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Results for “Splicing Life”

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At least 55 records · Page 3Linked to original sources

PubMed2000

Utilization of the bovine papillomavirus type 1 late-stage-specific nucleotide 3605 3' splice site is modulated by a novel exonic bipartite regulator but not by an intronic purine-rich element.

Bovine papillomavirus type 1 (BPV-1) late gene expression is regulated at both transcriptional and posttranscriptional levels. Maturation of the capsid protein (L1) pre-mRNA requires a switch in 3' splice site utilization. This switch involves activation of the nucleotide (nt) 3605 3' splice site, which is utilized only in fully differentiated keratinocytes during late stages of the virus life cycle. Our previous studies of the mechanisms that regulate BPV-1 alternative splicing identified three cis-acting elements between these two splice sites. Two purine-rich exonic splicing enhancers, SE1 and SE2, are essential for preferential utilization of the nt 3225 3' splice site at early stages of the virus life cycle. Another cis-acting element, exonic splicing suppressor 1 (ESS1), represses use of the nt 3225 3' splice site. In the present study, we investigated the late-stage-specific nt 3605 3' splice site and showed that it has suboptimal features characterized by a nonconsensus branch point sequence and a weak polypyrimidine track with interspersed purines. In vitro and in vivo experiments showed that utilization of the nt 3605 3' splice site was not affected by SE2, which is intronically located with respect to the nt 3605 3' splice site. The intronic location and sequence composition of SE2 are similar to those of the adenovirus IIIa repressor element, which has been shown to inhibit use of a downstream 3' splice site. Further studies demonstrated that the nt 3605 3' splice site is controlled by a novel exonic bipartite element consisting of an AC-rich exonic splicing enhancer (SE4) and an exonic splicing suppressor (ESS2) with a UGGU motif. Functionally, this newly identified bipartite element resembles the bipartite element composed of SE1 and ESS1. SE4 also functions on a heterologous 3' splice site. In contrast, ESS2 functions as an exonic splicing suppressor only in a 3'-splice-site-specific and enhancer-specific manner. Our data indicate that BPV-1 splicing regulation is very complex and is likely to be controlled by multiple splicing factors during keratinocyte differentiation.

3' Untranslated Regions↗

PubMed1993

Order of intron removal during splicing of endogenous adenine phosphoribosyltransferase and dihydrofolate reductase pre-mRNA.

Using a strategy based on reverse transcription and the polymerase chain reaction, we have determined the order of splicing of the four introns of the endogenous adenine phosphoribosyltransferase (aprt) gene in Chinese hamster ovary cells. The method involves a pairwise comparison of molecules that retain one intron and have either retained or spliced another intron(s). A highly preferred order of removal was found: intron 3 > 2 > 4 = 1. This order did not represent a linear progression from one end of the transcript to the other, nor did it correlate with the conformity of the splice site sequences to the consensus sequences or to the calculated energy of duplex formation with U1 small nuclear RNA. By using actinomycin D to inhibit RNA synthesis, the in vivo rate of the first step in splicing was estimated for all four introns; a half-life of 6 min was found for introns 2, 3, and 4. Intron 1 was spliced more slowly, with a 12-min half-life. A substantial amount of RNA that retained intron 1 as the sole intron was exported to the cytoplasm. In the course of these experiments, we also determined that intron 3, but not intron 4, is spliced before 3'-end formation is complete, probably on nascent transcripts. This result is consistent with the idea that polyadenylation is required for splicing of the 3'-most intron. We applied a similar strategy to determine the last intron to be spliced in a very large transcript, that of the endogenous dihydrofolate reductase (dhfr) gene in Chinese hamster ovary cells (25 kb). Here again, intron 1 was the last intron to be spliced.

Adenine Phosphoribosyltransferase↗

PubMed2005

Regulation of apoptosis by alternative pre-mRNA splicing.

Apoptosis, a phenomenon that allows the regulated destruction and disposal of damaged or unwanted cells, is common to many cellular processes in multicellular organisms. In humans more than 200 proteins are involved in apoptosis, many of which are dysregulated or defective in human diseases including cancer. A large number of apoptotic factors are regulated via alternative splicing, a process that allows for the production of discrete protein isoforms with often distinct functions from a common mRNA precursor. The abundance of apoptosis genes that are alternatively spliced and the often antagonistic roles of the generated protein isoforms strongly imply that alternative splicing is a crucial mechanism for regulating life and death decisions. Importantly, modulation of isoform production of cell death proteins via pharmaceutical manipulation of alternative splicing may open up new therapeutic avenues for the treatment of disease.

