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PubMed2026

Elevated intron retention implicates neuroinflammation in brains of individuals with alcohol use disorder.

Intron retention, a form of alternative RNA splicing, can occur as part of normal gene regulation or result from disruption of the splicing machinery. Retained introns can potentially form double-stranded RNA, activating innate immune sensors and inflammation. This mechanism has been implicated in cancer but has not been studied in neuropsychiatric diseases like alcohol use disorder. We systematically analysed transcriptome-wide intron retention events in post-mortem brain tissue from 142 individuals (66 with alcohol use disorder and 76 controls), encompassing 320 region-specific samples from the superior frontal cortex, nucleus accumbens, central nucleus and basolateral amygdala. Analyses were adjusted for demographic, technical and biological covariates. Validation was performed in alcohol-preferring (P) rats using long-read sequencing. In complementary experiments, immunofluorescent staining was used to detect double-stranded RNA in rat brain tissue, while single-cell RNA-sequencing was performed to test activation of double-stranded RNA-sensing pathways in human brains. Brains from individuals with alcohol use disorder showed significantly higher total intron retention compared with controls, independent of age, with females showing greater increases than males. A total of 368 introns were positively associated with alcohol use disorder, and these introns were significantly longer and had weaker splice acceptor sites compared with non-associated introns. Genes harbouring these intron retention events were enriched in Purkinje neurons, visual cortex neurons and oligodendrocytes. Computational predictions indicated these long introns could form duplex RNA structures. Increased double-stranded RNA was confirmed experimentally in multiple brain regions of alcohol-consuming rats, where it co-localized primarily with neuronal nuclei and dendrites. In individuals with alcohol use disorder, we found that multiple pathways including double-stranded RNA responses, neuroinflammation, interferon and NF-κB signalling, adaptive immunity and apoptosis were activated. In addition, NeuN-positive neuronal counts significantly decreased in both the prefrontal and visual cortices. Furthermore, single-cell analysis demonstrated upregulation of TICAM1, the target of double-stranded RNA sensor TLR3, in oligodendrocytes, as well as widespread activation of downstream inflammatory pathways across glial and neuronal cell types. These findings provide the first evidence that chronic alcohol consumption promotes an overall increase of intron retention in the brain and is associated with the presence of double-stranded RNA. Furthermore, the double-stranded RNA may contribute to neuronal loss and brain pathology by activating a neuroinflammatory response.

alcohol use disorder↗
PubMed1993

Identification of two new nucleotide mutations (HPRTUtrecht and HPRTMadrid) in exon 3 of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene.

Mutations in the X-linked hypoxanthine-guanine phosphoribosyl transferase gene (HPRT) result in deficiencies of HPRT enzyme activity, which may cause either a severe form of gout or Lesch-Nyhan syndrome depending on the residual enzyme activity. Mutations leading to these diseases are heterogeneous and include DNA base substitutions, DNA deletions, DNA base insertions and errors in RNA splicing. Identification of mutations has been performed at the RNA and DNA level. Sequencing genomic DNA of the HPRT gene offers the possibility of direct diagnostic analysis independent on the expression of the mature HPRT mRNA. We describe a Dutch and a Spanish family, in which the Lesch-Nyhan syndrome and a severe partial HPRT-deficient phenotype, respectively, were diagnosed. Direct sequencing of the exons coding for the HPRT gene was performed in both families. Two new exon 3 mutations have been identified. At position 16676, the normally present G was substituted by an A in the Dutch kindred (HPRTUtrecht), and led to an arginine for glycine change at residue 70. At position 16680, the G was substituted by a T in the Spanish family (HPRTMadrid); this substitutes a valine for glycine at residue 71. These new mutations are located within one of the clusters of hotspots in exon 3 of the HPRT gene in which HPRTYale and HPRTNew Haven have previously been identified.

Amino Acid Sequence↗
PubMed1978

Polyoma virus giant RNAs contain tandem repeats of the nucleotide sequence of the entire viral genome.

