scholarly journals A novel TREM2 splice isoform lacking the ligand binding is expressed in brain and shares localization

2021 ◽  
Author(s):  
Benjamin C Shaw ◽  
Henry C Snider ◽  
Andrew K Turner ◽  
Diana J Zajac ◽  
James F Simpson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Splice Variants ◽  
Ectopic Expression ◽  
Anterior Cingulate ◽  
Splice Isoforms ◽  
Similar Frequency ◽  
Exon 2 ◽  
Reading Frame ◽  
Brain Expression ◽  
Splice Isoform

Background: Genetic variants in TREM2 are strongly associated with Alzheimer's Disease (AD) risk but alternative splicing in TREM2 transcripts has not been comprehensively described. Objective: Recognizing that alternative splice variants can result in reduced gene expression and/or altered function, we sought to fully characterize splice variation in TREM2. Methods: Human blood and anterior cingulate autopsy tissue from 61 donors were used for genotyping and cDNA synthesis followed by both end-point and quantitative PCR to identify and quantify novel TREM2 isoforms. Results: In addition to previously described transcripts lacking exon 3 or exon 4, or retaining part of intron 3, we identified novel isoforms lacking exon 2, along with isoforms lacking multiple exons. Isoforms lacking exon 2 were predominant at approximately 10% of TREM2 mRNA in the brain. Expression of TREM2 and frequency of exon 2 skipping did not differ between AD samples and non-AD controls (p = 0.1268 and p = 0.4909, respectively). Further, these novel splice isoforms were also observed across multiple tissues (brain, liver, lung, kidney, heart, aorta, skeletal muscle) with similar frequency (range 5.3 - 13.0%). Using ectopic expression, we found that the exon 2 skipped isoform D2-TREM2 is translated to protein and localizes similarly to full-length TREM2 protein, and that both D2-TREM2 and FL-TREM2 proteins are primarily retained in the Golgi complex. Conclusion: Since the TREM2 ligand binding domain is encoded by exon 2, and skipping this exon retains reading frame while conserving localization, we hypothesize that D2-TREM2 acts as an inhibitor of TREM2 and that targeting TREM2 splicing may be a novel therapeutic pathway for AD.

scholarly journals Polypurine Reverse-Hoogsteen Hairpins as a Tool for Exon Skipping at the Genomic Level in Mammalian Cells

2021 ◽  
Vol 22 (7) ◽  
pp. 3784
Author(s):  
Véronique Noé ◽  
Carlos J. Ciudad
Keyword(s):  
Dna Sequencing ◽  
Rare Diseases ◽  
Mammalian Cells ◽  
Mrna Level ◽  
Exon Skipping ◽  
Selective Medium ◽  
Rt Pcr ◽  
Exon 2 ◽  
Reading Frame ◽  
Additional Exon

Therapeutic strategies for rare diseases based on exon skipping are aimed at mediating the elimination of mutated exons and restoring the reading frame of the affected protein. We explored the capability of polypurine reverse-Hoogsteen hairpins (PPRHs) to cause exon skipping in NB6 cells carrying a duplication of exon 2 of the DHFR gene that causes a frameshift abolishing DHFR activity. Methods: Different editing PPRHs were designed and transfected in NB6 cells followed by incubation in a DHFR-selective medium lacking hypoxanthine and thymidine. Surviving colonies were analyzed by DNA sequencing, RT-PCR, Western blotting and DHFR enzymatic activity. Results: Transfection of editing PPRHs originated colonies in the DHFR-selective medium. DNA sequencing results proved that the DHFR sequence in all these colonies corresponded to the wildtype sequence with just one copy of exon 2. In the edited colonies, the skipping of the additional exon was confirmed at the mRNA level, the DHFR protein was restored, and it showed high levels of DHFR activity. Conclusions: Editing-PPRHs are able to cause exon skipping at the DNA level and could be applied as a possible therapeutic tool for rare diseases.

scholarly journals Differential Expression of BARD1 Isoforms in Melanoma

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 320
Author(s):  
Lorissa I. McDougall ◽  
Ryan M. Powell ◽  
Magdalena Ratajska ◽  
Chi F. Lynch-Sutherland ◽  
Sultana Mehbuba Hossain ◽  
...  
Keyword(s):  
Long Range ◽  
Patient Outcomes ◽  
Splice Variants ◽  
Significant Proportion ◽  
Nanopore Sequencing ◽  
Rna Seq ◽  
Splice Isoforms ◽  
Tissue Samples ◽  
Multiple Transcripts ◽  
Mrna Variants