Alternative Splicing↗

PubMed1995

Splicing of cauliflower mosaic virus 35S RNA is essential for viral infectivity.

A splicing event essential for the infectivity of a plant pararetrovirus has been characterized. Transient expression experiments using reporter constructs revealed a splice donor site in the leader sequence of the cauliflower mosaic virus (CaMV) 35S RNA and three additional splice donor sites within open reading frame (ORF) I. All four donors use the same splice acceptor within ORF II. Splicing between the leader and ORF II produces an mRNA from which ORF III and, in the presence of the CaMV translational transactivator, ORF IV can be translated efficiently. The other three splicing events produce RNAs encoding ORF I-II in-frame fusions. All four spliced CaMV RNAs were detected in CaMV-infected plants. Virus mutants in which the splice acceptor site in ORF II is inactivated are not infectious, indicating that splicing plays an essential role in the CaMV life cycle. The results presented here suggest a model for viral gene expression in which RNA splicing is required to provide appropriate substrate mRNAs for the specialized translation mechanisms of CaMV.

Base Sequence↗

PubMed2004

Conservation and developmental control of alternative splicing in maebl among malaria parasites.

Genes of malaria parasites and other unicellular organisms have larger exons with fewer and smaller introns than metaozoans. Such differences in gene structure are perceived to extend to simpler mechanisms for transcriptional control and mRNA processing. Instead, we discovered a surprisingly complex level of post-transcriptional mRNA processing in analysis of maebl transcripts in several Plasmodium species. Mechanisms for internal alternative cis-splicing and exon skipping were active in multiple life cycle stages to change exon structure in the deduced coding sequence (CDS). The major alternatively spliced transcript utilized a less favorable acceptor splice site, which shifted codon triplet usage to a different CDS with a hydrophilic C terminus, changing the canonical type I membrane MAEBL product to a predicted soluble isoform. We found that developmental control of the alternative splicing pattern was distinct from the canonical splicing pattern. Western blot analysis indicated that MAEBL expression was better correlated with the appearance of the canonical ORF1 transcript. Together these data reveal that RNA metabolism in unicellular eukaryotes like Plasmodium is more sophisticated than believed and may have a significant role regulating gene expression in Plasmodium.

Alternative Splicing↗

PubMed2002

Induction of alternatively spliced spermidine/spermine N1-acetyltransferase mRNA in the human kidney cells infected by venezuelan equine encephalitis and tick-borne encephalitis viruses.

293 and RH cells derived from human embryo kidney were infected by Venezuelan equine encephalitis and tick-borne encephalitis viruses and cDNA libraries representing cellular mRNAs induced or suppressed due to the infection were prepared using suppressive subtractive hybridization. Among the up-regulated clones the RT-PCR and Northern analyses revealed an unusual transcript of the spermidine/spermine N1-acetyltransferase (SSAT) gene that was shown to be an alternatively spliced form containing an additional 110-bp exon. The alternatively spliced transcript is polyadenylated and can be expected to yield only a truncated 71 amino acid polypeptide. This first evidence of the host gene alternatively spliced mRNA induction by RNA viruses raises the questions of its biological role, regulation mechanisms of alternative splicing, and significance for the virus life cycle.

Acetyltransferases↗

PubMed2006

Linking splicing to Pol II transcription stabilizes pre-mRNAs and influences splicing patterns.

RNA processing is carried out in close proximity to the site of transcription, suggesting a regulatory link between transcription and pre-mRNA splicing. Using an in vitro transcription/splicing assay, we demonstrate that an association of RNA polymerase II (Pol II) transcription and pre-mRNA splicing is required for efficient gene expression. Pol II-synthesized RNAs containing functional splice sites are protected from nuclear degradation, presumably because the local concentration of the splicing machinery is sufficiently high to ensure its association over interactions with nucleases. Furthermore, the process of transcription influences alternative splicing of newly synthesized pre-mRNAs. Because other RNA polymerases do not provide similar protection from nucleases, and their RNA products display altered splicing patterns, the link between transcription and RNA processing is RNA Pol II-specific. We propose that the connection between transcription by Pol II and pre-mRNA splicing guarantees an extended half-life and proper processing of nascent pre-mRNAs.

DNA-Directed RNA Polymerases↗

PubMed2006

Chloride channelopathy in myotonic dystrophy resulting from loss of posttranscriptional regulation for CLCN1.

