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PubMed2005

In vivo transcriptional profile analysis reveals RNA splicing and chromatin remodeling as prominent processes for adult neurogenesis.

Neural stem cells and neurogenesis persist in the adult mammalian brain subventricular zone (SVZ). Cells born in the rodent SVZ migrate to the olfactory bulb (Ob) where they differentiate into interneurons. To determine the gene expression and functional profile of SVZ neurogenesis, we performed three complementary sets of transcriptional analysis experiments using Affymetrix GeneChips: (1) comparison of adult mouse SVZ and Ob gene expression profiles with those of the striatum, cerebral cortex, and hippocampus; (2) profiling of SVZ stem cells and ependyma isolated by fluorescent-activated cell sorting (FACS); and (3) analysis of gene expression changes during in vivo SVZ regeneration after anti-mitotic treatment. Gene Ontology (GO) analysis of data from these three separate approaches showed that in adult SVZ neurogenesis, RNA splicing and chromatin remodeling are biological processes as statistically significant as cell proliferation, transcription, and neurogenesis. In non-neurogenic brain regions, RNA splicing and chromatin remodeling were not prominent processes. Fourteen mRNA splicing factors including Sf3b1, Sfrs2, Lsm4, and Khdrbs1/Sam68 were detected along with 9 chromatin remodeling genes including Mll, Bmi1, Smarcad1, Baf53a, and Hat1. We validated the transcriptional profile data with Northern blot analysis and in situ hybridization. The data greatly expand the catalogue of cell cycle components, transcription factors, and migration genes for adult SVZ neurogenesis and reveal RNA splicing and chromatin remodeling as prominent biological processes for these germinal cells.

Animals↗

PubMed2003

Reprogramming alternative pre-messenger RNA splicing through the use of protein-binding antisense oligonucleotides.

Alternative pre-messenger RNA splicing is a major contributor to proteomic diversity in higher eukaryotes and represents a key step in the control of protein function in a large variety of biological systems. As a means of artificially altering splice site choice, we have investigated the impact of positioning proteins in the vicinity of 5' splice sites. We find that a recombinant GST-MS2 protein interferes with 5' splice site use, most efficiently when it binds upstream of that site. To broaden the use of proteins as steric inhibitors of splicing, we have tested the activity of antisense oligonucleotides carrying binding sites for the heterogeneous nuclear ribonucleoprotein A1/A2 proteins. In a HeLa cell extract, tailed oligonucleotides complementary to exonic sequences elicit strong shifts in 5' splice site selection. In four different human cell lines, an interfering oligonucleotide carrying A1/A2 binding sites also shifted the alternative splicing of the Bcl-x pre-mRNA more efficiently than oligonucleotides acting through duplex formation only. The use of protein-binding oligonucleotides that interfere with U1 small nuclear ribonucleoprotein binding therefore represents a novel and powerful approach to control splice site selection in cells.

Alternative Splicing↗

PubMed1998

Crystal structure and evolution of a transfer RNA splicing enzyme.

The splicing of transfer RNA precursors is similar in Eucarya and Archaea. In both kingdoms an endonuclease recognizes the splice sites and releases the intron, but the mechanism of splice site recognition is different in each kingdom. The crystal structure of the endonuclease from the archaeon Methanococcus jannaschii was determined to a resolution of 2.3 angstroms. The structure indicates that the cleavage reaction is similar to that of ribonuclease A and the arrangement of the active sites is conserved between the archaeal and eucaryal enzymes. These results suggest an evolutionary pathway for splice site recognition.

Amino Acid Sequence↗

PubMed1995

Binding of a cell-type-specific RNA splicing factor to its target regulatory sequence.

The transcript of the Saccharomyces cerevisiae MER2 gene is spliced efficiently during meiosis but not during vegetative growth. Efficient splicing of the wild-type MER2 transcript requires the Mer1 protein, which is produced only in meiotic cells. Analysis of deletion and substitution mutations in the MER2 5' exon demonstrates that the unusually large size of this exon plays an important role in splicing regulation. The cis-acting sequences essential for Mer1-dependent splicing of MER2 RNA were determined by the analysis of MER2 deletion mutants and hybrid genes. The 80-base MER2 intron is sufficient for Mer1-dependent splicing in vivo, but sequences in the 5' exon enhance splicing efficiency. The Mer1 protein contains the KH motif found in some RNA-binding proteins, and RNA gel mobility shift assays demonstrate that Mer1 binds specifically to MER2 RNA. Both the transcript derived from the intronless MER2 gene and the transcript consisting only of the intron are able to bind to Mer1 in vitro, but neither has as high affinity for the protein as the intact substrate. RNase T1 footprinting indicates that the Mer1 protein contacts MER2 RNA at several points in the 5' exon and in the intron. Thus, Mer1 interacts directly with a regulatory element in MER2 RNA and promotes splicing.