Melanoma comprises <5% of cutaneous malignancies, yet it causes a significant proportion of skin cancer-related deaths worldwide. While new therapies for melanoma have been developed, not all patients respond well. Thus, further research is required to better predict patient outcomes. Using long-range nanopore sequencing, RT-qPCR, and RNA sequencing analyses, we examined the transcription of BARD1 splice isoforms in melanoma cell lines and patient tissue samples. Seventy-six BARD1 mRNA variants were identified in total, with several previously characterised isoforms (γ, φ, δ, ε, and η) contributing to a large proportion of the expressed transcripts. In addition, we identified four novel splice events, namely, Δ(E3_E9), ▼(i8), IVS10+131▼46, and IVS10▼176, occurring in various combinations in multiple transcripts. We found that short-read RNA-Seq analyses were limited in their ability to predict isoforms containing multiple non-contiguous splicing events, as compared to long-range nanopore sequencing. These studies suggest that further investigations into the functional significance of the identified BARD1 splice variants in melanoma are warranted.

Coding sequence and expression of the homeobox gene Hox 1.3

Development ◽  
1988 ◽  
Vol 102 (2) ◽  
pp. 349-359 ◽  
Author(s):  
M. Fibi ◽  
B. Zink ◽  
M. Kessel ◽  
A.M. Colberg-Poley ◽  
S. Labeit ◽  
...  
Keyword(s):  
Homeobox Gene ◽  
Cell System ◽  
T Antigen ◽  
Open Reading Frame ◽  
Homeodomain Protein ◽  
Chromosome 11 ◽  
3T3 Cells ◽  
Exon 2 ◽  
Reading Frame ◽  
3T3 Fibroblasts

We have characterized Hox 1.3 (previously described as m2), a murine homeobox-containing gene, which is a member of the Hox 1 cluster located on chromosome 6. A cloned cDNA was isolated from an Okayama-Berg library generated from the chemically transformed cell line MB66 MCA ACL6. The protein sequence of 270 amino acids was deduced from the nucleotide sequence of an open reading frame containing the homeobox. The open reading frame is interrupted at the genomic level by a 960 bp intron and is organized in two exons. The Hox 1.3 protein was found to contain extensive sequence homology with the murine homeodomain protein Hox 2.1, which is encoded on chromosome 11. There are two homology with the regions in the first exon, i.e. a hexapeptide conserved in many homeobox-containing genes and the N-terminal domain, which was found to be homologous only to Hox 2.1. Furthermore, in exon 2 the homologies of the homeodomain regions are extended up to the carboxy terminus of Hox 1.3 and Hox 2.1. During prenatal murine development, maximal expression of Hox 1.3 is observed in 12-day embryonic tissue. The two transcripts carrying the Hox 1.3 homeobox are 1.9 kb and about 4 kb in length. An abundant Hox 1.3-specific 1.9 kb RNA is also found in F9 cells which were induced for parietal endoderm differentiation, whereas F9 teratocarcinoma stem cells do not stably express this specific RNA. Induction of the transcript occurs immediately after retinoic acid/cAMP treatment and the RNA level remains high for 5 days. Thus, the kinetics are different from the previously described homeobox transcripts Hox 1.1 and Hox 3.1. Interestingly, by analogy to the F9 cell system a negative correlation between transformation and Hox 1.3 expression is observed in 3T3 fibroblasts also. Untransformed 3T3 cells carry abundant 1.9 kb Hox 1.3 RNA, whereas the methylcholanthrene-transformed MB66 and LTK- cells or 3T3 cells transformed by the oncogenes src, fos or SV40 T antigen express only low levels.

Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus

2021 ◽  
Vol 221 (2) ◽  
Author(s):  
Daniel Crosby ◽  
Melissa R. Mikolaj ◽  
Sarah B. Nyenhuis ◽  
Samantha Bryce ◽  
Jenny E. Hinshaw ◽  
...  
Keyword(s):  
Network Formation ◽  
Fusion Activity ◽  
Amphipathic Helix ◽  
Alternate Splicing ◽  
Charge Reversal ◽  
Splice Isoforms ◽  
C Terminus ◽  
Splice Isoform ◽  
Inhibitory Domain ◽  
Α Helix

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. Here, we report successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion.

