

Vertebrate eye development and homoeostasis critically depend on the regulation of proliferation of cells forming the retinal pigment epithelium (RPE). Previous results indicated that the death-associated protein like-1 DAPL1 cell autonomously suppresses RPE proliferation in vivo and in vitro. Here, we show in human RPE cell lines that the pigment cell transcription factor MITF regulates RPE cell proliferation by upregulating DAPL1 expression. DAPL1 regulation by MITF is, however, mediated predominantly by (-) MITF, one of two alternative splice isoforms of MITF that lacks six residues located upstream of the DNA-binding basic domain. Furthermore, we find that the regulation of DAPL1 by MITF is indirect in that (-) MITF stimulates the transcription of Musashi homolog-2 (MSI2), which negatively regulates the processing of the anti-DAPL1 microRNA miR-7. Our results provide molecular insights into the regulation of RPE cell proliferation and quiescence and may help us understand the mechanisms of normal RPE maintenance and of eye diseases associated with either RPE hyperproliferation or the lack of regenerative proliferation.

Identification and expression of CAMTA genes in Populus trichocarpa under biotic and abiotic stress

The calmodulin-binding transcription activators (CAMTAs) transcription factor family plays an important role in normal plant growth and development, as well as in biotic and abiotic stress resistance. In this study, we identified seven CAMTA genes across the whole genome of Populus trichocarpa and analyzed the expression patterns of PtCAMTAs in the root and leaf tissues. Promoter cis-element analysis indicated that most CAMTA genes contained stress- or phytohormone-related cis-elements. Quantitative real-time reverse transcription-PCR (qRT-PCR) indicated indicated that PtCAMTAs were induced by mannitol, NaCl, cold stress, pathogenic infection with A. alternata, and phytohormone treatments with abscisic acid, salicylic acid, and methyl jasmonate. We analyzed the expression of homologous genes between P. trichocarpa and P. ussuriensis and alternative splicing forms of PtCAMTA genes under cold stress. We also performed a network interaction analysis for PtCAMTA proteins to predict their interactions and associations. The results of the present study serve as a basis for future functional studies on the Populus CAMTA family.

Defense against Reactive Carbonyl Species Involves at Least Three Subcellular Compartments Where Individual Components of the System Respond to Cellular Sugar Status.

Methylglyoxal (MGO) and glyoxal (GO) are toxic reactive carbonyl species generated as by-products of glycolysis. The pre-emption pathway for detoxification of these products, the glyoxalase (GLX) system, involves two consecutive reactions catalyzed by GLXI and GLXII. In Arabidopsis thaliana, the GLX system is encoded by three homologs of GLXI and three homologs of GLXII, from which several predicted GLXI and GLXII isoforms can be derived through alternative splicing. We identified the physiologically relevant splice forms using sequencing data and demonstrated that the resulting isoforms have different subcellular localizations. All three GLXI homologs are functional in vivo, as they complemented a yeast GLXI loss-of-function mutant. Efficient MGO and GO detoxification can be controlled by a switch in metal cofactor usage. MGO formation is closely connected to the flux through glycolysis and through the Calvin Benson cycle; accordingly, expression analysis indicated that GLXI is transcriptionally regulated by endogenous sugar levels. Analyses of Arabidopsis loss-of-function lines revealed that the elimination of toxic reactive carbonyl species during germination and seedling establishment depends on the activity of the cytosolic GLXI;3 isoform. The Arabidopsis GLX system involves the cytosol, chloroplasts, and mitochondria, which harbor individual components that might be used at specific developmental stages and respond differentially to cellular sugar status.

Implementation of CsLIS/NES in linalool biosynthesis involves transcript splicing regulation in Camellia sinensis.