Base Sequence↗

PubMed2006

Proinsulin is encoded by an RNA splice variant in human blood myeloid cells.

Genes for peripheral tissue-restricted self-antigens are expressed in thymic and hematopoietic cells. In thymic medullary epithelial cells, self-antigen expression imposes selection on developing autoreactive T cells and regulates susceptibility to autoimmune disease in mouse models. Less is known about the role of self-antigen expression by hematopoietic cells. Here we demonstrate that one of the endocrine self-antigens expressed by human blood myeloid cells, proinsulin, is encoded by an RNA splice variant. The surface expression of immunoreactive proinsulin was significantly decreased after transfection of monocytes with small interfering RNA to proinsulin. Furthermore, analogous to proinsulin transcripts in the thymus, the abundance of the proinsulin RNA splice variant in blood cells corresponded with the length of the variable number of tandem repeats 5' of the proinsulin gene, known to be associated with type 1 diabetes susceptibility. Self-antigen expression by peripheral myeloid cells extends the umbrella of "immunological self" and, by analogy with the thymus, may be implicated in peripheral immune tolerance.

Autoantigens↗

PubMed1984

Spliced RNA from the IR1-U2 region of Epstein-Barr virus: presence of an open reading frame for a repetitive polypeptide.

We have constructed a cDNA library from the cytoplasmic RNAs of Raji cells, a Burkitt's lymphoma cell line latently infected with Epstein-Barr virus. We report here the characterization of a cDNA representing a spliced RNA transcribed from the IR1-U2 region of the viral genome. The cDNA is 1007 bp long. The 5' region contains three tandem repeats of two exons, 66 and 132 bp, which are transcribed from the IR1 repeats. The 3' region is formed from four exons transcribed from U2. An open reading frame extends from the 5' end to position 784, and includes the repeats. This reading frame presumably corresponds to the carboxy-terminal 261 amino acids of a polypeptide containing several repeats of a 66 amino acid sequence. Since it would be encoded by the IR1-U2 region of the viral genome, the putative polypeptide might be involved in the process of growth-transformation of B-lymphocytes.

Amino Acid Sequence↗

PubMed2006

Analysis of RNA splicing defects in PITX2 mutants supports a gene dosage model of Axenfeld-Rieger syndrome.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is associated with mutations in the PITX2 gene that encodes a homeobox transcription factor. Several intronic PITX2 mutations have been reported in Axenfeld-Rieger patients but their effects on gene expression have not been tested. METHODS: We present two new families with recurrent PITX2 intronic mutations and use PITX2c minigenes and transfected cells to address the hypothesis that intronic mutations effect RNA splicing. Three PITX2 mutations have been analyzed: a G>T mutation within the AG 3' splice site (ss) junction associated with exon 4 (IVS4-1G>T), a G>C mutation at position +5 of the 5' (ss) of exon 4 (IVS4+5G>C), and a previously reported A>G substitution at position -11 of 3'ss of exon 5 (IVS5-11A>G). RESULTS: Mutation IVS4+5G>C showed 71% retention of the intron between exons 4 and 5, and poorly expressed protein. Wild-type protein levels were proportionally expressed from correctly spliced mRNA. The G>T mutation within the exon 4 AG 3'ss junction shifted splicing exclusively to a new AG and resulted in a severely truncated, poorly expressed protein. Finally, the A>G substitution at position -11 of the 3'ss of exon 5 shifted splicing exclusively to a newly created upstream AG and resulted in generation of a protein with a truncated homeodomain. CONCLUSION: This is the first direct evidence to support aberrant RNA splicing as the mechanism underlying the disorder in some patients and suggests that the magnitude of the splicing defect may contribute to the variability of ARS phenotypes, in support of a gene dosage model of Axenfeld-Rieger syndrome.

Abnormalities, Multiple↗

PubMed1986

Defective RNA splicing in the absence of adenovirus-associated RNAI.