scholarly journals Cloning and functional characterization of the human fractalkine receptor promoter regions

2002 ◽  
Vol 368 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Alexandre GARIN ◽  
Philippe PELLET ◽  
Philippe DETERRE ◽  
Patrice DEBRÉ ◽  
Christophe COMBADIÈRE
Keyword(s):  
Functional Characterization ◽  
Cell Types ◽  
Promoter Regions ◽  
Exon 2 ◽  
Reading Frame ◽  
Fractalkine Receptor ◽  
A Cell ◽  
Exon 4 ◽  
Coronary Events

We have previously shown that reduced expression of the fractalkine receptor, CX3CR1, is correlated with rapid HIV disease progression and with reduced susceptibility to acute coronary events. In order to elucidate the mechanisms underlying transcriptional regulation of CX3CR1 expression, we structurally and functionally characterized the CX3CR1 gene. It consists of four exons and three introns spanning over 18kb. Three transcripts are produced by splicing the three untranslated exons with exon 4, which contains the complete open reading frame. The transcript predominantly found in leucocytes corresponds to the splicing of exon 2 with exon 4. Transcripts corresponding to splicing of exons 1 and 4 are less abundant in leucocytes and splicing of exons 3 and 4 are rare longer transcripts. A constitutive promoter activity was found in the regions extending upstream from untranslated exons 1 and 2. Interestingly, exons 1 and 2 enhanced the activity of their respective promoters in a cell-specific manner. These data show that the CX3CR1 gene is controlled by three distinct promoter regions, which are regulated by their respective untranslated exons and that lead to the transcription of three mature messengers. This highly complex regulation may allow versatile and precise expression of CX3CR1 in various cell types.

Three differentially expressed survivin cDNA variants encode proteins with distinct antiapoptotic functions

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1435-1442 ◽  
Author(s):  
Edward M. Conway ◽  
Saskia Pollefeyt ◽  
Jan Cornelissen ◽  
Inky DeBaere ◽  
Marta Steiner-Mosonyi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Messenger Rna ◽  
Coiled Coil ◽  
Cdna Clones ◽  
Exon 2 ◽  
Reading Frame ◽  
Coiled Coil Domain ◽  
Acid Protein ◽  
Apoptosis Protein

Survivin is a member of the inhibitor of apoptosis protein (IAP) family that is believed to play a role in oncogenesis. To elucidate further its physiologic role(s), we have characterized the murinesurvivin gene and complementary DNA (cDNA). The structural organization of the survivin gene, located on chromosome 11E2, is similar to that of its human counterpart, both containing 4 exons. Surprisingly, 3 full-length murine survivin cDNA clones were isolated, predicting the existence of 3 distinct survivin proteins. The longest open reading frame, derived from all 4 exons, predicts a 140-amino acid residue protein, survivin140, similar to human survivin, which contains a single IAP repeat and a COOH-terminal coiled-coil domain that links its function to the cell cycle. A second cDNA, which retains intron 3, predicts the existence of a 121-amino acid protein, survivin121 that lacks the coiled-coil domain. Removal of exon 2-derived sequences by alternative pre-messenger RNA (mRNA) splicing results in a third 40-amino acid residue protein, survivin40, lacking the IAP repeat and coiled-coil structure. Predictably, only recombinant survivin140 and survivin121 inhibited caspase-3 activity. All 3 mRNA species were variably expressed during development from 7.5 days postcoitum. Of the adult tissues surveyed, thymus and testis accumulated high levels of survivin140 mRNA, whereas survivin121-specific transcripts were detected in all tissues, while those representing survivin40 were absent. Human counterparts to the 3 survivin mRNA transcripts were identified in a study of human cells and tissues. The presence of distinct isoforms of survivin that are expressed differentially suggests that survivin plays a complex role in regulating apoptosis.

scholarly journals The Histone Variant MacroH2A1 Regulates Key Genes for Myogenic Cell Fusion in a Splice-Isoform Dependent Manner

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1109
Author(s):  
Sarah Hurtado-Bagès ◽  
Melanija Posavec Marjanovic ◽  
Vanesa Valero ◽  
Roberto Malinverni ◽  
David Corujo ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Ex Vivo ◽  
Histone Variants ◽  
Histone Variant ◽  
C2c12 Cells ◽  
Dependent Manner ◽  
Chronic Injury ◽  
Splice Isoforms ◽  
Splice Isoform