Transmembrane chloride ion conductance in skeletal muscle increases during early postnatal development. A transgenic mouse model of myotonic dystrophy type 1 (DM1) displays decreased sarcolemmal chloride conductance. Both effects result from modulation of chloride channel 1 (CLCN1) expression, but the respective contributions of transcriptional vs. posttranscriptional regulation are unknown. Here we show that alternative splicing of CLCN1 undergoes a physiological splicing transition during the first 3 wk of postnatal life in mice. During this interval, there is a switch to production of CLCN1 splice products having an intact reading frame, an upregulation of CLCN1 mRNA encoding full-length channel protein, and an increase of CLCN1 function, as determined by patch-clamp analysis of single muscle fibers. In a transgenic mouse model of DM1, however, the splicing transition does not occur, CLCN1 channel function remains low throughout the postnatal interval, and muscle fibers display myotonic discharges. Thus alternative splicing is a posttranscriptional mechanism regulating chloride conductance during muscle development, and the chloride channelopathy in a transgenic mouse model of DM1 results from a failure to execute a splicing transition for CLCN1.

Alternative Splicing↗

PubMed2004

Protein inhibitor of activated STAT Y (PIASy) and a splice variant lacking exon 6 enhance sumoylation but are not essential for embryogenesis and adult life.

Protein inhibitor of activated STAT Y (PIASy) is the shortest member of the PIAS family and has been reported to modulate the transcriptional activities of STAT1, lymphoid enhancer factor 1 (LEF-1), and the androgen receptor. PIAS proteins have also been identified as E3 ligases for the small ubiquitin-like modifier (SUMO) proteins. PIASy in particular has been reported to mediate SUMO-2/3 modification of LEF-1, sequestering it into nuclear bodies, and SUMO-1 ligation to c-Myb, modulating its transcriptional activation properties. We have cloned murine Piasy and a splice variant which omits exon 6, containing the nuclear retention PINIT motif. Cell culture studies indicate that both the full length and the splice variant are localized in the nucleus but differentially enhance SUMO ligation. To further understand the functions of PIASy, we have generated PIASy-deficient mice. Surprisingly, Piasy(-/-) mice appear phenotypically normal. Activation of STAT1 is not significantly perturbed in Piasy(-/-) cells, and sumoylation patterns for SUMO-1 or SUMO-3 modification are similar when comparing tissues and embryonic fibroblasts from wild-type and knockout mice. Our study demonstrates that at steady state, PIASy is either dispensable or compensated for by other PIAS family members or by other mechanisms when deleted.

Alternative Splicing↗

PubMed1985

RNA catalysis and the origin of life.

Until the discovery of catalytic RNAs, first the self-splicing intron in Tetrahymena and then the bacterial RNAse P, cellular enzymes had always seemed to be protein in nature. The recognition that RNA can catalytically make and break phosphodiester bonds simplifies some of the assumptions required of a rudimentary self-replicating entity. Available information on the chemistry of RNA-catalyzed reactions is reviewed, with particular attention to self-splicing introns and tRNA processing by RNase P. An explicit model for a self-replicating RNA is described. The model postulates a nucleotide binding/polymerization site in the RNA, and takes advantage of intrinsic fluidity in RNA higher order structure to dissociate parent and progeny complementary strands.

Animals↗

PubMed2000

A cellular protein, hnRNP H, binds to the negative regulator of splicing element from Rous sarcoma virus.

Incomplete RNA splicing is a key feature of the retroviral life cycle. This is in contrast to the processing of most cellular pre-mRNAs, which are usually spliced to completion. In Rous sarcoma virus, splicing control is achieved in part through a cis-acting RNA element termed the negative regulator of splicing (NRS). The NRS is functionally divided into two parts termed NRS5' and NRS3', which bind a number of splicing factors. The U1 and U11 small nuclear ribonucleoproteins interact with sequences in NRS3', whereas NRS5' binds several proteins including members of the SR [corrected] family of proteins. Among the proteins that specifically bind NRS5' is a previously unidentified 55-kDa protein (p55). In this report we describe the isolation and identification of p55. The p55 binding site was localized by UV cross-linking to a 31-nucleotide segment, and a protein that binds specifically to it was isolated by RNA affinity selection and identified by mass spectrometry as hnRNP H. Antibodies against hnRNP H immunoprecipitated cross-linked p55 and induced a supershift of a p55-containing complex formed in HeLa nuclear extract. Furthermore, UV cross-linking and electrophoretic mobility shift assays indicated that recombinant hnRNP H specifically interacts with the p55 binding site, confirming that hnRNP H is p55. The possible roles of hnRNP H in NRS function are discussed.

Avian Sarcoma Viruses↗

PubMed2005

Tissue transglutaminase during mouse central nervous system development: lack of alternative RNA processing and implications for its role(s) in murine models of neurotrauma and neurodegeneration.