We have analyzed late gene expression in 293 cells infected with an adenovirus type 5 mutant dl331, which is defective in production of the low molecular weight virus-associated (VA) RNAI. The results show that several steps in late gene expression are affected. In addition to the previously characterized defect in late mRNA translation, mutant infected cells also show an aberrant selection of RNA splice sites and a substantially reduced L2, L3, and L5 mRNA accumulation. Normal or even slightly elevated amounts of mRNA from region L1 are produced. However, the L1 pre-mRNA is spliced only to generate the mRNA encoding the Mr 52,000-55,000 polypeptide and no detectable mRNA for polypeptide IIIa. Cotransfection of a plasmid encoding VA RNAI complemented the splicing defect in trans, suggesting that the abnormalities are due to the absence of VA RNAI, rather than to a cis-acting change in the nuclear precursor RNA. In a HeLa cell variant, which allows late protein synthesis also in the absence of VA RNAI, because of a lack of eukaryotic initiation factor 2 alpha kinase expression, a normal repertoire of late mRNA was produced. We conclude that a soluble factor, most likely a late viral protein, controls differential RNA splicing and late mRNA accumulation during an adenovirus infection.

Adenoviruses, Human↗

PubMed1991

Tissue- and development-specific alternative RNA splicing regulates expression of multiple isoforms of erythroid membrane protein 4.1.

Protein 4.1, a multifunctional structural protein originally described as an 80-kDa component of the erythroid membrane skeleton, exhibits tissue- and development-specific heterogeneity in molecular weight, subcellular localization, and primary amino acid sequence. Earlier reports suggested that some of this impressive heterogeneity is generated by alternative RNA splicing (Conboy, J. G., Chan, J., Mohandas, N., and Kan, Y. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9062-9065; Tang, T. K., Leto, T., Marchesi, V. T., and Benz, E. J. (1990) J. Cell Biol. 110, 617-624). We have now completed a systematic analysis of 4.1 mRNA isoforms expressed in erythroid cells, and have generated an "alternative splicing map" which summarizes diagrammatically a multitude of polypeptide isoforms potentially generated by combinatorial splicing of nine alternative exons. Complex 5' splicing events yield mRNA isoforms that may initiate translation at different sites and thus generate elongated or truncated NH2 termini; elongated approximately 135-kDa and prototypical approximately 80-kDa species were detected in both erythrocytes and T-lymphocytes, but in very different ratios. Among the functional domains of 4.1 responsible for interaction with other membrane skeletal elements, four variants of the 10-kDa spectrin-actin-binding region and four variants of the putative 30-kDa glycophorin-binding region are predicted. Developmentally controlled alternative RNA splicing in the spectrin-actin-binding region may help regulate remodeling of membrane architecture and mechanical properties that occur during erythropoiesis.

Amino Acid Sequence↗

PubMed1986

RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis.

The transcripts of most eukaryotic genes contain intervening sequences and must be spliced to yield functional messenger RNA. We report that a brief severe heat shock blocks the processing of intervening sequences in Drosophila cells and that this block persists for at least 2 hr after cells are returned to normal temperatures. If a mild heat shock, which induces the synthesis of heat shock proteins, is administered prior to the severe heat shock, processing occurs under otherwise restrictive conditions. When heat shock protein synthesis is inhibited, this protection is not observed. We suggest that the disruption of intron processing contributes to heat-induced lethality and developmental abnormalities and that one function of the heat shock proteins is to protect processing from heat-induced disruption.

Animals↗

PubMed1987

Mechanism and requirements of in vitro RNA splicing of the primary transcript from the T4 bacteriophage thymidylate synthase gene.

The splicing of a procaryotic precursor RNA transcribed from the T4 phage thymidylate synthase (td) gene with SP6 RNA polymerase was investigated in vitro. The intron excision-cyclization reaction increased progressively to 60 degrees C. Exon ligation, though barely detectable at the lower temperatures, was greatly enhanced at 60 degrees C. Both reactions required Mg2+. The addition of guanosine to the 5' end of an intron-exon II intermediate via a 3',5'-phosphodiester bond was essential for the ligation of exon I to exon II. The added guanosine and the first intron-encoded uridine are subsequently lost as a dinucleotide from the 5' end during cyclization of the linear form of the excised intron RNA. Exon ligation is intramolecular and occurs more readily in the nascent RNA molecule (cotranscriptionally) than in the finished transcript (posttranscriptionally). These data and the identification of various structural elements (P, Q, R, S, E, E') in the td intron that are found typically in eucaryotic class I introns firmly establish the td intron as the first example of class I intron of procaryotic origin.

DNA Restriction Enzymes↗

PubMed2005

Effects of RNA splicing and post-transcriptional regulation on HIV-1 growth: a quantitative and integrated perspective.