MacroH2A histone variants have functions in differentiation, somatic cell reprogramming and cancer. However, at present, it is not clear how macroH2As affect gene regulation to exert these functions. We have parted from the initial observation that loss of total macroH2A1 led to a change in the morphology of murine myotubes differentiated ex vivo. The fusion of myoblasts to myotubes is a key process in embryonic myogenesis and highly relevant for muscle regeneration after acute or chronic injury. We have focused on this physiological process, to investigate the functions of the two splice isoforms of macroH2A1. Individual perturbation of the two isoforms in myotubes forming in vitro from myogenic C2C12 cells showed an opposing phenotype, with macroH2A1.1 enhancing, and macroH2A1.2 reducing, fusion. Differential regulation of a subset of fusion-related genes encoding components of the extracellular matrix and cell surface receptors for adhesion correlated with these phenotypes. We describe, for the first time, splice isoform-specific phenotypes for the histone variant macroH2A1 in a physiologic process and provide evidence for a novel underlying molecular mechanism of gene regulation.

scholarly journals Old gene, new phenotype: splice-altering variants in CEACAM16 cause recessive non-syndromic hearing impairment

2018 ◽  
Vol 55 (8) ◽  
pp. 555-560 ◽  
Author(s):  
Kevin T Booth ◽  
Kimia Kahrizi ◽  
Hossein Najmabadi ◽  
Hela Azaiez ◽  
Richard JH Smith
Keyword(s):  
Hearing Loss ◽  
Hearing Impairment ◽  
Loss Of Function ◽  
Bioinformatics Pipeline ◽  
Exon 2 ◽  
Reading Frame ◽  
Sequencing Platform ◽  
Syndromic Hearing Impairment ◽  
Generation Sequencing

BackgroundHearing loss is a genetically and phenotypically heterogeneous disorder.ObjectivesThe purpose of this study was to determine the genetic cause underlying the postlingual progressive hearing loss in two Iranian families.MethodsWe used OtoSCOPE, a next-generation sequencing platform targeting >150 genes causally linked to deafness, to screen two deaf probands. Data analysis was completed using a custom bioinformatics pipeline, and variants were functionally assessed using minigene splicing assays.ResultsWe identified two homozygous splice-altering variants (c.37G>T and c.662–1G>C) in the CEACAM16 gene, segregating with the deafness in each family. The minigene splicing results revealed the c.37G>T results in complete skipping of exon 2 and loss of the AUG start site. The c.662–1G>C activates a cryptic splice site inside exon 5 resulting in a shift in the mRNA reading frame.ConclusionsThese results suggest that loss-of-function mutations in CEACAM16 result in postlingual progressive hearing impairment and further support the role of CEACAM16 in auditory function.

scholarly journals REGIA, An EU Project on Functional Genomics of Transcription Factors fromArabidopsis thaliana

2002 ◽  
Vol 3 (2) ◽  
pp. 102-108 ◽  
Author(s):  
Javier Paz-Ares ◽  
The REGIA Consortium
Keyword(s):  
Transcription Factors ◽  
Expression Patterns ◽  
Regulatory Gene ◽  
Gene Array ◽  
Ectopic Expression ◽  
Phenotypic Analysis ◽  
Systematic Analysis ◽  
Biotechnological Potential ◽  
Reading Frame ◽  
Eu Project

Transcription factors (TFs) are regulatory proteins that have played a pivotal role in the evolution of eukaryotes and that also have great biotechnological potential. REGIA (REgulatory Gene Initiative in Arabidopsis) is an EU-funded project involving 29 European laboratories with the objective of determining the function of virtually all transcription factors from the model plant,Arabidopsis thaliana. REGIA involves: 1. the definition ofTFgene expression patterns inArabidopsis; 2. the identification of mutations atTFloci; 3. the ectopic expression of TFs (or derivatives) inArabidopsisand in crop plants; 4. phenotypic analysis of the mutants and mis-expression lines, including both RNA and metabolic profiling; 5. the systematic analysis of interactions between TFs; and 6. the generation of a bioinformatics infrastructure to access and integrate all this information. We expect that this programme will establish the full biotechnological potential of plant TFs, and provide insights into hierarchies, redundancies, and interdependencies, and their evolution. The project involves the preparation of both aTFgene array for expression analysis and a normalised full length open reading frame (ORF) library of TFs in a yeast two hybrid vector; the applications of these resources should extend beyond the scope of this programme.

Sign in / Sign up

Export Citation Format

Share Document