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PubMed1983

Close relationship between certain nuclear and mitochondrial introns. Implications for the mechanism of RNA splicing.

We present the first indication of a direct relationship between a nuclear and a mitochondrial splicing system. The intron in the precursor of the large, nuclearly coded ribosomal RNA of two species of Tetrahymena possesses all the features of a class of fungal mitochondrial introns. Sequences conserved in mitochondrial introns of different fungal species are also found in the same order in these Tetrahymena nuclear introns, and the intron RNA can be folded to form a secondary structure similar to that proposed for mitochondrial introns by Davies et al. (1982). This "core" secondary structure brings the ends of the intron together. Furthermore, the first intron in the precursor of the large, nuclearly coded rRNA of Physarum polycephalum also has the characteristic conserved sequences and core RNA secondary structure. The limited sequence data available suggest that the intron in the large rRNA of chloroplasts in Chlamydomonas reinhardtii also resembles the mitochondrial introns. Tetrahymena large nuclear rRNA introns also have an internal sequence that can act as an adaptor by pairing with upstream and downstream exon sequences adjacent to the splice junctions to precisely align the splice junctions. These nuclear introns therefore fit the model of the role of intron RNA in the splicing process that was proposed by Davies et al. (1982), suggesting that the mechanisms of splicing may be very similar in these apparently diverse systems. It is therefore probable that the RNA secondary structures for which there is good evidence in the case of mitochondrial introns will be found to form the basis of active site structure and precise alignment in splicing and cyclization of the Tetrahymena intron "ribozyme".

Animals↗
PubMed1987

Regulation of the expression of proto-oncogene c-src by alternative RNA splicing in chicken skeletal muscle.

Expression of the c-src locus in skeletal muscle of the chicken was found to be different from other tissues. A 4 kilobases (kb) cellular src (c-src) mRNA, which most likely codes for pp60c-src, is expressed in various chicken tissues examined, and most of its approximately 2.2 kb non-coding sequences are present at the 3' end of the RNA molecule. Whereas the 4 kb RNA is also expressed in embryonic chicken muscle, it disappears shortly before hatching and is replaced by a smaller-sized class of c-src mRNAs of 2.8 to 3.3 kb. The transition of expression from the 4 kb to the 2.8-3.3 kb RNA in skeletal muscle occurs shortly before hatching of the chicken and persists into adulthood. The two classes of the c-src mRNA share sequences both within the 5' coding and the 3' non-coding regions of pp60c-src, however, the 2.8 to 3.3 kb RNA lacks most of the kinase domain, but it contains upstream c-src sequences missing in the 4 kb RNA. The two classes of c-src mRNA most likely are generated by alternative splicing, and the smaller c-src RNA most likely codes for a product other than tyrosine protein kinase.

Age Factors↗
PubMed1990

A set of U1 snRNA-complementary sequences involved in governing alternative RNA splicing of the kininogen genes.

The rat K and T kininogen genes show different modes of mRNA production. The K gene encodes two distinct mRNAs for high molecular weight (HMW) and low molecular weight (LMW) kininogens. These two mRNAs are generated by differential usage of the 3'-terminal exon (LMW exon) and the exon next to and upstream from the LMW exon (HMW exon) through alternative splicing and polyadenylation. In contrast, the T gene generates one mRNA by using selectively the LMW exon, although the T gene is extremely homologous to the K gene. In this study, we constructed a series of chimeric kininogen genes by not only exchanging equivalent restriction fragments of the two genes but also replacing nucleotides that differ between the two genes. We then examined the sequences and the mechanisms governing the different expression patterns of the two genes by transfecting the chimeric genes into heterologous COS cells. The results indicated that the different expression patterns of the K and T genes are governed by two separate internal sequences of the HMW and LMW exons. The internal HMW sequence contains a set of five repetitive sequences, and these repetitive sequences are highly complementary to the 5' portion of U1 snRNA. Furthermore, the nucleotide differences in the U1 snRNA-complementary sequences between the K and T genes have marked effects on the relative formation of the HMW and LMW mRNAs; this indicates that the repetitive sequences complementary to U1 snRNA play a crucial role in determining the relative expression of the two mRNAs. Based on these findings, we discuss a novel mechanism for alternative RNA processing, in which splicing efficiency is controlled by the interaction of U1 small nuclear ribonucleoproteins and the U1 snRNA-complementary repetitive sequences of the kininogen pre-mRNA.

