GAGAG Motifs Research Articles

Complete reconstitution of bypass and blocking functions in a minimal artificial Fab-7 insulator from Drosophila bithorax complex

Boundaries in the bithorax complex (BX-C) delimit autonomous regulatory domains that drive parasegment-specific expression of the Hox genes Ubx, abd-A, and Abd-B The Fab-7 boundary is located between the iab-6 and iab-7 domains and has two key functions: blocking cross-talk between these domains and at the same time promoting communication (boundary bypass) between iab-6 and the Abd-B promoter. Using a replacement strategy, we found that multimerized binding sites for the architectural proteins Pita, Su(Hw), and dCTCF function as conventional insulators and block cross-talk between the iab-6 and iab-7 domains; however, they lack bypass activity, and iab-6 is unable to regulate Abd-B Here we show that an ∼200-bp sequence of dHS1 from the Fab-7 boundary rescues the bypass defects of these multimerized binding sites. The dHS1 sequence is bound in embryos by a large multiprotein complex, Late Boundary Complex (LBC), that contains the zinc finger proteins CLAMP and GAF. Using deletions and mutations in critical GAGAG motifs, we show that bypass activity correlates with the efficiency of recruitment of LBC components CLAMP and GAF to the artificial boundary. These results indicate that LBC orchestrates long-distance communication between the iab-6 regulatory domain and the Abd-B gene, while the Pita, Su(Hw), and dCTCF proteins function to block local cross-talk between the neighboring regulatory domains iab-6 and iab-7.

Frameshifted β-Amyloid Precursor Protein (APP+1) Is a Secretory Protein, and the Level of APP+1 in Cerebrospinal Fluid Is Linked to Alzheimer Pathology

Molecular misreading of the beta-amyloid precursor protein (APP) gene generates mRNA with dinucleotide deletions in GAGAG motifs. The resulting truncated and partly frameshifted APP protein (APP+1) accumulates in the dystrophic neurites and the neurofibrillary tangles in the cortex and hippocampus of Alzheimer patients. In contrast, we show here that neuronal cells transfected with APP+1 proficiently secreted APP+1. Because various secretory APP isoforms are present in cerebrospinal fluid (CSF), this study aimed to determine whether APP+1 is also a secretory protein that can be detected in CSF. Post-mortem CSF was obtained at autopsy from 50 non-demented controls and 122 Alzheimer patients; all subjects were staged for neuropathology (Braak score). Unexpectedly, we found that the APP+1 level in the CSF of non-demented controls was much higher (1.75 ng/ml) than in the CSF of Alzheimer patients (0.51 ng/ml) (p < 0.001), and the level of APP+1 in CSF was inversely correlated with the severity of the neuropathology. Moreover the earliest neuropathological changes are already reflected in a significant decrease of the APP+1 level in CSF. These data show that APP+1 is normally secreted by neurons, preventing intra-neuronal accumulation of APP+1 in brains of non-demented controls without neurofibrillary pathology.

Deficient protein kinase a in systemic lupus erythematosus: a disorder of T lymphocyte signal transduction.

Systemic lupus erythematosus (SLE) is an idiopathic autoimmune disease characterized by impaired T lymphocyte immune effector functions. We have identified a disorder of signal transduction in SLE T cells involving the cyclic AMP/protein kinase A (cAMP/PKA) pathway. Cyclic AMP-stimulated PKA-catalyzed protein phosphorylation is markedly diminished owing to profound deficiencies of both type I (PKA-I) and type II (PKA-II) isozyme activities. Deficient PKA-I isozyme is characterized by a significant reduction in the amount of type I regulatory beta subunit (RI beta) steady state mRNA by competitive polymerase chain reaction. This is associated with a 30% decrease in RI alpha protein and a 65% reduction in RI beta protein. Indeed, T cells from approximately 25% of SLE subjects have no detectable RI beta protein. Transient transfection of T cells not expressing RI beta protein with autologous SLE RI beta cDNA bypassed the block in translation, reconstituting PKA activity and augmenting IL-2 production. Of importance was the initial identification of novel RI alpha mRNA mutations characterized by heterogeneous transcript mutations, including deletions, transitions, and transversions. Most mutations are clustered adjacent to GAGAG motifs and CT repeats. By contrast, deficient PKA-II activity is the result of spontaneous dissociation of the cytosolic RII beta(2)C(2) holoenzyme, aberrant RII beta translocation to the nucleus from the cytosol, and retention of RII beta in the nucleus. In conclusion, distinct mechanisms account for deficient PKA-I and PKA-II isozyme activities in SLE T cells.