The bulk of late virus-specific RNA synthesized in polyoma virus-infected mouse cells is larger than a single strand of poloma DNA. The arrangement of viral nucleotide sequences in these giant polyoma RNAs was studied by electron microscopy of hybrids between purified high molecular weight viral RNA and the HindII-1 fragment of polyoma DNA, which contains 91% of the viral genome. Hybrid molecules containing a short single-stranded gap (corresponding to the 9% of viral sequences not present in HindII-1), flanked by double-stranded regions, were photographed and measured. The majority of hybrid molecules contained no single-stranded loops or branches, showing that all viral sequences are transcribed contiguously and that no nonviral sequences are present in the RNA. Hybrid molecules, containing RNA up to 3.5 times the genome length, had a repeating structure of single-stranded gaps 8% of genome length interspersed with double-stranded regions 89% of genome length, showing that giant polyoma RNAs contain tandem repeats of the nucleotide sequence of the entire viral DNA. A small proportion of hybrid molecules contained single-stranded branches or deletion loops in characteristic positions, indicating that RNA "splicing" may occur on high molecular weight nuclear polyoma RNA.

Cell Nucleus↗
PubMed1994

In vitro selection of antisense oligonucleotides targeted to a hairpin structure.

Antisense oligonucleotides are widely used to selectively prevent pre-RNA splicing, mRNA translation or cDNA synthesis from a retroviral RNA template. However, intramolecular folding of the RNA chain can sequester the target sequence into a stable structure. Consequently, the antisense effect can be greatly reduced or even abolished. Hydrogen donor and acceptor sites are still available on nucleic acid bases involved in secondary structures. However, the rational design of antisense sequences able to recognize the three dimensional array of these sites is not available. We used an in vitro selection procedure to fish out aptastrucs, i.e., oligomers able ("apte") to bind to a structure. A population of randomly synthesized oligonucleotides was mixed with the structure of interest and oligodeoxynucleotide sequences bound to the target were selected and amplified. The selection involves the destruction of the unbound candidates by a restriction enzyme. This procedure can be used both for RNA and DNA target structures and does not require the purification of the bound oligonucleotides at each cycle of selection. Several cycles of selection-amplification, followed by cloning and sequencing, allowed us to identify three oligonucleotides able to form a complex with a DNA hairpin. Due to the sequence of the selected candidates, these aptastruc-hairpin complexes involve very likely non-canonical interactions between the two partners.

DNA↗
PubMed1991

Accumulation of MVM gene products is differentially regulated by transcription initiation, RNA processing and protein stability.

The accumulation and stability of minute virus of mice (MVMp) RNA and protein as well as comparative strengths of the two viral promoters have been analyzed in highly synchronous infections of murine A9 fibroblasts. Results indicate that there is a temporal phasing of the accumulation of MVM RNA and protein: the RNA products of the P4 promoter appear prior to the products of the P38 promoter and NS1 and NS2 appear prior to the capsid proteins. Total and cytoplasmic spliced RNA accumulate similarly, although there is a lag in cytoplasmic accumulation of about 2 hr. Total RNA contains abundant unspliced R1 and R3 which are confined to the nucleus. There is no detectable difference in the ratio of the various spliced versions of each RNA species throughout infection. R2 accumulates faster and in a greater amount than R1 in both total and cytoplasmic RNA even though they are generated from the same promoter. During this same period, however, NS1 and NS2 accumulate to similar levels during 1-hr pulses. The stabilities of all MVM RNA species produced at both 9 and 12 hr postrelease are equivalent. Late in infection R3 accumulates faster and in greater amounts than the combined products of the P4 promoter, by approximately two- to threefold. This increase can be accounted for by an increase in the frequency of initiation from the P38 promoter, relative to P4, as assayed by nuclear run-on experiments. Therefore, the steady-state levels of the individual viral proteins during infection is controlled by specific regulation at the level of the initiation of transcription, RNA processing, and protein stability.

Animals↗
PubMed1988

Mitochondrial biogenesis: recent developments and insights.

Biosynthesis of a functional mitochondrion requires the coordinate expression of genes in both mitochondrial and nuclear DNAs. In yeast, three mitochondrial genes are split and RNA splicing plays a pivotal role in their expression. The recent finding that some introns are capable of self-splicing activity in vitro has permitted analysis of the mechanisms involved in RNA catalysis and may eventually shed light on the evolution of splicing mechanisms in general. Most mitochondrial proteins are encoded by nuclear genes, synthesized in the cytoplasm and imported by the organelle. The availability of cloned genes coding for several constituent subunits of the ubiquinol-cytochrome c reductase, which are imported by mitochondria, has allowed study of selected steps in the addressing of proteins to mitochondria and their intercompartmental sorting within the organelle. Recent developments are discussed.

DNA, Mitochondrial↗
PubMed2004

The Drosophila U1-70K protein is required for viability, but its arginine-rich domain is dispensable.

