scholarly journals Variants in the 5′UTR reduce SHOX expression and contribute to SHOX haploinsufficiency

2020 ◽  
Vol 29 (1) ◽  
pp. 110-121
Author(s):  
Deepak Babu ◽  
Silvia Vannelli ◽  
Antonella Fanelli ◽  
Simona Mellone ◽  
Ave Maria Baffico ◽  
...  
Keyword(s):  
Point Mutations ◽  
Mrna Splicing ◽  
Luciferase Assay ◽  
Transcriptional Level ◽  
Coding Region ◽  
Exon Trapping ◽  
Branch Site ◽  
Uncertain Significance ◽  
Negative Effect

AbstractSHOX haploinsufficiency causes 70–90% of Léri-Weill dyschondrosteosis (LWD) and 2–10% of idiopathic short stature (ISS). Deletions removing the entire gene or enhancers and point mutations in the coding region represent a well-established cause of haploinsufficiency. During diagnostic genetic testing on ISS/LWD patients, in addition to classic SHOX defects, five 5′UTR variants (c.-58G > T, c.-55C > T, c.-51G > A, c.-19G > A, and c.-9del), were detected whose pathogenetic role was unclear and were thus classified as VUS (Variants of Uncertain Significance). The purpose of the present study was to investigate the role of these noncoding variations in SHOX haploinsufficiency. The variants were tested for their ability to interfere with correct gene expression of a regulated reporter gene (luciferase assay). The negative effect on the mRNA splicing predicted in silico for c.-19G > A was assayed in vitro through a minigene splicing assay. The luciferase assay showed that c.-51G > A, c.-19G > A, and c.-9del significantly reduce luciferase activity by 60, 35, and 40% at the homozygous state. Quantification of the luciferase mRNA showed that c.-51G > A and c.-9del might interfere with the correct SHOX expression mainly at the post-transcriptional level. The exon trapping assay demonstrated that c.-19G > A determines the creation of a new branch site causing an aberrant mRNA splicing. In conclusion, this study allowed us to reclassify two of the 5′UTR variants identified during SHOX diagnostic screening as likely pathogenic, one remains as a VUS, and two as likely benign variants. This analysis for the first time expands the spectrum of the genetic causes of SHOX haploinsufficiency to noncoding variations in the 5′UTR.

SHQ1 Regulates MYC mRNA Splicing and Promotes T Cell Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 440-440
Author(s):  
Hexiu Su ◽  
Yufeng Hu ◽  
Jue Jiang ◽  
Zhichao Chen ◽  
Hudan Liu
Keyword(s):  
Cell Proliferation ◽  
T Cell ◽  
Functional Role ◽  
Conflicts Of Interest ◽  
Mrna Splicing ◽  
Transcriptional Level ◽  
Normal Peripheral Blood ◽  
Genome Wide ◽  
T Cell Leukemogenesis

