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Results for “RNA Splicing”

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PubMed1996

Differential use of protein 4.1 translation initiation sites during erythropoiesis: implications for a mutation-induced stage-specific deficiency of protein 4.1 during erythroid development.

Expression of multiple protein 4.1 isoforms in erythroid progenitors and in a variety of nonerythroid tissues results from alternative pre-mRNA splicing. In 4.1 pre-mRNA, several translation initiation sites are present; synthesis of isoforms larger than 80 kD occurs when an upstream 5' AUG is spliced in, whereas the 80-kD mature erythroid isoform is produced when the upstream AUG is spliced out and translation is initiated at the downstream AUG. During erythropoiesis, this splicing switch is developmentally regulated. We studied this developmental switch in hereditary elliptocytosis 4.1Alg, in which a DNA rearrangement involving the exon containing the downstream AUG results in loss of coding capacity for the 80-kD 4.1, leading to mature red blood cells deficient in 4.1 with decreased membrane mechanical stability. Analysis of erythroblast RNA by reverse transcriptase-polymerase chain reaction showed that, although it retained the upstream AUG, its coding region was approximately 2.2 kb, compared with approximately 2.5 kb of normal 4.1 mRNA, because of the deletion of exons, including the one that codes for the downstream AUG. Immunofluorescent microscopy and Western blot analysis documented protein 4.1 expression in HE 4.1Alg erythroblasts. These studies emphasize the crucial role of differentiation-regulated RNA splicing because, within the same erythroid tissue, the HE 4.1Alg phenotype did not appear until after the differentiation-associated splicing event.

Base Sequence↗

PubMed1984

Fast skeletal muscle myosin light chains 1 and 3 are produced from a single gene by a combined process of differential RNA transcription and splicing.

Fast myosin light chains (MLCf) 1 and 3 are proteins associated with the thick filament in vertebrate fast muscle fibers. MLC1f and MLC3f have complete sequence homology for the first 141 amino acids from their COO- end, but they differ in length and amino acid sequence at their NH+3 ends (MLC1f = 49 amino acids, MLC3f = 8 amino acids), and they are translated from different mRNAs. To elucidate the structural relationship between mammalian fast myosin light chain 1 and 3 isoforms, cDNA clones have been isolated from rat skeletal muscle and their primary nucleotide sequences were determined. MLC1f and MLC3f mRNAs display complete sequence identity in the 3' untranslated sequences (285 base pairs) and in the codons specifying the 142 carboxyl-terminal amino acids. In contrast, the sequences encoding the amino-terminal 49 and 8 amino acids of MLC1f and MLC3f, respectively, as well as their 5' untranslated regions, are highly divergent. Using the cloned cDNAs as probes we have isolated the single gene locus encoding both MLC1f and MLC3f in four overlapping genomic clones spanning over approximately 25 kilobase pairs (kb) of DNA. The sequences encoding the common body of MLC1f and MLC3f are distributed in 4 separate exons at the 3' end of the gene. The start of transcription site and 5' untranslated region of MLC3f is found 5 kb upstream from the common body while the MLC1f mRNA transcription start site and the exon specifying the 5' untranslated region and amino acid 1-40 of MLC1f lies about 10 kb further upstream. Strikingly, a mini-exon that specifies amino acids 41-49 of MLC1f is located downstream from the two MLC3f-specific exons (5' untranslated and amino acids 1-8) and is separated from the upstream MLC1f exons by 12 kb of DNA. This bizarre gene organization implies a novel form of alternative promoter utilization and RNA splicing that is tissue-specific and developmentally regulated in order to generate the mature MLC1f and MLC3f mRNAs from a single gene.

Amino Acid Sequence↗

PubMed1983

Saccharomyces cerevisiae actin--Escherichia coli lacZ gene fusions: synthetic-oligonucleotide-mediated deletion of the 309 base pair intervening sequence in the actin gene.