The conserved spliceosomal U1-70K protein is thought to play a key role in RNA splicing by linking the U1 snRNP particle to regulatory RNA-binding proteins. Although these protein interactions are mediated by repeating units rich in arginines and serines (RS domains) in vitro, tests of this domain's importance in intact multicellular organisms have not been carried out. Here we report a comprehensive genetic analysis of U1-70K function in Drosophila. Consistent with the idea that U1-70K is an essential splicing factor, we find that loss of U1-70K function results in lethality during embryogenesis. Surprisingly, and contrary to the current view of U1-70K function, animals carrying a mutant U1-70K protein lacking the arginine-rich domain, which includes two embedded sets of RS dipeptide repeats, have no discernible mutant phenotype. Through double-mutant studies, however, we show that the U1-70K RS domain deletion no longer supports viability when combined with a viable mutation in another U1 snRNP component. Together our studies demonstrate that while the protein interactions mediated by the U1-70K RS domain are not essential for viability, they nevertheless contribute to an essential U1 snRNP function.

Amino Acid Sequence↗
PubMed1995

cis-splicing and polyadenylation of actin RNA can precede 5' trans-splicing in nematodes.

In nematodes many precursor RNAs undergo both cis-splicing (intron removal) and trans-splicing (spliced leader addition) via molecular mechanisms thought to be very similar. We examined the temporal order of trans- and cis-splicing by analyzing cDNAs of partially processed transcripts from an actin gene of the parasitic nematode, Onchocerca volvulus, which has five short introns. We found that in this model gene system trans-splicing can follow cis-splicing and polyadenylation, and we obtained no evidence for alternative pathways of splicing. This result is in contrast to the tubulin genes of African trypanosomes where trans-splicing precedes polyadenylation (1).

Actins↗
PubMed1998

Rat embryonic hippocampal neurons express a new class A calcium channel variant.

The aim of this study was to investigate, using a RT-PCR strategy, rat voltage-gated class A calcium channel (alpha1A) splice variants during rat hippocampus development. Results demonstrate the presence of multiple alpha1A mRNAs with the hippocampus formation and revealed a new variant of the rat alpha1A subunit (alpha1A-EFe) that diverges from alpha1A-a in the EF-hand domain. alpha1A-EFe expression in hippocampal neurons is restricted to the embryonic period. This in vivo developmental program is recapitulated in dissociated cultures of E17 embryonic hippocampal neurons. These data demonstrate that rat hippocampus neurons express a unique alpha1A splice variant during the embryonic period and suggest that alternative RNA splicing may modulate neuronal calcium channel properties during development.

Aging↗
PubMed1991

Alternative splicing of beta-tropomyosin pre-mRNA: cis-acting elements and cellular factors that block the use of a skeletal muscle exon in nonmuscle cells.

The rat beta-tropomyosin (beta-TM) gene encodes both skeletal muscle beta-TM and fibroblast TM-1 by an alternative RNA-splicing mechanism. This gene contains 11 exons. Exons 1-5, 8, and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts as well as smooth muscle cells, whereas exons 7 and 10 are used in skeletal muscle cells. In this study we have carried out an extensive mutational analysis to identify cis-acting elements that block the use of the skeletal muscle-specific exon 7 in nonmuscle cells. These studies localize the critical elements for regulated alternative splicing to sequences within exon 7 and the adjacent upstream intron. In addition, mutations that inactivate the 5'- or 3'-splice sites of exon 6 do not result in the use of the skeletal muscle-specific exon 7 in nonmuscle cells, suggesting that splice-site selection in vivo is not regulated by a simple cis-acting competition mechanism but, rather, by a mechanism that inhibits the use of exon 7 in certain cellular environments. In support of this hypothesis we have identified sequence-specific RNA-binding proteins in HeLa cell nuclear extracts using native gel electrophoresis and binding competition assays. Mutations in the pre-mRNA that result in the use of the skeletal muscle exon in vivo also disrupt the binding of these proteins to the RNA in vitro. We propose that the binding of these proteins to the pre-mRNA is involved in regulated alternative splicing and that this interaction is required for blocking the use of the skeletal muscle exon in nonmuscle cells.

Base Sequence↗
PubMed2001

Nonsense-mediated decay of mutant waxy mRNA in rice.