Despite major advances over the last two decades in our understanding of RNA splicing and (post-) transcriptional regulation in human immunodeficiency virus type-1 (HIV-1), debate continues on the mechanisms and effects of Rev protein on HIV-1 growth. Moreover, arguments that HIV-1 has been optimised for growth have been largely based on speculation. Here, we begin systematically to address these issues by developing a detailed kinetic model for HIV-1 intracellular development. The model accounts for transcription, successive steps in RNA splicing, nuclear export of mRNAs, translation and shuttling of Rev and Tat, Tat-mediated transactivation of transcription, thresholds on Rev in its effects on nuclear export of mRNA, and inhibitory effects of Rev on splicing. Using the model, we found that inefficient splicing of HIV-1 mRNA was generally beneficial for HIV-1 growth, but that an excessive reduction in the splicing efficiency could be detrimental, suggesting that there exists a splicing efficiency that optimises HIV-1 growth. Further, we identified two key contributors to splicing efficiency, the intrinsic splicing rate and the extent of Rev-mediated splicing inhibition, and we showed how these should be balanced for HIV-1 to optimise its growth. Finally, we found that HIV-1 growth is relatively insensitive to different levels of the Rev export threshold, and we suggest that this mechanism evolved to delay viral growth, perhaps to enable evasion of host defensive responses. In summary, our model provides a quantitative and qualitative framework for probing how constituent mechanisms contribute to the complex, yet logical, process of HIV-1 growth.

Computer Simulation↗

PubMed1995

Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymatic properties pertinent to splicing.

The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in the splicing of group I introns. Here, bacterially expressed CYT-18 protein, purified by a new procedure involving polyethyleneimine precipitation to remove tightly bound nucleic acids, was used to characterize properties pertinent to RNA splicing. Analytical ultracentrifugation and other methods showed that the CYT-18 protein is an asymmetric homodimer. The measured frictional ratio, f/fo = 1.55, corresponds to an axial ratio of 10 for a prolate ellipsoid or 12 for an oblate ellipsoid. Like bacterial TyrRSs, the CYT-18 protein exhibits half-sites reactivity, each homodimer having one active site for tyrosyl adenylation and RNA splicing. The splicing activity of CYT-18 was unaffected by aminoacylation substrates at concentrations used in aminoacylation reactions, whereas the TyrRS activity was inhibited by physiological concentrations of the splicing cofactor GTP, as well as CTP or UTP, or by low concentrations of a group I intron RNA. Kinetic measurements suggest that the binding of CYT-18 to a group I intron substrate is a two-step process, with an initial biomolecular step that is close to diffusion limited (3.24 +/- 0.03 x 10(7) M-1s-1) followed by a slower conformational change (0.54 +/- 0.07 s-1). After CYT-18 binding, splicing occurs at a rate of 0.0025 s-1, within 6-fold of the rate of self-splicing of the Tetrahymena large rRNA intron in vitro. The Kd for the complex between the CYT-18 protein and a group I intron substrate, calculated from koff/kon, was < 0.3 pM, substantially lower than determined by presumed equilibrium measurements [Guo, Q., & Lambowitz, A. M. (1992) Genes Dev. 6, 1357-1372]. As a result of this tight binding, the CYT-18 protein functions stoichiometrically in in vitro splicing reactions due to its extremely slow dissociation from the excised intron RNA. The very tight binding of the CYT-18 protein to the intron RNA raises the possibility that specific mechanisms exist for dissociating the protein from the excised intron in vivo.

Cloning, Molecular↗

PubMed1996

Alternative RNA splicing of the human endothelin-A receptor generates multiple transcripts.

In order to elucidate the regulatory mechanisms of expression of the human endothelin-A receptor (hET-AR) gene, we characterized hET-AR transcripts using reverse transcriptase (RT)-PCR analysis in a variety of human tissues. RT-PCR of lung mRNA using a set of primers from exons 2 and 5 showed two lower-molecular-mass transcripts in addition to the expected fragment. When RT-PCR with primers from exons 4 and 8 was performed, no transcripts other than the expected one were detected. PCR cloning utilizing a set of primers from exons 2 and 8 which covered the entire coding sequence revealed that the cDNA clones corresponding to the two novel transcripts contained deletions of 199 bp and 327 bp respectively compared with the previously described hET-AR cDNA. Comparison of their sequences with that of the hET-AR gene showed that the deleted sequences correspond exactly to exon 4 and exons 3 and 4 respectively, indicating that these lower-molecular-mass ET-AR transcripts results from alternative RNA splicing (designated ET-AR delta 4 and ET-AR delta 3,4 respectively). Alternative splicing of exon 4 results in a transcript which would be translated into a C-terminal truncated protein containing the first, second and third transmembrane domains, while the splicing out of exons 3 and 4 would produce a protein with five membrane-spanning domains but lacking the third and fourth domains present in the ET-AR protein. An RNase protection assay revealed that ET-AR delta 4 and ET-AR delta 3,4 as well as ET-AR, transcripts were observed in various human tissues, including the lung, aorta, atrium, kidney and placenta, which are known to express ET-AR abundantly. Thus we have isolated the cDNAs of novel transcripts of hET-AR which are generated by alternative RNA splicing, and these results suggest that this alternative RNA splicing might contribute to the regulation of ET-AR gene expression.