Animals↗
PubMed1994

Nuclear-mitotic apparatus protein: a structural protein interface between the nucleoskeleton and RNA splicing.

Vertebrate splicing factors are localized to discrete domains within the nuclei of somatic cells. The mechanism whereby such nuclear domains, identified as speckles by immunofluorescence microscopy, are generated is unclear. Recent studies suggest that the spatial order within the nucleus is maintained by nuclear matrix factors. Here we show that a protein in the nuclear matrix and mitotic apparatus [nuclear-mitotic apparatus protein, NuMA; Lydersen, B. & Pettijohn, D. (1980) Cell 22, 489-499] colocalizes with splicing factors in interphase nuclei and is associated with small nuclear ribonucleoproteins in a complex immunoprecipitated from HeLa extract with small nuclear ribonucleoprotein antibodies. Moreover, NuMA associates with splicing complexes that are reconstituted in vitro using wild-type pre-mRNA, but not with nonspecific RNA. Cumulatively, these observations suggest a function of NuMA or NuMA-like proteins in interphase cells in providing a bridge between RNA processing and the nucleoskeleton.

Antigens, Nuclear↗
PubMed2001

Function of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase in RNA splicing. Role of the idiosyncratic N-terminal extension and different modes of interaction with different group I introns.

The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) promotes the splicing of group I introns by helping the intron RNA fold into the catalytically active structure. The regions required for splicing include an idiosyncratic N-terminal extension, the nucleotide-binding fold domain, and the C-terminal RNA-binding domain. Here, we show that the idiosyncratic N-terminal region is in fact comprised of two functionally distinct parts: an upstream region consisting predominantly of a predicted amphipathic alpha-helix (H0), which is absent from bacterial tyrosyl-tRNA synthetases (TyrRSs), and a downstream region, which contains predicted alpha-helices H1 and H2, corresponding to features in the X-ray crystal structure of the Bacillus stearothermophilus TyrRS. Bacterial genetic assays with libraries of CYT-18 mutants having random mutations in the N-terminal region identified functionally important amino acid residues and supported the predicted structures of the H0 and H1 alpha-helices. The function of N and C-terminal domains of CYT-18 was investigated by detailed biochemical analysis of deletion mutants. The results confirmed that the N-terminal extension is required only for splicing activity, but surprisingly, at least in the case of the N. crassa mitochondrial (mt) large ribosomal subunit (LSU) intron, it appears to act primarily by stabilizing the structure of another region that interacts directly with the intron RNA. The H1/H2 region is required for splicing activity and TyrRS activity with the N. crassa mt tRNA(Tyr), but not for TyrRS activity with Escherichia coli tRNA(Tyr), implying a somewhat different mode of recognition of the two tyrosyl-tRNAs. Finally, a CYT-18 mutant lacking the N-terminal H0 region is totally defective in binding or splicing the N. crassa ND1 intron, but retains substantial residual activity with the mt LSU intron, and conversely, a CYT-18 mutant lacking the C-terminal RNA-binding domain is totally defective in binding or splicing the mt LSU intron, but retains substantial residual activity with the ND1 intron. These findings lead to the surprising conclusion that CYT-18 promotes splicing via different sets of interactions with different group I introns. We suggest that these different modes of promoting splicing evolved from an initial interaction based on the recognition of conserved tRNA-like structural features of the group I intron catalytic core.

Adenosine Monophosphate↗
PubMed1993

Higher level organization of individual gene transcription and RNA splicing.

Visualization of fibronectin and neurotensin messenger RNAs within mammalian interphase nuclei was achieved by fluorescence hybridization with genomic, complementary DNA, and intron-specific probes. Unspliced transcripts accumulated in one or two sites per nucleus. Fibronectin RNA frequently accumulated in elongated tracks that overlapped and extended well beyond the site of transcription. Splicing appears to occur directly within this RNA track, as evidenced by an unambiguous spatial separation of intron-containing and spliced transcripts. Excised introns for neurotensin RNA appear free to diffuse. The transcription and processing site of the fibronectin gene localized to the nuclear interior and was associated with larger transcript domains in over 88 percent of the cells. These results support a view of nuclear function closely integrated with structure.