Two rice (Oryza sativa) waxy mutations of the Japonica background were shown to contain approximately 20% of the fully spliced mRNA relative to the wild type. Sequencing analysis of the entire waxy genes of the two mutants revealed the presence of premature translation termination codons in exon 2 and exon 7. These results indicated that the lower accumulation of fully spliced RNA in the mutants was caused by nonsense-mediated decay (NMD), which is an RNA surveillance system universally found in eukaryotes. It is interesting that levels of RNA retaining intron 1 were not changed by premature nonsense codons, suggesting that splicing may be linked with NMD in plants, as previously found in mammalian cells. Measurements of the half-lives of waxy RNAs in transfected rice protoplasts indicated that the half-life of waxy RNA with a premature nonsense codon was 3.3 times shorter than that without a premature nonsense codon. Because the wild-type waxy transcripts, which are derived from the Wx(b) gene predominantly distributed among Japonica rice, have been shown to be less efficiently spliced and their alternative splicing has been documented, we examined whether these splicing properties influenced the efficiency of NMD. However, no effects were observed. These results established that NMD occurs in rice waxy RNA containing a premature nonsense codon.

Alternative Splicing↗
PubMed2005

A deoxyribozyme that synthesizes 2',5'-branched RNA with any branch-site nucleotide.

RNA molecules with internal 2',5'-branches are intermediates in RNA splicing, and branched RNAs have recently been proposed as retrotransposition intermediates. A broadly applicable in vitro synthetic route to branched RNA that does not require self-splicing introns or spliceosomes would substantially improve our ability to study biochemical processes that involve branched RNA. We recently described 7S11, a deoxyribozyme that was identified by in vitro selection and has general RNA branch-forming ability. However, an important restriction for 7S11 is that the branch-site RNA nucleotide must be a purine (A or G), because a pyrimidine (U or C) is not tolerated. Here, we describe the compact 6CE8 deoxyribozyme (selected using a 20 nt random region) that synthesizes 2',5'-branched RNA with any nucleotide at the branch site. The Mn2+-dependent branch-forming ligation reaction is between an internal branch-site 2'-hydroxyl nucleophile on one RNA substrate with a 5'-triphosphate on another RNA substrate. The preference for the branch-site nucleotide is U > C congruent with A > G, although all four nucleotides are tolerated with useful ligation rates. Nearly all other nucleotides elsewhere in both RNA substrates allow ligation activity, except that the sequence requirement for the RNA strand with the 5'-triphosphate is 5'-pppGA, with 5'-pppGAR (R = purine) preferred. These characteristics permit 6CE8 to prepare branched RNAs of immediate practical interest, such as the proposed branched intermediate of Ty1 retrotransposition. Because this branched RNA has two strands with identical sequence that emerge from the branch site, we developed strategies to control which of the two strands bind with the deoxyribozyme during the branch-forming reaction. The ability to synthesize the proposed branched RNA of Ty1 retrotransposition will allow us to explore this important biochemical pathway in greater detail.

Catalysis↗
PubMed1998

MyoD is indispensable for muscle-specific alternative splicing in mouse mitochondrial ATP synthase gamma-subunit pre-mRNA.

Muscle-specific alternative RNA splicing is an essential step during myogenesis. In this paper, we report that a muscle-specific transcription factor, MyoD, plays a central role in the induction of muscle-specific alternative splicing during myogenesis. Recently, we reported that muscle and nonmuscle isoforms of the mitochondrial ATP synthase gamma-subunit (F1gamma) were generated by alternative splicing and that acidic stimulation promoted this muscle-specific alternative splicing (Endo, H., Matsuda, C., and Kagawa, Y. (1994) J. Biol. Chem. 269, 12488-12493). In this report, mouse myoblasts are shown to express the muscle-specific isoform of F1gamma after induction with low-serum medium (differentiation medium) or acidic medium, although myotube formation was not detected after acidic induction. RNA blot analysis revealed that the expression levels of both MEF2 and myogenin were increased by low-serum induction, but not by acidic induction. High expression of MyoD mRNA was observed after both types of induction. Overexpression of exogenous MyoD in fibroblasts showed that MyoD was necessary for muscle-specific alternative splicing in both types of induction. Exogenous Id, a negative regulator of MyoD, blocked muscle-specific alternative splicing of F1gamma pre-mRNA by both types of induction. In addition, MyoD induced several muscle-specific alternative splicings, including structural protein pre-mRNAs such as beta-tropomyosin and neural-cell adhesion molecule and transcriptional protein pre-mRNAs such as MEF2A and MEF2D. Our analysis of the two induction systems shows a common MyoD-dependent mechanism of muscle-specific alternative splicing in several genes, independent of MEF2 and myogenin.