Volatile terpenoids produced in tea plants (Camellia sinensis) are airborne signals interacting against other ecosystem members, but also pleasant odorants of tea products. Transcription regulation (including transcript processing) is pivotal for plant volatile terpenoid production. In this study, a terpene synthase gene CsLIS/NES was recovered from tea plants (C.sinensis cv. "Long-Men Xiang"). CsLIS/NES transcription regulation resulted in 2 splicing forms: CsLIS/NES-1 and CsLIS/NES-2 lacking a 305bp-fragment at N-terminus, both producing (E)-nerolidol and linalool in vitro. Transgenic tobacco studies and a gene-specific antisense oligo-deoxynucleotide suppression applied in tea leaves indicated that CsLIS/NES-1, localized in chloroplasts, acted as linalool synthase, whereas CsLIS/NES-2 localized in cytosol, functioned as a potential nerolidol synthase, but not linalool synthase. Expression patterns of the 2 transcript isoforms in tea were distinctly different and responded differentially to the application of stress signal molecule methyl jasmonate. Leaf expression of CsLIS/NES-1, but not CsLIS/NES-2, was significantly induced by methyl jasmonate. Our data indicated that distinct transcript splicing regulation patterns, together with subcellular compartmentation of CsLIS/NE-1 and CsLIS/NE-2 implemented the linalool biosynthesis regulation in tea plants in responding to endogenous and exogenous regulatory factors.

Intron retention enhances gene regulatory complexity in vertebrates

BackgroundWhile intron retention (IR) is now widely accepted as an important mechanism of mammalian gene expression control, it remains the least studied form of alternative splicing. To delineate conserved features of IR, we performed an exhaustive phylogenetic analysis in a highly purified and functionally defined cell type comprising neutrophilic granulocytes from five vertebrate species spanning 430 million years of evolution.ResultsOur RNA-sequencing-based analysis suggests that IR increases gene regulatory complexity, which is indicated by a strong anti-correlation between the number of genes affected by IR and the number of protein-coding genes in the genome of individual species. Our results confirm that IR affects many orthologous or functionally related genes in granulocytes. Further analysis uncovers new and unanticipated conserved characteristics of intron-retaining transcripts. We find that intron-retaining genes are transcriptionally co-regulated from bidirectional promoters. Intron-retaining genes have significantly longer 3′ UTR sequences, with a corresponding increase in microRNA binding sites, some of which include highly conserved sequence motifs. This suggests that intron-retaining genes are highly regulated post-transcriptionally.ConclusionsOur study provides unique insights concerning the role of IR as a robust and evolutionarily conserved mechanism of gene expression regulation. Our findings enhance our understanding of gene regulatory complexity by adding another contributor to evolutionary adaptation.

De novo transcriptome sequencing and assembly from apomictic and sexual Eragrostis curvula genotypes

A long-standing goal in plant breeding has been the ability to confer apomixis to agriculturally relevant species, which would require a deeper comprehension of the molecular basis of apomictic regulatory mechanisms. Eragrostis curvula (Schrad.) Nees is a perennial grass that includes both sexual and apomictic cytotypes. The availability of a reference transcriptome for this species would constitute a very important tool toward the identification of genes controlling key steps of the apomictic pathway. Here, we used Roche/454 sequencing technologies to generate reads from inflorescences of E. curvula apomictic and sexual genotypes that were de novo assembled into a reference transcriptome. Near 90% of the 49568 assembled isotigs showed sequence similarity to sequences deposited in the public databases. A gene ontology analysis categorized 27448 isotigs into at least one of the three main GO categories. We identified 11475 SSRs, and several of them were assayed in E curvula germoplasm using SSR-based primers, providing a valuable set of molecular markers that could allow direct allele selection. The differential contribution to each library of the spliced forms of several transcripts revealed the existence of several isotigs produced via alternative splicing of single genes. The reference transcriptome presented and validated in this work will be useful for the identification of a wide range of gene(s) related to agronomic traits of E. curvula, including those controlling key steps of the apomictic pathway in this species, allowing the extrapolation of the findings to other plant species.

Quantitative RNA-seq meta-analysis of alternative exon usage in C. elegans.