Plasmids carrying gene fusions between the yeast (Saccharomyces cerevisiae) actin gene and an initiation-defective Escherichia coli lacZ (beta-galactosidase) gene have been constructed. Expression of beta-galactosidase in such fusion plasmids depends on transcription of the actin gene, and is possible only after the RNA-splicing machinery has removed from the primary RNA transcript the 309-bp intervening sequence (IVS) interrupting the actin coding region. Mutants deleting the actin IVS were constructed via synthetic oligonucleotide-mediated in vitro mutagenesis of the actin-beta-galactosidase fusion plasmid. A 17-base synthetic oligonucleotide was used to generate a 309-bp deletion which precisely removed the actin IVS. A partial deletion mutant was also constructed in which 272-bp, starting at the 5' end of the actin IVS, and including the 5' splice junction signal, were deleted. Both the complete and partial IVS-deletion mutants were transformed into yeast hosts. However, the partial deletion resulted in a greater than 98% reduction in beta-galactosidase activity. The precise deletion of the actin IVS did not reduce the levels of beta-galactosidase activity as compared with the parental fusion plasmid containing the intact IVS.

Actins↗

PubMed2006

Potent induction of Arabidopsis thaliana flowering by elevated growth temperature.

The transition to flowering is an important event in the plant life cycle and is modulated by several environmental factors including photoperiod, light quality, vernalization, and growth temperature, as well as biotic and abiotic stresses. In contrast to light and vernalization, little is known about the pathways that mediate the responses to other environmental variables. A mild increase in growth temperature, from 23 degrees C to 27 degrees C, is equally efficient in inducing flowering of Arabidopsis plants grown in 8-h short days as is transfer to 16-h long days. There is extensive natural variation in this response, and we identify strains with contrasting thermal reaction norms. Exploiting this natural variation, we show that FLOWERING LOCUS C potently suppresses thermal induction, and that the closely related floral repressor FLOWERING LOCUS M is a major-effect quantitative trait locus modulating thermosensitivity. Thermal induction does not require the photoperiod effector CONSTANS, acts upstream of the floral integrator FLOWERING LOCUS T, and depends on the hormone gibberellin. Analysis of mutants defective in salicylic acid biosynthesis suggests that thermal induction is independent of previously identified stress-signaling pathways. Microarray analyses confirm that the genomic responses to floral induction by photoperiod and temperature differ. Furthermore, we report that gene products that participate in RNA splicing are specifically affected by thermal induction. Above a critical threshold, even small changes in temperature can act as cues for the induction of flowering. This response has a genetic basis that is distinct from the known genetic pathways of floral transition, and appears to correlate with changes in RNA processing.

Arabidopsis↗

PubMed2005

Cooperativity of paired oligonucleotide probes for microarray hybridization assays.

Synthetic DNA probes attached to microarrays usually range in length from 25 to 70 nucleotides. There is a compromise between short probes with lower sensitivity, which can be accurately synthesized in higher yields, and long probes with greater sensitivity but lower synthesis yields. Described here are microarrays printed with spots containing a mixture of two short probes, each designed to hybridize at noncontiguous sites in the same targeted sequence. We have shown that, for a printed microarray, mixed probe spots containing a pair of 30mers show significantly greater hybridization than spots containing a single 30mer and can approach the amount of hybridization to spots containing a 60mer or a 70mer. These spots with mixed oligonucleotide probes display cooperative hybridization signals greater than those that can be achieved by either probe alone. Both the higher synthesis yields of short probes and the greater sensitivity of long oligonucleotides can be utilized. This strategy provides new design options for microarray hybridization assays to detect RNA abundance, RNA splice variants, or sequence polymorphisms.

Bacillus subtilis↗

PubMed1999

RNA secondary structure and compensatory evolution.