Alternative Splicing↗

PubMedDate not supplied

Tissue-specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing.

A novel mammalian neuropeptide, the tachykinin substance K, is specified by a discrete genomic segment. Alternative RNA splicing generates two distinct mRNAs encoding the neuropeptide substance P alone or with substance K from a single preprotachykinin gene. Relative amounts of the mRNAs vary in different tissues, suggesting that the substance K-encoding sequence is regulated in a tissue-specific manner.

Amino Acid Sequence↗

PubMed1987

A yeast nuclear gene, MRS1, involved in mitochondrial RNA splicing: nucleotide sequence and mutational analysis of two overlapping open reading frames on opposite strands.

We have cloned a 1.6-kb fragment of yeast nuclear DNA, which complements pet- mutant MK3 (mrs1). This mutant was shown to be defective in mitochondrial RNA splicing: the excision of intron 3 from the mitochondrial COB pre-RNA is blocked. The DNA sequence of the nuclear DNA fragment revealed two open reading frames (ORF1 with 1092 bp; ORF2 with 735 bp) on opposite strands, which overlap by 656 bp. As shown by in vitro mutagenesis, ORF1, but not ORF2, is responsible for complementation of the splice defect. Hence, ORF1 represents the nuclear MRS1 gene. Disruption of the gene (both ORFs) in the chromosomal DNA of the respiratory competent yeast strain DBY747 (long form COB gene) leads to a stable pet- phenotype and to the accumulation of the same mitochondrial RNA precursors as in strain MK3. The amino acid sequence of the putative ORF1 product does not exhibit any homology with other known proteins, except for a small region of homology with the gene product of another nuclear yeast gene involved in mitochondrial RNA splicing, CBP2. The function of the MRS1 (ORF1) gene in mitochondrial RNA splicing and the significance of the overlapping ORFs in this gene are discussed.

Base Sequence↗

PubMed1993

Pre-messenger RNA splicing of transcripts synthesized from human small nuclear RNA gene promoters.

In order to explore the coupling of transcription with splicing in mammalian cells we have prepared hybrid genes in which either the human U2 promoter, recognized by RNA polymerase II, or the human U6 promoter, recognized by RNA polymerase III, was fused to an intron-containing gene segment. Neither human small nuclear RNA gene contains an intron although U6 genes from some species of yeast contain a short intervening sequence. Following transfection of human cells and analysis of specific RNAs by primer extension we found that the chimeric U2 promoter-derived transcript was efficiently spliced but the RNA polymerase III transcript driven by the U6 promoter remained unspliced. Hence, the splicing apparatus differentiates between transcripts produced from two closely related promoters that are distinguished by RNA polymerase selectivity.

Base Sequence↗

PubMed2001

Oncogenic TLS/ERG and EWS/Fli-1 fusion proteins inhibit RNA splicing mediated by YB-1 protein.

The translocation liposarcoma protein TLS has recently been shown to function as an adapter molecule coupling gene transcription to RNA splicing. Here we demonstrate that YB-1, a protein known to play important roles in transcription and translation, interacts with the COOH-terminal domains of TLS and the structurally related Ewing's sarcoma protein EWS. Through this interaction, YB-1 is recruited to RNA polymerase II and promotes splicing of E1A pre-mRNA to the 13S isoform. This splicing function of YB-1 is inhibited by exogenous TLS/ERG or EWS/Fli-1 fusion proteins, which bind to RNA polymerase II but fail to recruit the YB-1 protein. In Ewing's sarcoma cells that express endogenous EWS/Fli-1, this linkage between YB-1 and RNA Pol II via EWS (or TLS) was found to be defective. Together, these results suggest that TLS and EWS fusion proteins may contribute to malignant transformation through disruption of RNA splicing mediated by TLS- and EWS-binding proteins such as YB-1.

3T3 Cells↗