Novel Frameshift Mutations near Short Simple Repeats

In patients with Alzheimer's disease or Down's syndrome, the cerebellar cortex exhibits protein deposits in neurofibrillary tangles and neuritic plaques. Recently, the deposits have been shown to contain protein fragments of ubiquitin-B and amyloid precursor protein (APP) with an aberrant carboxyl terminus resulting from frameshift mutations (dinucleotide deletions; DeltaGU or DeltaGA) in or adjacent to GAGAG motifs in their mRNAs, a process referred to as molecular misreading. We have now used a bacterial expression system with the green fluorescent protein as a reporter to screen gene transcripts from aged controls, Alzheimer's disease, and Down's syndrome for molecular misreading. Novel frameshift mutations at a number of locations in the transcripts of the ubiquitin-B and APP genes were discovered (DeltaGA, DeltaG, DeltaGU, DeltaGG, DeltaCA, DeltaAU, DeltaA, DeltaAA, DeltaC, DeltaU, and insertion of an A). Interestingly, most mutations were in close proximity of short simple repeats (GAGAG, GGUGGU, GAGACACACA, UCAUCAUCA, CAAACAAA, and GAAGAAGAA), demonstrating that the GAGAG motif does not constitute the only hot spot for transcriptional errors. Unlike the previously detected aberrant APP fragments, some of the novel ones have the potential to generate the neurotoxic peptide beta-amyloid. We conclude that during aging molecular misreading is a widespread phenomenon.

Molecular misreading: a new type of transcript mutation expressed during aging

Dinucleotide deletions (e.g. ΔGA, ΔGU) are created by molecular misreading in or adjacent to GAGAG motifs of neuronal mRNAs. As a result, the reading frame shifts to the +1 frame, and so-called “+1 proteins” are subsequently synthesized. +1 Proteins have a wild-type N-terminus, but an aberrant C-terminus downstream from the site of the dinucleotide deletion. Molecular misreading was discovered in the rat vasopressin gene associated with diabetes insipidus and subsequently in human genes linked to Alzheimer’s disease (AD), e.g. β amyloid precursor protein (βAPP) and ubiquitin-B (UBB). Furthermore, βAPP +1 and UBB +1 proteins accumulate in the neuropathological hallmarks (i.e. in the tangles, neuritic plaques, and neuropil threads) of AD. As these +1 proteins were also found in elderly nondemented controls, but not in younger ones (<51 years), molecular misreading in nondividing cells might act as a factor that only becomes manifest at an advanced age. Frameshift mutations (UBB +1) and pretangle staining (Alz-50 and MC1) seem to occur independently of each other during early stages of AD. We recently detected +1 proteins, not only in proliferating cells present in non-neuronal tissues such as the liver and epididymis, but also in neuroblastoma cell lines. These observations suggest that molecular misreading is a general source of transcript errors that are involved in cellular derangements in various age-related pathologies.

MRNA mutations of type I protein kinase A regulatory subunit α in T lymphocytes of a subject with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by multiple T lymphocyte immune effector dysfunctions. Protein kinase A (PKA) isozymes contribute to the regulation of T cell immune effector functions. In SLE T cells, there is a profound deficiency of PKA-I isozyme activity characterized by both reduced RI alpha transcript and RI alpha protein levels. To identify a molecular mechanism(s) for this isozyme deficiency, we utilized single-strand conformation polymorphism (SSCP) analysis to detect structural changes in the cDNA. Of 10 SLE subjects, cDNAs from a single subject revealed a shifted band. Sequence analyses demonstrated that a shifted SSCP band from SLE T cells carried heterogeneous transcript mutations, including deletions, transitions and transversions. Most of these transcript mutations are clustered adjacent to GAGAG motifs and CT repeats-regions that are susceptible to transcript editing and/or molecular misreading. By contrast, no genomic mutations were identified. These results suggest the occurrence of mRNA editing and/or defective function of RNA polymerase in a subject with SLE. Mutant RI alpha transcripts are pathophysiolgically significant, for they can encode diverse, aberrant RI alpha isoforms, including truncated, dominant-negative subunits, resulting in deficient PKA-I activity. We propose that deficient PKA-I isozyme activity contributes to the pathogenesis of SLE by hindering effective signal transduction and impairing T cell effector functions.

Molecular misreading in non‐neuronal cells

+1 Frame-shifted proteins such as amyloid precursor protein+1 and ubiquitin-B+1 have been identified in the neuropathological hallmarks of Alzheimer’s disease. These frameshifts are caused by dinucleotide deletions in GAGAG motifs of messenger RNA encoded by genes that have maintained the unchanged wild-type DNA sequence. This process is termed ‘molecular misreading’. A key question is whether this process is confined to neurons or whether it could also occur in non-neuronal cells. A transgenic mouse line (MV-B) carrying multiple copies of a rat vasopressin minigene as a reporter driven by the MMTV-LTR promotor was used to screen non-neuronal tissues for molecular misreading by means of detection of the rat vasopressin+1 protein and mutated mRNA. Molecular misreading was demonstrated to occur in several organs (e.g., epididymis and the parotid gland) where transgenic vasopressin expression is abundant, but its penetrance is variable both between and within tissues. This implies that non-neural tissues too...

Molecular misreading in non-neuronal cells.