Abstract T-acute lymphoblastic leukemias (T-ALLs) are aggressive hematologic tumors resulting from the malignant transformation of T cell progenitors. In this context, constitutive activation of NOTCH1 signaling is the most prominent oncogenic pathway in T cell transformation. Yet functional role(s) of NOTCH1 in T-ALL pathogenesis and precise mechanism(s) of action remain to be fully determined. SHQ1 is an essential assembly factor for H/ACA ribonucleoproteins which are required for spliceosomal small nuclear RNA (snRNA) maturation. We here identify SHQ1 as a NOTCH1 downstream target that plays a pivotal role in maintaining MYC splicing fidelity and promoting T-ALL cell proliferation in vitro and in vivo. We identified SHQ1 as a NOTCH1-regulated gene from multiple human T-ALL genome-wide expression studies. To validate this finding, we analyzed SHQ1 expression in a spectrum of human T-ALL cells upon NOTCH1 pathway inhibition. NOTCH1 inactivation caused a marked downregulation of SHQ1 in all T-ALL cell lines tested. We further demonstrated NOTCH1 bound to the canonical CSL binding sites in the SHQ1 promoter and directly activated the transcription. We next systematically analyzed the SHQ1 expression in 174 T-ALL primary samples and found that SHQ1 expression was significantly elevated in T-ALL compared with normal peripheral blood. To explore the functional role of SHQ1, we knocked it down in T-ALL using specific shRNAs. SHQ1 depletion profoundly inhibited T-ALL cell proliferation and induced massive apoptotic cell death. Consistent with these in vitro findings, SHQ1 depletion in a human T-ALL xenograft significantly delayed leukemia onset and prolonged survival. To decipher the molecular mechanism whereby SHQ1 contributes to T cell leukemogenesis, we performed genome-wide RNA-Seq and analyzed alternative splicing across the genome. Approximately 70% of genes exhibited abnormal intron retention upon SHQ1 depletion. Among those whose expression levels and mRNA splicing were prominently altered by SHQ1, MYC gained our attention because of its vital role in T-ALL. Depletion of SHQ1 resulted in marked downregulation of mature MYC mRNA and protein. Endogenous mRNA analysis and mini-gene experiments showed aberrant accumulation of MYC pre-mRNA in SHQ1-depleted cells. Expectedly, SHQ1 knockdown had minimal effects on T-ALL cells constitutively expressing a human MYC protein. In addition to the well-documented transcriptional control of MYC by NOTCH1, we herein report a previously unsuspected mechanism in which NOTCH1 modulates MYC splicing by activation of SHQ1. Our findings not only shed new insights in the molecular pathology of NOTCH1-induced T-ALL but also provide a new layer of regulation of oncogene MYC at the post-transcriptional level, mechanism of which may apply to other MYC involved tumors. Disclosures No relevant conflicts of interest to declare.

scholarly journals EvaluatingLRRK2Genetic Variants with Unclear Pathogenicity

2015 ◽  
Vol 2015 ◽  
pp. 1-6 ◽  
Author(s):  
Fathima Shaffra Refai ◽  
Shin Hui Ng ◽  
Eng-King Tan
Keyword(s):  
Cell Survival ◽  
Kinase Activity ◽  
Point Mutations ◽  
Protein Domain ◽  
Wild Type ◽  
Function Mechanism ◽  
Functional Assays ◽  
Functional Aspects ◽  
Negative Effect

Mutations in the leucine-rich repeat kinase 2 (LRRK2) have been known to be a major genetic component affecting Parkinson’s disease (PD). However, the pathogenicity of many of theLRRK2variants is unclear because they have been detected in single patients or also in patients and controls. Here, we selected 5 exonic variants (L1165P, T1410M, M1646T, L2063X, and Y2189C) from each of the protein domain ofLRRK2and analysed their possible association with pathogenicity usingin vitrofunctional assays. Point mutations representing each of these variants were incorporated into theLRRK2gene, and functional aspects such as the percentage of cell survival upon application of stress and kinase activity were measured. Our results showed that all 5 variants had a significantly negative effect on the survival of cells, in both presence and absence of stress, as compared to the wild-type. In addition, there was also a slight increase in kinase activity in most of the variants in comparison to the wild-type. A negative correlation between cell survival and kinase activity was observed. These data suggest that most of the variants despite being located in different domains ofLRRK2appear to exert a potential pathogenic effect possibly through an increased kinase activity, supporting a gain of function mechanism.

scholarly journals Molecular cloning and characterization of human caspase-activated DNase

1998 ◽  
Vol 95 (16) ◽  
pp. 9123-9128 ◽  
Author(s):  
Naomi Mukae ◽  
Masato Enari ◽  
Hideki Sakahira ◽  
Yoji Fukuda ◽  
Johji Inazawa ◽  
...  
Keyword(s):  
Dna Fragmentation ◽  
Cell Lines ◽  
Point Mutations ◽  
Leukemic Cell ◽  
Coding Region ◽  
Chromosomal Dna ◽  
Caspase Activated Dnase