Almost 20 years after the completion of the C. elegans genome sequence, gene structure annotation is still an ongoing process with new evidence for gene variants still being regularly uncovered by additional in-depth transcriptome studies. While alternative splice forms can allow a single gene to encode several functional isoforms, the question of how much spurious splicing is tolerated is still heavily debated. Here we gathered a compendium of 1682 publicly available C. elegans RNA-seq data sets to increase the dynamic range of detection of RNA isoforms, and obtained robust measurements of the relative abundance of each splicing event. While most of the splicing reads come from reproducibly detected splicing events, a large fraction of purported junctions is only supported by a very low number of reads. We devised an automated curation method that takes into account the expression level of each gene to discriminate robust splicing events from potential biological noise. We found that rarely used splice sites disproportionately come from highly expressed genes and are significantly less conserved in other nematode genomes than splice sites with a higher usage frequency. Our increased detection power confirmed trans-splicing for at least 84% of C. elegans protein coding genes. The genes for which trans-splicing was not observed are overwhelmingly low expression genes, suggesting that the mechanism is pervasive but not fully captured by organism-wide RNA-seq. We generated annotated gene models including quantitative exon usage information for the entire C. elegans genome. This allows users to visualize at a glance the relative expression of each isoform for their gene of interest.

PGC-1α functions as a co-suppressor of XBP1s to regulate glucose metabolism

ObjectivePeroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) promotes hepatic gluconeogenesis by activating HNF4α and FoxO1. PGC-1α expression in the liver is highly elevated in obese and diabetic conditions, leading to increased hepatic glucose production. We previously showed that the spliced form of X-box binding protein 1 (XBP1s) suppresses FoxO1 activity and hepatic gluconeogenesis. The shared role of PGC-1α and XBP1s in regulating FoxO1 activity and gluconeogenesis led us to investigate the probable interaction between PGC-1α and XBP1s and its role in glucose metabolism. MethodsWe investigated the biochemical interaction between PGC-1α and XBP1s and examined the role of their interaction in glucose homeostasis using animal models. ResultsWe show that PGC-1α interacts with XBP1s, which plays an anti-gluconeogenic role in the liver by suppressing FoxO1 activity. The physical interaction between PGC-1α and XBP1s leads to suppression of XBP1s activity rather than its activation. Upregulating PGC-1α expression in the liver of lean mice lessens XBP1s protein levels, and reducing PGC-1α levels in obese and diabetic mouse liver restores XBP1s protein induction. ConclusionsOur findings reveal a novel function of PGC-1α as a suppressor of XBP1s function, suggesting that hepatic PGC-1α promotes gluconeogenesis through multiple pathways as a co-activator for HNF4α and FoxO1 and also as a suppressor for anti-gluconeogenic transcription factor XBP1s.

ASXL gain-of-function truncation mutants: defective and dysregulated forms of a natural ribosomal frameshifting product?

BackgroundProgrammed ribosomal frameshifting (PRF) is a gene expression mechanism which enables the translation of two N-terminally coincident, C-terminally distinct protein products from a single mRNA. Many viruses utilize PRF to control or regulate gene expression, but very few phylogenetically conserved examples are known in vertebrate genes. Additional sex combs-like (ASXL) genes 1 and 2 encode important epigenetic and transcriptional regulatory proteins that control the expression of homeotic genes during key developmental stages. Here we describe an ~150-codon overlapping ORF (termed TF) in ASXL1 and ASXL2 that, with few exceptions, is conserved throughout vertebrates.ResultsConservation of the TF ORF, strong suppression of synonymous site variation in the overlap region, and the completely conserved presence of an EH[N/S]Y motif (a known binding site for Host Cell Factor-1, HCF-1, an epigenetic regulatory factor), all indicate that TF is a protein-coding sequence. A highly conserved UCC_UUU_CGU sequence (identical to the known site of +1 ribosomal frameshifting for influenza virus PA-X expression) occurs at the 5′ end of the region of enhanced synonymous site conservation in ASXL1. Similarly, a highly conserved RG_GUC_UCU sequence (identical to a known site of −2 ribosomal frameshifting for arterivirus nsp2TF expression) occurs at the 5′ end of the region of enhanced synonymous site conservation in ASXL2.ConclusionsDue to a lack of appropriate splice forms, or initiation sites, the most plausible mechanism for translation of the ASXL1 and 2 TF regions is ribosomal frameshifting, resulting in a transframe fusion of the N-terminal half of ASXL1 or 2 to the TF product, termed ASXL-TF. Truncation or frameshift mutants of ASXL are linked to myeloid malignancies and genetic diseases, such as Bohring-Opitz syndrome, likely at least in part as a result of gain-of-function or dominant-negative effects. Our hypothesis now indicates that these disease-associated mutant forms represent overexpressed defective versions of ASXL-TF.ReviewersThis article was reviewed by Laurence Hurst and Eugene Koonin.