Adenosine Triphosphatases↗
PubMed1997

Spontaneous occurrence of early region 1A reiteration mutants of type 5 adenovirus in persistently infected human T-lymphocytes.

Mutants of type 5 adenovirus (Ad5) with reiterated DNA sequences in the E1a region appeared in a human T-lymphocyte cell line, Molt-4, persistently infected with H5sub304, a deletion/substitution mutant that has a wild-type phenotype in viral replication. Endonuclease analyses and DNA sequencing revealed DNA reiteration in each mutant. In the four representative mutants investigated, the DNA reiterations all started within a six-base-pair consensus sequence, G(or C)CTGTG, located in the second exon of the E1a region (at nt 1333, 1367, or 1419). There was not any DNA homology between the breakpoints in the second exon and the inserting sequences (starting at nt 532, 710, or 792). Northern analyses suggested that the reiterated splicing sites of the representative mutants were all used in RNA splicing, and the closest donor and recipient joints were used most frequently. These observations imply that during persistent infection Ad5 underwent spontaneous mutations by sequence-specific breakage and nonhomologous end-end joining recombination events. These E1a reiteration mutants could be propagated in HeLa, A549, and KB cells; they were genetically stable; and they killed CREF cells at a strikingly high frequency. Preliminary observations tend to correlate this CREF cell killing with the accumulation of the early viral proteins and/or viral DNA in the infected cells. This degree of cell damage was not observed in Ad5wt or H5sub304 infection of CREF cells. The observed E1a reiterations provide a model to gain insight into understanding the evolutionary events of some, if not all, adenovirus types during many years of symbiotic, persistent relationship in human tonsils and adenoids and possibly other lymphoid organs.

Adenovirus E1A Proteins↗
PubMed1985

Evidence for age-dependent impairment of ovalbumin heterogeneous nuclear RNA (HnRNA) processing in hen oviduct.

The expression of the ovalbumin gene in hen oviduct decreases during ageing. Northern transfer of electrophoretically fractionated total RNA from oviducts of mature egg-laying and old non-egg-laying hens, and hybridization to nick-translated plasmid pOV230 (ovalbumin complementary DNA cloned into pMB9) revealed with age increasing concentrations of distinct high-molecular-weight ovalbumin pre-mRNA species as compared with that of functional 18S ovalbumin mRNA. The accumulation of ovalbumin sequence-containing processing intermediates in old hen oviducts indicates lowered rates of RNA splicing and/or altered steady state concentrations of splicing intermediates during post-transcriptional maturation of heterogeneous nuclear RNA in old animals.

Age Factors↗
PubMed1990

Cells nonproductively infected with HIV-1 exhibit an aberrant pattern of viral RNA expression: a molecular model for latency.

U1 and ACH-2 cells are subclones of HIV-1-infected monocyte/macrophage-like and T lymphocyte cell lines, respectively, which express the HIV-1 genome at very low levels. We have examined whether they might provide a model of HIV-1 latency. The patterns of HIV-1-specific RNA expressed in these cells consisted of singly and multiply spliced RNA species, with little or no full-length genomic RNA. Upon stimulation with agents that activate the HIV-1 long terminal repeat in these cells, a marked rise in the amount of small mRNAs, encoding the viral regulatory proteins, preceded the increase in the unspliced RNA. Thus, U1 and ACH-2 cells maintain HIV-1 in a state equivalent to the early phase of a lytic infection and, after stimulation, recapitulate the events of a single cycle infection of highly susceptible target cells.

Blotting, Northern↗
PubMed1986

Leader sequences of murine coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription.

Mouse hepatitis virus (MHV), which replicates in cytoplasm of infected cells, contains an identical leader RNA sequence at the 5' end of each of the virus-specific mRNAs. Previous studies suggested that the synthesis of these mRNAs does not involve conventional RNA splicing and may instead require priming by a free leader RNA. In this communication, we demonstrate that, during a mixed infection with two different MHVs, the leader RNA sequences from one virus could be detected on the mRNAs of the coinfecting virus at a high frequency, as if the leader sequence and mRNAs were joined together from two randomly segregating RNA segments. This finding demonstrates that MHV mRNA transcription utilizes independently transcribed leader RNA species that possess the trans-acting property. This study thus provides further evidence in support of the unique model of "leader-primed transcription" for coronavirus mRNA synthesis.

Murine hepatitis virus↗