Caspase-activated DNase (CAD) cleaves chromosomal DNA during apoptosis. Here, we report isolation of two classes of human CAD cDNAs from a human KT-3 leukemic cell cDNA library. One class of cDNA encoded a protein comprising 338 amino acids, which showed a marked similarity to its murine counterpart. In vitro transcription and translation of this cDNA resulted in a functional CAD protein when the protein was synthesized in the presence of its inhibitor (inhibitor of CAD). The other cDNA class contained many deletions, insertions, and point mutations in the sequence corresponding to the coding region, suggesting that it is derived from a pseudogene. The functional CAD gene was localized to human chromosome 1p36.3 by fluorescent in situ hybridization. The CAD mRNA was expressed in a limited number of human tissues, including pancreas, spleen, prostate, and ovary. The expression of the CAD mRNA in human cell lines correlated with their ability to show DNA fragmentation during apoptosis. Overexpression of CAD potentiated DNA fragmentation by apoptotic stimuli in these cell lines, indicating that CAD is responsible for the apoptotic DNA degradation.

scholarly journals PglB function and glycosylation efficiency is temperature dependent when the pgl locus is integrated in the Escherichia coli chromosome

2022 ◽  
Vol 21 (1) ◽  
Author(s):  
Vanessa S. Terra ◽  
Marta Mauri ◽  
Thippeswamy H. Sannasiddappa ◽  
Alexander A. Smith ◽  
Mark P. Stevens ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rapid Identification ◽  
Effect Of Temperature ◽  
Limiting Factor ◽  
Transcriptional Level ◽  
Coding Region ◽  
Temperature Dependent ◽  
Live Attenuated Vaccine ◽  
E Coli

Abstract Background Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni. Results We generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain. Conclusions We determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level.

scholarly journals SilentMutations (SIM): a tool for analyzing long-range RNA-RNA interactions in viral genomes and structured RNAs

2018 ◽  
Author(s):  
Daniel Desirò ◽  
Martin Hölzer ◽  
Bashar Ibrahim ◽  
Manja Marz
Keyword(s):  
Long Range ◽  
Influenza A ◽  
Graphical Representation ◽  
Point Mutations ◽  
Multiple Point ◽  
Coding Region ◽  
Viral Rnas ◽  
Viral Genomes ◽  
Single Nucleotide Change

ABSTRACTBackgroundA single nucleotide change in the coding region can alter the amino acid sequence of a protein. In consequence, natural or artificial sequence changes in viral RNAs may have various effects not only on protein stability, function and structure but also on viral replication.In recent decades, several tools have been developed to predict the effect of mutations in structured RNAs such as viral genomes or non-coding RNAs. Some tools use multiple point mutations and also take coding regions into account. However, none of these tools was designed to specifically simulate the effect of mutations on viral long-range interactions.ResultsHere, we developedSilentMutations (SIM), an easy-to-use tool to analyze the effect of multiple point mutations on the secondary structures of two interacting viral RNAs. The tool can simulate disruptive and compensatory mutants of two interacting single-stranded RNAs. This allows a fast and accurate assessment of key regions potentially involved in functional long-range RNA-RNA interactions and will eventually help virologists and RNA-experts to design appropriate experiments.SIMonly requires two interacting single-stranded RNA regions as input. The output is a plain text file containing the most promising mutants and a graphical representation of all interactions.ConclusionWe applied our tool on two experimentally validated influenza A virus and hepatitis C virus interactions and we were able to predict potential double mutants forin vitrovalidation experiments.AvailabilityThe source code and documentation ofSIMare freely available at github.com/desiro/silentMutations.

scholarly journals Two isoforms of the kidney androgen-regulated protein are encoded by two alleles of a single gene in OFl mice

1992 ◽  
Vol 59 (2) ◽  
pp. 117-124 ◽  
Author(s):  
E. Melanitou ◽  
D. Tronik ◽  
F. Rougeon
Keyword(s):  
Amino Acid ◽  
Mouse Genome ◽  
Single Gene ◽  
Point Mutations ◽  
Apparent Molecular Weight ◽  
Analysis Data ◽  
Size Difference ◽  
Cdna Clones ◽  
Coding Region