The classic concept of epistatic fitness interactions between genes has been extended to study interactions within gene regions, especially between nucleotides that are important in maintaining pre-mRNA/mRNA secondary structures. It is shown that the majority of linkage disequilibria found within the Drosophila Adh gene are likely to be caused by epistatic selection operating on RNA secondary structures. A recently proposed method of RNA secondary structure prediction based on DNA sequence comparisons is reviewed and applied to several types of RNAs, including tRNA, rRNA, and mRNA. The patterns of covariation in these RNAs are analyzed based on Kimura's compensatory evolution model. The results suggest that this model describes the substitution process in the pairing regions (helices) of RNA secondary structures well when the helices are evolutionarily conserved and thermodynamically stable, but fails in some other cases. Epistatic selection maintaining pre-mRNA/mRNA secondary structures is compared to weak selective forces that determine features such as base composition and synonymous codon usage. The relationships among these forces and their relative strengths are addressed. Finally, our mutagenesis experiments using the Drosophila Adh locus are reviewed. These experiments analyze long-range compensatory interactions between the 5' and 3' ends of Adh mRNA, the different constraints on secondary structures in introns and exons, and the possible role of secondary structures in RNA splicing.

Alcohol Dehydrogenase↗

PubMed1991

Isolation and characterization of a cDNA encoding a chicken beta thyroid hormone receptor.

We have isolated and characterized a cDNA encoding a chicken beta homolog of c-erbA, or thyroid hormone receptor (TR). Chicken liver cDNA libraries were screened with a rat TR beta-1 cDNA probe, and several cDNA inserts were isolated and characterized. The sequence of one cDNA predicts a 369-amino-acid open reading frame (ORF), with a protein sequence that possesses 96% identity with that of rat TR beta-1, but only 88% identity with chicken TR alpha. These data indicate that the cDNA likely encodes a beta form of TR that has the expected putative DNA and T3 binding domains. The chicken TR beta (chTR beta) in vitro translated protein binds T3 with high affinity, and binds both the thyroid hormone response element (TRE) from the rat growth hormone gene and the Xenopus vitellogenin A2 gene estrogen response element (ERE), similarly to that of the rat TR beta-1. Northern blot analysis revealed the expression of a 7.0-kb RNA in several tissues including cerebellum, pituitary, kidney, and liver. This chicken liver TR beta cDNA sequence varies in both the 5' and 3' untranslated regions from the chicken kidney TR beta cDNA sequence recently reported (Forrest et al., 1990). The 5' untranslated cDNA sequence divergence occurs near a potential splice site junction of the human TR beta gene, suggesting that this chicken liver cDNA may represent an alternatively spliced RNA product of the chicken TR beta gene.

Amino Acid Sequence↗

PubMed2002

Signal-dependent regulation of splicing via phosphorylation of Sam68.

Evolution of human organismal complexity from a relatively small number of genes--only approximately twice that of worm or fly--is explained mainly by mechanisms generating multiple proteins from a single gene, the most prevalent of which is alternative pre-messenger-RNA splicing. Appropriate spatial and temporal generation of splice variants demands that alternative splicing be subject to extensive regulation, similar to transcriptional control. Activation by extracellular cues of several cellular signalling pathways can indeed regulate alternative splicing. Here we address the link between signal transduction and splice regulation. We show that the nuclear RNA-binding protein Sam68 is a new extracellular signal-regulated kinase (ERK) target. It binds exonic splice-regulatory elements of an alternatively spliced exon that is physiologically regulated by the Ras signalling pathway, namely exon v5 of CD44. Forced expression of Sam68 enhanced ERK-mediated inclusion of the v5-exon sequence in mRNA. This enhancement was impaired by mutation of ERK-phosphorylation sites in Sam68, whereas ERK phosphorylation of Sam68 stimulated splicing of the v5 exon in vitro. Finally, Ras-pathway-induced alternative splicing of the endogenous CD44-v5 exon was abolished by suppression of Sam68 expression. Our data define Sam68 as a prototype regulator of alternative splicing whose function depends on protein modification in response to extracellular cues.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Complex expression pattern of RPGR reveals a role for purine-rich exonic splicing enhancers.