Maternal provision of non-sex-specific transformer messenger RNA in sex determination of the wasp Asobara tabida.

In many insect species maternal provision of sex-specifically spliced messenger RNA (mRNA) of sex determination genes is an essential component of the sex determination mechanism. In haplodiploid Hymenoptera, maternal provision in combination with genomic imprinting has been shown for the parasitoid Nasonia vitripennis, known as maternal effect genomic imprinting sex determination (MEGISD). Here, we characterize the sex determination cascade of Asobara tabida, another hymenopteran parasitoid. We show the presence of the conserved sex determination genes doublesex (dsx), transformer (tra) and transformer-2 (tra2) orthologues in As. tabida. Of these, At-dsx and At-tra are sex-specifically spliced, indicating a conserved function in sex determination. At-tra and At-tra2 mRNA is maternally provided to embryos but, in contrast to most studied insects, As. tabida females transmit a non-sex-specific splice form of At-tra mRNA to the eggs. In this respect, As. tabida sex determination differs from the MEGISD mechanism. How the paternal genome can induce female development in the absence of maternal provision of sex-specifically spliced mRNA remains an open question. Our study reports a hitherto unknown variant of maternal effect sex determination and accentuates the diversity of insect sex determination mechanisms.

MicroRNA 122, Regulated by GRLH2, Protects Livers of Mice and Patients From Ethanol-Induced Liver Disease

Chronic, excessive alcohol consumption leads to alcoholic liver disease (ALD) characterized by steatosis, inflammation, and eventually cirrhosis. The hepatocyte specific microRNA 122 (MIR122) regulates hepatocyte differentiation and metabolism. We investigated whether an alcohol-induced decrease in level of MIR122 contributes to development of ALD. We obtained liver samples from 12 patients with ALD and cirrhosis and 9healthy individuals (controls) and analyzed them by histology and immunohistochemistry. C57Bl/6 mice were placed on a Lieber-DeCarli liquid diet, in which they were fed ethanol for 8weeks, as a model of ALD, or a control diet. These mice were also given injections of CCl4, to increase liver fibrosis, for 8weeks. On day 28, mice with ethanol-induced liver disease and advanced fibrosis, and controls, were given injections of recombinant adeno-associated virus 8 vector that expressed the primary miR-122 transcript (pri-MIR122, to overexpress MIR122 in hepatocytes) or vector (control). Two weeks before ethanol feeding, some mice were given injections of a vector that expressed an anti-MIR122, to knock down its expression. Serum and liver tissues were collected; hepatocytes and liver mononuclear cells were analyzed by histology, immunoblots, and confocal microscopy. We performed in silico analyses to identify targets of MIR122 and chromatin immunoprecipitation quantitative polymerase chain reaction analyses in Huh-7 cells. Levels of MIR122 were decreased in liver samples from patients with ALD and mice on the Lieber-DeCarli diet, compared with controls. Transgenic expression of MIR122 in hepatocytes of mice with ethanol-induced liver disease and advanced fibrosis significantly reduced serum levels of alanine aminotransferase (ALT) and liver steatosis and fibrosis, compared with mice given injections of the control vector. Ethanol feeding reduced expression of pri-MIR122 by increasing expression of the spliced form of the transcription factor grainyhead like transcription factor 2 (GRHL2) in liver tissues from mice. Levels of GRHL2 also were increased in livertissues from patients with ALD, compared with controls; increases correlated with decreases in levels of MIR122 in human liver. Mice given injections of the anti-MIR122 before ethanol feeding had increased steatosis, inflammation, and serum levels of alanine aminotransferase compared with micegiven a control vector. Levels of hypoxia-inducible factor1 alpha (HIF1α) mRNA, a target of MIR122, were increased inliver tissues from patients and mice with ALD, compared withcontrols. Mice with hepatocyte-specific disruption of Hif1αdeveloped less-severe liver injury following administration of ethanol, injection of anti-MIR122, or both. Levels of MIR122 decrease in livers from patients with ALD and mice with ethanol-induced liver disease, compared with controls. Transcription of MIR122 is inhibited by GRHL2, which is increased in livers of mice and patients with ALD. Expression of an anti-MIR122 worsened the severity of liver damage following ethanol feeding in mice. MIR122 appears to protect the liver from ethanol-induced damage by reducing levels of HIF1α. These processes might be manipulated to reduce the severity of ALD in patients.