SummaryTwo cDNA clones coding for two forms of the mouse kidney androgen-regulated protein (KAP) distinguished by their electrophoretic mobilities on SDS gel electrophoresis have been isolated from libraries prepared from strains of mice having one (BALB/c) or two (OFl) forms of the KAP protein. The corresponding mRNAs have identical sizes, as well as identical sequences in their 5' non-translated regions. The size difference observed between the two proteins is due to two point mutations in the coding region of the KAP mRNA, leading to two amino-acid changes one of which resulted in the substitution of a glycine for a glutamic acid. As shown by in vitro transcription/translation experiments, these two amino-acid differences are responsible for the shift in the apparent molecular weight of the protein on SDS gels. Both forms of the protein are more abundant in males than in females.In vitro translation of kidney RNAs isolated from six different strains and species of mice revealed the presence of other forms of the KAP protein, characterized by small variations of their molecular weights. Southern blot analysis data are consistent with the presence of only one kap gene in the mouse genome. A restriction fragment length polymorphism has been observed, which does not correlate with the protein polymorphism, indicating the presence of another allele in the OF1 mouse genome.

scholarly journals Biological properties and relative fitness of inter-subgroup cucumber mosaic virus RNA 3 recombinants produced in vitro

2007 ◽  
Vol 88 (10) ◽  
pp. 2852-2861 ◽  
Author(s):  
Olivier Pierrugues ◽  
Laurent Guilbaud ◽  
Isabelle Fernandez-Delmond ◽  
Frédéric Fabre ◽  
Mark Tepfer ◽  
...  
Keyword(s):  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Point Mutations ◽  
Biological Properties ◽  
Open Reading Frames ◽  
Relative Fitness ◽  
Coding Region ◽  
Virus Rna ◽  
Viral Cdna

In vitro reverse transcription of a mixture of total RNA from plants infected with the I17F or R strains of cucumber mosaic virus (CMV), representative of subgroups IA and II, respectively, results in viral cDNA populations including rare recombinant RNA 3 molecules, some of which also have point mutations. The biological properties of 17 recombinants in the capsid gene or the 3′ non-coding region of RNA 3 were evaluated when associated with I17F RNAs 1 and 2. Six viruses displayed deficiencies (non-viability, deficiencies for movement and/or replication, delayed infection, loss of aphid transmissibility). Nine induced symptoms close to those of I17F-CMV on tobacco and pepper plants. All recombinants bearing the movement protein (MP) of R-CMV and part or most of the capsid protein (CP) of I17F-CMV, as well as the recombinant created in vitro by exchanging the corresponding open reading frames, also induced filiformism on tobacco, but induced only faint symptoms on melon. Two recombinants induced atypically severe symptoms on both tobacco and pepper. Most of the recombinants generally accumulated to lower levels than the wild-type I17F strain in tobacco. Three recombinants, however, including one responsible for severe symptoms, accumulated to generally higher levels than I17F-CMV. When two of these were tested in co-infection experiments with I17F RNA 3, they proved to be poorly competitive, suggesting that they would be unlikely to emerge in the field.

Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis

1992 ◽  
Vol 67 (01) ◽  
pp. 063-065 ◽  
Author(s):  
Sherryl A M Taylor ◽  
Jacalyn Duffin ◽  
Cherie Cameron ◽  
Jerome Teitel ◽  
Bernadette Garvey ◽  
...  
Keyword(s):  
Nucleotide Substitution ◽  
Novel Mutation ◽  
Factor Ix ◽  
Causative Mutation ◽  
Coding Region ◽  
Body Of Knowledge ◽  
Polymerase Chain ◽  
Splice Junctions

SummaryChristmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

The depressing (negative) effect of oestradiol benzoate on the in vitro secretion of LH and FSH by the pituitary gland of the LRH-pretreated long-term ovariectomized rat changes into the augmentative (positive) effect after discontinuation of the LRH-pretreatment