PURPOSE: To examine the mechanism underlying transcript heterogeneity in the gene for the retinitis pigmentosa GTPase regulator (RPGR). METHODS: Transcript heterogeneity was analyzed by reverse transcription-polymerase chain reactions (RT-PCR), rapid amplification of cDNA ends (RACE), and transient expression of minigene constructs. Protein variants were identified by immunoblot analysis and by immunocytochemistry. RESULTS: RPGR transcripts terminated either uniformly at the end of exon 19, producing the constitutive transcript with few variants, or at variable sites downstream from exon 15. The latter transcripts resembled the previously described open reading frame (ORF)14/15 variant, but the ORF14/15 exon was not found in full length. Instead, various portions of a purine-rich region were removed as introns. Numerous splice site combinations were used, giving rise to innumerable variants. Analysis of the purine-rich region found multiple exonic splicing enhancers (ESEs) known to promote splicing through interaction with serine-arginine repeat (SR) proteins. Antibodies targeting different regions of RPGR detected a multitude of RPGR proteins in photoreceptors, concentrated in the connecting cilium. Predominant ORF14/15-encoded RPGR polypeptides migrated at approximately 200 kDa and were photoreceptor specific. CONCLUSIONS: The exceptional heterogeneity in RPGR transcript processing results primarily from a novel form of alternative RNA splicing mediated by multiple exonic splicing enhancers. RPGR is composed of a population of proteins with a constant N-terminal core encompassing the RCC1 homology domain followed by a C-terminal portion of variable lengths and sequences.

Animals↗

PubMed1997

The genome and genes of Neurospora crassa.

Neurospora crassa is an organism with a 7-decade contribution to genetic research. in a genome of 42.9 Mb and just over 1000 map units, to date over 800 different genes have been identified by phenotype and/or map location, and 222 genes have been characterized by sequencing. Methods by which analysis of the genome has been carried out are discussed, including linkage, RFLP, and chromosome walking. Characterized centomeres, telomeres, the nucleolar organizer and the dispersed 5S rRNA genes are discussed. Analysis of the protein-encoding genes is undertaken, using new software for the querying of standard sequence databases. Gene analysis includes consensus sequences for transcription and RNA splicing and new insights into codon usage.

Chromosome Mapping↗

PubMed1998

Matrin CYP, an SR-rich cyclophilin that associates with the nuclear matrix and splicing factors.

We report the identification and cloning of a nuclear matrix protein termed matrin cyclophilin or matrin CYP. The derived sequence of matrin cyp encodes a protein of 752 amino acids with a predicted mass of 88 kDa. A 172-residue stretch at the amino terminus shows high identity with the ubiquitous family of cyclophilins. Clustered throughout the carboxyl half of the protein are a series of serine-arginine (SR) repeats that are a characteristic feature of many RNA splicing factors. Antibodies raised against matrin CYP recognize a 106-kDa antigen that is detected in isolated nuclei and quantitatively subfractionates in the nuclear matrix. Laser scanning confocal microscopy localizes most of the anti-matrin CYP-specific antigen within the nucleus in a pattern of large bright speckles that co-localize with splicing factors and diffuse nucleoplasmic staining. A strikingly similar pattern of staining is observed in cells extracted for in situ nuclear matrices. A fusion protein containing the cyclophilin domain of matrin CYP exhibits cyclosporin A (CsA)-sensitive, peptidylprolyl cis-trans-isomerase activity that is characteristic of native cyclophilins. Although total rat liver nuclei contains predominantly CsA-resistant PPIase activity, the corresponding activity in the nuclear matrix is largely CsA-sensitive.

Amino Acid Sequence↗

PubMed1988

Structure of the rat insulin-like growth factor II transcriptional unit: heterogeneous transcripts are generated from two promoters by use of multiple polyadenylation sites and differential ribonucleic acid splicing.