Role of reinforcement couplers in serviceability performance of concrete members

Connection of reinforcing bars by couplers is a common form of reinforcement splicing. However, the variation of stiffness at the location of couplers and the potentially excessive residual slips are suspected to cause adverse impact on the serviceability, especially for structural members subjected to repeated loading. This paper studies the role of couplers in the serviceability performance of concrete members. Relevant provisions in design codes are reviewed and compared. Laboratory tests are conducted to investigate the slip behaviour of couplers. A section analysis approach based on equivalent stiffness model is proposed to account for the effects of couplers, and formulations of crack width calculation are explored for use in structural design.

Mild stress induces brain region-specific alterations of selective ER stress markers’ mRNA expression in Wfs1-deficient mice

In this work, the effect of mild stress (elevated plus maze test, EPM) on the expression of endoplasmic reticulum (ER) stress markers in different brain areas of wild type (WT) and Wfs1-deficient (Wfs1KO) mice was investigated. The following ER stress markers were studied: activating transcription factor 6α (Atf6α), protein kinase-like ER kinase (Perk), X-box binding protein 1 (Xbp1) and its spliced form (Xbp1s), 78-kilodalton glucose regulated protein (Grp78), 94-kilodalton glucose regulated protein (Grp94), C/EBP homologous protein (Chop). Wfs1KO and WT mice, not exposed to EPM, had similar patterns of ER stress markers in the studied brain areas. The exploratory activity of Wfs1KO mice in the EPM was inhibited compared to WT mice, probably reflecting increased anxiety in genetically modified mice. In response to the EPM, activation of inositol-requiring transmembrane kinase and endonuclease 1α (Ire1α) ER stress pathway was seen in both genotypes, but in different brain areas. Such a brain region-specific Ire1α activation was linked with dominant behavioural trends in these mice as more anxious, neophobic Wfs1KO mice had increased ER stress markers expression in the temporal lobe, the brain region related to anxiety, and more curious WT mice had ER stress markers increased in the ventral striatum which is related to the exploratory drive. The molecular mechanism triggering respective changes in ER stress markers in these brain regions is likely related to altered levels of monoamine neurotransmitters (serotonin, dopamine) in Wfs1KO mice.

Contribution of major FLM isoforms to temperature-dependent flowering in Arabidopsis thaliana.

FLOWERING LOCUS M (FLM), a component of the thermosensory flowering time pathway in Arabidopsis thaliana, is regulated by temperature-dependent alternative splicing (AS). The main splicing variant, FLM-β, is a well-documented floral repressor that is down-regulated in response to increasing ambient growth temperature. Two hypotheses have been formulated to explain how flowering time is modulated by AS of FLM. In the first model a second splice variant, FLM-δ, acts as a dominant negative isoform that competes with FLM-β at elevated ambient temperatures, thereby indirectly promoting flowering. Alternatively, it has been suggested that the induction of flowering at elevated temperatures is caused only by reduced FLM-β expression. To better understand the role of the two FLM splice forms, we employed CRISPR/Cas9 technology to specifically delete the exons that characterize each splice variant. Lines that produced repressive FLM-β but were incapable of producing FLM-δ were late flowering. In contrast, FLM-β knockout lines that still produced FLM-δ flowered early, but not earlier than the flm-3 loss of function mutant, as would be expected if FLM-δ had a dominant-negative effect on flowering. Our data support the role of FLM-β as a flower repressor and provide evidence that a contribution of FLM-δ to the regulation of flowering time in wild-type A. thaliana seems unlikely.