+1 Frame-shifted proteins such as amyloid precursor protein(+1) and ubiquitin-B(+1) have been identified in the neuropathological hallmarks of Alzheimer's disease. These frameshifts are caused by dinucleotide deletions in GAGAG motifs of messenger RNA encoded by genes that have maintained the unchanged wild-type DNA sequence. This process is termed 'molecular misreading'. A key question is whether this process is confined to neurons or whether it could also occur in non-neuronal cells. A transgenic mouse line (MV-B) carrying multiple copies of a rat vasopressin minigene as a reporter driven by the MMTV-LTR promotor was used to screen non-neuronal tissues for molecular misreading by means of detection of the rat vasopressin(+1) protein and mutated mRNA. Molecular misreading was demonstrated to occur in several organs (e.g., epididymis and the parotid gland) where transgenic vasopressin expression is abundant, but its penetrance is variable both between and within tissues. This implies that non-neural tissues too, could be affected by cellular derangements caused by molecular misreading.

Molecular misreading. A new type of transcript mutation in gerontology.

Molecular misreading is a novel process that causes mutations in neuronal transcripts. It is defined as the inaccurate conversion of genomic information from DNA into nonsense transcripts and the subsequent translation into mutant proteins. As a result of dinucleotide deletions (delta GA, delta GU, delta CU) in and around GAGAG motifs in mRNA the reading frame shifts to the +1 frame, and subsequently the so-called +1 proteins are synthetized. +1 Proteins have a wild-type NH2 terminus and from the site of the dinucleotide deletion onwards an aberrant, nonfunctional COOH terminus. Molecular misreading was found in the rat vasopressin gene associated with diabetes insipidus and in the human genes linked to Alzheimer's disease (AD), that is, beta-amyloid precursor protein (beta APP) and ubiquitin-B (UBB). Moreover, beta APP+1 and UBB+1 proteins accumulate in the neuropathological hallmarks of AD. Inasmuch as these +1 proteins were also found in elderly, nondemented control patients, but not in younger ones (< 72 years), molecular misreading may act as a factor that becomes manifest in aged people. A hotspot for dinucleotide deletions is GAGAG motifs. Because statistically an average of 2.1 GAGAG motifs per gene can be expected, other genes expressed in other tissues may undergo molecular misreading as well. Indeed, we recently detected +1 proteins in proliferating cells present in tissues such as the liver, epididymis, parotid gland, and neuroblastoma cell lines. Therefore, molecular misreading can be regarded as a general biological source of transcript errors that may be involved in cellular derangements in numerous age-related pathologic conditions apart from Alzheimer's disease.

Molecular misreading of genes in Down syndrome as a model for the Alzheimer type of neurodegeneration.

The occurrence of +1 frameshifted proteins, such as amyloid precursor protein (APP+1) and ubiquitin-B (UBB+1) in Down syndrome (DS) has been linked to the onset of Alzheimer's disease (AD). In DS and AD patients, but also in elderly non-demented persons, these co-called +1 proteins accumulate in the neuropathological hallmarks (neurofibrillary tangles, dystrophic neurites of the neuritic plaques and neuropil threads) and may have deleterious effects on neuronal function. Frameshifts are caused by dinucleotide deletions in GAGAG motifs in messenger RNA and are now thought to be the result of unfaithful transcription of normal DNA by a novel process termed "molecular misreading". In the present review some of the critical events in molecular misreading are discussed, the emphasis being on DS.

Immunocytochemical evidence for the presence of vasopressin in intermediate sized neurosecretory granules of solitary neurohypophyseal terminals in the homozygous brattleboro rat

A single base deletion (delta G) in the vasopressin gene is the cause of diabetes insipidus in the homozygous Brattleboro rat (di/di). The resulting frameshift leads to the expression of an aberrant vasopressin precursor which is unable to enter the secretory pathway, thereby preventing vasopressin biosynthesis. In a small number of solitary magnocellular hypothalamic neurons within the supraoptic and paraventricular nuclei, the reading frame is restored by a dinucleotide (delta GA) frameshift mutation, at two separate GAGAG motifs downstream of the original G-deletion. This results in two + 1 di-vasopressin precursors that are still partially mutated within the neurophysin region. The present study provides immunocytochemical evidence which demonstrates that, within magnocellular solitary neurons of the supraoptic and paraventricular nuclei of the di/di rat, the + 1 di-vasopressin precursors can enter the secretory pathway followed by their enzymatic processing into vasopressin during axonal transport to the neural lobe. However, the cellular characteristics of biosynthesis are different from those of wild-type rats. Immunoelectron microscopical localization of vasopressin gene products in the neural lobe of did/di rats revealed their presence in neurosecretory granules, the diameter of which is intermediate (116 nm) between those of the neurosecretory granules in the di/di (80-100 nm) and wild-type (160 nm) rats.

Frameshift mutations at two hotspots in vasopressin transcripts in post-mitotic neurons.

Mutations in DNA underlie carcinogenesis, inherited pathology, and aging and are generally thought to be introduced during meiosis and mitosis. Here we report that in post-mitotic neurons specific frameshift mutations occur at high frequency. These mutations were identified in vasopressin transcripts in magnocellular neurons of the homozygous Brattleboro rat and predominantly consist of a GA deletion in GAGAG motifs. Immunocytochemistry provides evidence for similar events in wild-type rats. However, the diseased state of the Brattleboro rat, resulting in a permanent activation of vasopressin neurons, enhanced the mutational rate. These data reveal hitherto unrecognized somatic mutations in nondividing neurons.