The rat insulin-like growth factor II (rIGF-II) gene, which exists as a single copy in the genome, is expressed as a multitranscript family of mRNA molecules ranging in size from 4.6 to 1 kilobases. Part of this heterogeneity can be ascribed to the presence of two different promoters, each transcribing alternative 5'-noncoding regions which are spliced to common coding exons. In the present study we use a combination of DNA sequence analysis of the gene, mapping of the mRNA molecules by Northern analysis and ribonuclease protection experiments, and DNA sequence analysis of cDNA clones complementary to different regions of the genome to establish the structure of several rIGF-II mRNA species. These results indicate that RNA heterogeneity also arises from the use of different polyadenylation sites. In addition, a variant 2 kilobases RNA was observed that was colinear with the distal 1700 base pairs of the 3147 base pair long exon 3, and may arise by alternative RNA splicing. These posttranscriptional modifications of RNAs arising from the rIGF-II transcription unit may generate molecules with different functional potential.

Animals↗

PubMed1994

Mice lacking Snrpn expression show normal regulation of neuronal alternative splicing events.

The SmN protein is closely related to the constitutively expressed RNA splicing protein SmB but is expressed only in brain and heart tissue. Mice which lack expression of SmN die shortly after birth suggesting a critical role for this protein possibly in the regulation of neuronal-specific alternative splicing events. We show here however that the neuronal-specific alternative splicing of the RNAs encoding several different classes of protein proceeds normally in mice lacking SmN expression. The potential role of SmN and the reasons for the lethal effect observed in non-expressing mice are discussed.

Alternative Splicing↗

PubMed1991

Amino acid sequence of mouse tenascin and differential expression of two tenascin isoforms during embryogenesis.

We have isolated cDNA clones for mouse tenascin and analyzed expression of tenascin mRNAs during embryonic development of the kidney and gut. The deduced amino acid sequence of the mouse tenascin cDNAs shows a modular structure of repeats similar to chicken and human tenascin. In mouse there are 14.5 cysteine-rich repeats with similarity to the EGF repeat, followed by several repeats with similarity to the type III repeat of fibronectin. A longer variant contains 13 fibronectin type III repeats, whereas a shorter splice variant of mouse tenascin lacks the 5 type III repeats that occur directly after the fifth repeat in the longer variant. Contrary to the chicken and human sequences, mouse tenascin does not contain an RGD sequence in the third type III repeat implicated in cell attachment, or in any other positions. In Northern hybridizations to RNA from primary embryonic fibroblasts, the cDNA clone M 20/1 detects two mRNAs with sizes close to 6 and 8 kb. This, and the other data presented here suggest that the two major mouse tenascin polypeptides arise through an alternative RNA splicing. The two major mRNAs are differentially expressed during development. The 8-kb mRNA is more prominent than the 6-kb mRNA throughout prenatal kidney development, but during postnatal development the ratio of the two mRNAs changes. A different expression pattern is seen in the developing gut where the 6-kb mRNA predominates during embryogenesis with the 8-kb mRNA appearing later. The mRNA data of the developing gut correspond with previous protein data, which showed that the shorter Mr 210,000 polypeptide predominates during earlier developmental stages and the larger Mr 260,000 polypeptide appears later in the embryonic gut (Aufderheide, E., and P. Ekblom. 1988. J. Cell Biol. 107:2341-2349).

Amino Acid Sequence↗

PubMed1992

Protein splicing removes intervening sequences in an archaea DNA polymerase.