1985 ◽  
Vol 110 (3) ◽  
pp. 329-337 ◽  
Author(s):  
G. A. Schuiling ◽  
H. Moes ◽  
T. R. Koiter
Keyword(s):  
Depressing Effect ◽  
Ovx Rats ◽  
Gonadotrophin Secretion ◽  
Oestradiol Benzoate ◽  
Negative Effect ◽  
Positive Effect ◽  
Perifusion System

Abstract. The effect of pretreatment in vivo with oestradiol benzoate on in vitro secretion of LH and FSH was studied in long-term ovariectomized (OVX) rats both at the end of a 5-day continuous in vivo pretreatment with LRH and 4-days after cessation of such LRH pretreatment. Rats were on day 0 sc implanted with osmotic minipumps which released LRH at the rate of 250 ng/h. Control rats were implanted with a piece of silicone elastomer with the dimensions of a minipump. On days 2 and 4 the rats were injected with either 3 μg EB or with oil. On day 5 part of the rats were decapitated and the in vitro autonomous (i.e. non-LRH-stimulated) and 'supra-maximally' LRHstimulated release of LH and FSH was studied using a perifusion system. From other rats the minipumps were removed on day 5 and perifusion was performed on day 9. On the 5th day of the in vivo LRH pretreatment the pituitary LH/FSH stores were partially depleted; the pituitaries of the EB-treated rats more so than those of the oil-injected rats. EB alone had no significant effect on the content of the pituitary LH- and FSH stores. On day 9, i.e. 4 days after removal of the minipumps, the pituitary LH and FSH contents had increased in both the oil- and the EB injected rats, but had not yet recovered to control values. In rats not subjected to the 5-days pretreatment with LRH EB had a positive effect on the supra-maximally LRH-stimulated secretion of LH and FSH as well as on the non-stimulated secretion of LH. EB had no effect on the non-stimulated secretion of FSH. After 5 days of in vivo pretreatment with LRH only, the in vitro non-stimulated and supra-maximally LRH-stimulated secretion of both LH and FSH were strongly impaired, the effect correlating well with the LRH-induced depletion of the pituitary LH/FSH stores. In such LRH-pretreated rats EB had on day 5 a negative effect on the (already depressed) LRH-stimulated secretion of LH (not on that of FSH). EB had no effect on the non-stimulated LH/FSH secretion. It could be demonstrated that the negative effect of the combined LRH/EB pretreatment was mainly due to the depressing effect of this treatment on the pituitary LH and FSH stores: the effect of oestradiol on the pituitary LRH-responsiveness (release as related to pituitary gonadotrophin content) remained positive. In LRH-pretreated rats, however, this positive effect of EB was smaller than in rats not pretreated with LRH. Four days after removal of the minipumps there was again a positive effect of EB on the LRH-stimulated secretion of LH and FSH as well as on the non-stimulated secretion of LH. The positive effect of EB on the pituitary LRH-responsiveness was as strong as in rats which had not been exposed to exogenous LRH. The non-stimulated secretion of FSH was again not affected by EB. The results demonstrate that the effect of EB on the oestrogen-sensitive components of gonadotrophin secretion consists of two components: an effect on the pituitary LRH-responsiveness proper, and an effect on the pituitary LH/FSH stores. The magnitude of the effect of EB on the LRH-responsiveness is LRH dependent: it is very weak (almost zero) in LRH-pretreated rats, but strong in rats not exposed to LRH as well as in rats of which the LRH-pretreatment was stopped 4 days previously. Similarly, the effect of EB on the pituitary LH and FSH stores is LRH-dependent: in the absence of LRH, EB has no influence on the contents of these stores, but EB can potentiate the depleting effect of LRH on the LH/FSH-stores. Also this effect disappear after cessation of the LRH-pretreatment.

Sign in / Sign up

Export Citation Format

Share Document