Tissue transglutaminase (tTG) is a member of a multigene family principally involved in catalyzing the formation of protein cross-links. Unlike other members of the transglutaminase family, tTG is multifunctional since it also serves as a guanosine triphosphate (GTP) binding protein (Galpha(h)) and participates in cell adhesion. Different isoforms of tTG can be produced by proteolysis or alternative splicing. We find that tTG mRNA is expressed at low levels in the mouse CNS relative to other tissues, and at lower levels in the CNS of mouse in comparison to that of human or rat. tTG mRNA levels are higher in the heart compared to the CNS, for example, and much higher in the liver. Within the CNS, tTG message is lowest in the adult cerebellum and thalamus and highest in the frontal cortex and striatum. In the hippocampus, tTG expression is highest during embryonic development and falls off dramatically after 1 week of life. We did not find alternative splicing of the mouse tTG. At the protein level, the predominant isoform is approximately 62 kDa. In summary, tTG, an important factor in neuronal survival, is expressed at low levels in the mouse CNS and, unlike rat and human tTG, does not appear to be regulated by alternative splicing. These findings have implications for analyses of rodent tTG expression in human neurodegenerative and neurotrauma models where alternative processing may be an attractive pathogenetic mechanism. They further impact on drug discovery paradigms, where modulation of activity may have therapeutic value.

Alternative Splicing↗

PubMed2004

Spliced spleen necrosis virus vector RNA is not encapsidated: implications for retroviral replication and vector design.

RNA splicing is a complex event in the retroviral life cycle and can involve multiple steps, as well as cis-acting sequences, to maintain a proper balance of spliced and unspliced viral RNA for translation and encapsidation. The retroviral RNA can be processed by cellular machinery and enables the removal of intronic sequences. We aimed to utilize the removal of a synthetic intron for targeted gene expression. To analyze intron removal and gene expression, we have constructed a novel self-inactivating gene-activating (SIGA) vector for potential universal gene therapy. New vectors for gene therapy are necessary for safe and effective gene delivery in humans. The SIGA vector is derived from spleen necrosis virus (SNV), which is an avian reticuloendotheliosis virus. The vector was designed so that expression of a therapeutic gene is blocked in helper cell lines due to an intervening sequence containing various blocks in transcription and translation. However, after one round of retroviral replication, the intervening sequence should be removed by the cellular machinery and the therapeutic gene will be selectively expressed in target cells. Our studies show that the intervening sequence in SIGA vector RNA is partially spliced. However, spliced vector RNA was not transduced to target cells. Previous studies showed that an infectious SNV vector enabled transduction of spliced RNA. However, yet-undefined differences in infectious and replication-deficient retroviral replication may have an effect on the transduction of spliced RNA. The results of this study present key information on spliced RNA and its encapsidation, as well as data for the construction of a new generation of SNV-derived retroviral vectors.

Animals↗

PubMed2004

Stress-induced alternative gene splicing in mind-body medicine.

Recent research documents how psychosocial stress can alter the expression of the acetylcholinesterase gene to generate at least 3 alternative proteins that are implicated in a wide variety of normal mind-body functions, as well as pathologies. These range from early embryological development, plasticity of the brain in adulthood, post-traumatic stress disorder (PTSD), and stress-associated dysfunctions of the central nervous, endocrine, and immune systems, to age-related neuropathologies. Such stress-induced alternative gene splicing is proposed here as a major mind-body pathway of psychosocial genomics-the modulation of gene expression by creative psychological, social, and cultural processes. We explore the types of research that are now needed to investigate how stress-induced alternative splicing of the acetylcholinesterase gene may play a pivotal role in the deep psychobiology of psychotherapy, meditation, spiritual rituals, and the experiencing of positive humanistic values that have been associated with mind-body medicine, such as compassion, beneficence, serenity, forgiveness, and gratitude.

Acetylcholinesterase↗

PubMed1995

A portable regulatory element directs specific expression of the Caenorhabditis elegans ubiquitin gene ubq-2 in the somatic gonad.