The Vent DNA polymerase gene from Thermococcus litoralis contains two in-frame insertions that must be spliced out to form the mature polymerase. Primer extension and cDNA PCR revealed no evidence of spliced RNA to account for this editing. In contrast, pulse-chase analysis indicated that expression constructs lacking the first insertion produced a protein precursor in Escherichia coli that was processed post-translationally to form polymerase and I-TliI, the endonuclease protein that is the product of the second insertion. At least one intermediate, which migrated more slowly than the precursor and may be branched, was also detected. Amino acid substitutions at the splice junction slowed or blocked the protein splicing reaction. Processing occurs in several heterologous systems, indicating either self-splicing or ubiquitous splicing factors. Processing occurs in a mutant lacking I-TliI endonuclease activity, establishing the independence of splicing and endonuclease activities.

Amino Acid Sequence↗

PubMed1986

Introns as relict retrotransposons: implications for the evolutionary origin of eukaryotic mRNA splicing mechanisms.

A model is presented for the evolutionary origin of intron sequences within eukaryotic protein-coding genes. We propose that introns are the vestiges of transposable elements and, specifically, that they represent a novel class of retrovirus-like transposons. The attraction of the retrotransposon model is that it gives the RNA splicing mechanism a central role in the evolution of introns. There is a growing body of evidence to suggest that several aspects of splicing are intron-encoded. Consequently, it is reasonable to look for evolutionary explanations of the splicing mechanism in the context of the evolution of the intron sequences themselves. According to this model the ancestral intron genomes were replicated into RNA copies simply because of their insertion within transcriptionally active regions of the host genome. Splicing was necessary not only to minimize their negative effects on host gene expression, but also, and perhaps more importantly, to generate new copies of the intron genome free of flanking exon sequences. These spliced intron copies were then available for reverse transcription and reinsertion elsewhere in the genome. Thus, splicing can be seen as an essential step in the intron replication cycle. Most modern introns have probably lost the majority of their original genetic content and may be considered as degenerate evolutionary relicts. An exception to this degeneracy is the set of splicing signals which must be retained because of its continued importance to host cell survival.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Evolution↗

PubMed2005

Comparison of the expression patterns of five neural RNA binding proteins in the Xenopus retina.

An increasing body of evidence indicates that gene expression can be modulated by posttranscriptional mechanisms. RNA binding proteins, for instance, control gene expression at many regulatory levels including RNA splicing, transport, stability, and translation. Although numerous RNA binding proteins have been identified, very few have been studied extensively in the context of developmental processes. We focused our study on five neural RNA binding proteins: one Musashi homolog, Nrp-1, one member of the Bruno gene family, BruL-1 (also known as Etr-1), and three members of the ELAV/Hu family, ElrB, ElrC, and ElrD. As an initial step in addressing their function during Xenopus neurogenesis, we used in situ hybridization to determine their expression patterns during retinal development. We found that RNA binding proteins belonging to different families have distinct spatio-temporal expression. These combinatorial expression patterns are reminiscent of previously described cell type-specific expression patterns of transcription factors during retinal development. The distribution of RNA binding proteins within the retina suggests that these regulators of posttranscriptional events may play important roles in multiple steps of retinogenesis.

Animals↗

PubMed2005

Genetic immunization with codon-optimized equine infectious anemia virus (EIAV) surface unit (SU) envelope protein gene sequences stimulates immune responses in ponies.

In the context of DNA vaccines the native equine infectious anemia virus (EIAV)-envelope gene has proven to be an extremely weak immunogen in horses probably because the RNA transcripts are poorly expressed owing to an unusual codon-usage bias, the possession of multiple RNA splice sites and potential adenosine-rich RNA instability elements. To overcome these problems a synthetic version of sequences encoding the EIAV surface unit (SU) envelope glycoprotein was produced (SYNSU) in which the codon-usage bias was modified to conform to that of highly expressed horse and human genes. In transfected COS-1 cell cultures, the steady state expression levels of SYNSU were at least 30-fold greater than equivalent native SU sequences. More importantly, EIAV-specific humoral and lymphocyte proliferation responses were induced in ponies immunized with a mammalian expression vector encoding SYNSU. However, these immunological responses were unable to confer protection against infection with a virulent EIAV strain.

Amino Acid Sequence↗