The Caenorhabditis elegans ubq-2 gene encodes a fusion of ubiquitin and a 52-amino-acid ribosomal protein. This single copy gene is both cis- and trans-spliced. It is expressed in all life stages of the worm and its transcript abundance is unaffected by heat stress. Transgenic analysis shows that expression of ubq-2 is regulated by an upstream promoter and a downstream element. The downstream element is required for ubq-2 promoter activity in embryos and in cells of the somatic gonad, including the distal tip cells, sheath cells, spermathecal cells, and cells of the uterus. The gonad-specific activity of the downstream regulator is transferable to a stress gene promoter such that heat-inducible expression of the transgene occurs in the somatic gonad. Stress-inducible beta-galactosidase expression in the gonad does not occur in all life stages, but is initiated late in the second or early in the third larval stage, when differentiation of gonadal tissues begins. Expression of a beta-galactosidase fusion protein from constructs containing the downstream ubq-2 regulator causes abnormalities of the gonad and embryonic lethality. Gonad abnormalities include arrested development and general disorganization. These abnormalities may be related to the overexpression of ubiquitin in the gonad.

Amino Acid Sequence↗

PubMed2004

Introns: mighty elements from the RNA world.

The discovery of RNA-based enzymatic activity by Thomas Cech's and Sidney Altman's laboratories was a momentous event that led Walter Gilbert to the concept of an "RNA world"--a primitive ancient stage of life that existed before the appearance of DNA and protein molecules. A year later, Gilbert formulated "the exon theory of genes," which hypothesized that introns are very ancient genetic elements present at the earliest stages of life in the RNA world. This theory has been fiercely debated and still has vigorous supporters and opponents. In this communication, we explore peculiarities in the RNA-protein world and their effect on intron-exon structures. We demonstrate that these peculiarities, which exist in the absence of DNA, could shed light on introns' original functions as well as the important role they might have played in the origin of life. For ancient DNA-lacking cells, a crucial problem existed in distinguishing two distinct subsets of RNAs: those messenger molecules coding for proteins and those heritable genetic molecules complementary to messenger RNAs that propagate the genetic information through generations. We propose that ancient introns could act as markers of RNA subsets, directing them to different functions.

Animals↗

PubMed2005

Two different mechanisms for tRNA ribose methylation in Archaea: a short survey.

The biogenesis of tRNA involves multiple reactions including post-transcriptional modifications and pre-tRNA splicing. Among the three domains of life, only Archaea have two different mechanisms for tRNA ribose methylation: site-specific 2'-O-methyltransferases and C/D guided-RNA machinery. Recently, the first archaeal tRNA 2'-O-methyltransferase, aTrm56, has been characterized. This enzyme is found in all archaeal genomes sequenced so far except one and belongs to the SPOUT family (class IV) of RNA methyltransferases. Its substrate is the conserved C56 in the T-loop of archaeal tRNAs. In the crenarchaeon Pyrobaculum aerophylum, in which no homologue of this methyltransferase is found, a box C/D guide sRNP insures the ribose methylation of C56. Moreover, a new twist on tRNA processing is the finding, in most euryarchaeal tRNAtrp genes, of a box C/D guide RNA within their intron specifying methylation at two sites. Modification of tRNA is an integral part of the complex maturation process of primary tRNA transcripts. In addition to their role in modification, both modification enzymes and C/D guide RNPs may have a chaperone function insuring the precise folding of the mature, functional tRNA.

Archaea↗

PubMed2004

From genomes to vaccines for leishmaniasis.

A total of 2183 clones derived from four life cycle stage-specific, spliced-leader cDNA libraries of Leishmania major LV39 Neal strain were randomly picked and sequenced to generate expressed sequence tags (ESTs). Then 1094 unique genes were identified, with 18.2% having BLAST hits with known genes/proteins and 81.8% failing to match genes currently deposited in public databases. Approximately 250 unique genes were obtained from a lesion-derived amastigote complementary DNA (cDNA) library, the form of the parasite that is infective to the mammalian host. Polymerase chain reaction (PCR)-amplified ESTs were spotted onto glass slides, and DNA microarray used to identify a further approx 100 unique cDNAs highly expressed in amastigotes. One hundred unique, randomly selected amastigote-expressed genes were PCR amplified to exclude the 5'-spliced leader, and the full-length genes subcloned into the TOPO-TA cloning vector. The genes were sequence-verified, excised using restriction enzyme digestion, and cloned upstream of the eukaryotic cytomegalovirus promoter into the expression vector pcDNA3. Expression plasmids were sequence-verified and large-scale, endotoxin-free plasmids prepared. Then 100 microg of each expression plasmid (DNA vaccine) was delivered subcutaneously to the rump of susceptible BALB/c mice, the mice boosted 4 wk later and then challenged 2 wk post-boost with 2 x 10(6) L. major LV39 parasites to the hind footpad. Infection was monitored on a weekly basis by measuring footpad depth with digital calipers. Protection was scored by comparing the footpad depth of mice receiving empty vector DNA to those immunized with DNA containing L. major amastigote-expressed genes.

Animals↗