Animals↗
PubMed1997

RNA splicing: out of the loop.

Recent functional analysis of catalytic and exon-binding domains from group II autocatalytic introns has revealed haunting similarities with small nuclear RNA sequences in the spliceosome.

Introns↗
PubMed2004

Differential recruitment of nuclear receptor coactivators may determine alternative RNA splice site choice in target genes.

The biological consequences of steroid hormone-mediated transcriptional activation of target genes might be difficult to predict because alternative splicing of a single neosynthesized precursor RNA can result in production of different protein isoforms with opposite biological activities. Therefore, an important question to address is the manner in which steroid hormones affect the splicing of their target gene transcripts. In this report, we demonstrate that individual steroid hormones had different and opposite effects on alternative splicing decisions, stimulating the production of different spliced variants produced from genes driven by steroid hormone-dependent promoters. Steroid hormone transcriptional effects are mediated by steroid hormone receptor coregulators that also modify alternative splicing decisions. Our data suggest that activated steroid hormone receptors recruit coregulators to the target promoter that participate in both the production and the splicing of the target gene transcripts. Because different coregulators activating transcription can have opposite effects on alternative splicing decisions, we conclude that the precise nature of the transcriptional coregulators recruited by activated steroid receptors, depending on the promoter and cellular contexts, may play a major role in regulating the nature of the spliced variants produced from certain target genes in response to steroid hormones.

Alternative Splicing↗
PubMed2006

Effect of photoreceptor degeneration on RNA splicing and expression of AMPA receptors.

PURPOSE: Glutamate is the most important neurotransmitter for excitatory synapses, and its ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor is widely expressed in the vertebrate retina. AMPA receptors are hetero-oligomers composed of subsets of four distinct subunits termed GluR1-4. This study was conducted to examine the developmental progression of the flip-to-flop alternative splicing switch of AMPA receptors in wild-type and rd mouse retina. METHODS: The flop:flip ratio and expression levels of GluR1-4 from postnatal day 8 (P8) to P40 were calculated using quantitative real-time polymerase chain reaction (PCR) analysis and immunoblot analysis. The time course of photoreceptor degeneration in rd mice was histologically analyzed. RESULTS: In wild-type mouse retina, the flop:flip ratio in GluR1, but not GluR2-4, dramatically increased between P16 and P20. In rd mice, photoreceptor degeneration progressed from P10 to P20. GluR1 flop:flip ratio in rd mice was normal compared with wild-type mice before P16, however, the dramatic increase between P16 and P20 was completely suppressed. The suppression in the later phase of retinal degeneration was specific to GluR1 and was not observed in GluR2-4. Moreover, the expression levels of GluR1, GluR3, and GluR4 were increased in rd mice. CONCLUSIONS: These results suggest that the inherited form of photoreceptor degeneration in rd mice contributes to the regulation of the flip-to-flop exon switch in GluR1 and the expression levels of AMPA receptors.

Aging↗
PubMed1989

Hepatitis B virus transcript produced by RNA splicing.

A new hepatitis B virus (HBV) transcript of about 2.2 kilobases was identified in HBV DNA-transfected human hepatoma cells. The 5' terminus of this viral RNA appears to map at one or more of the precore initiation sites, contains a deletion of 1,223 bases corresponding to the last codon of the core gene to the middle of the surface antigen gene, and terminates at the 3' polyadenylation site used by the other known HBV RNAs. The junction region of the deleted sequences showed the conserved splice donor and acceptor GT-AG sequences. Moreover, when a mutant HBV DNA in which the splice acceptor site was changed from AG to CG was transfected into human hepatoma cells, no 2.2-kilobase RNA was detected, further suggesting that this RNA represents a spliced transcript. The core gene, although an amino acid shorter, still encoded a functional viral core protein in complementation experiments. Sequence analysis of the cDNA of the 2.2-kilobase RNA suggests that this transcript can potentially encode a new protein that comprises the reverse transcriptase domain of HBV. However, genetic analysis using a transient DNA transfection system suggests that the gene product(s) of this transcript is not essential for viral replication. The function of this transcript remains to be studied.

Base Sequence↗
PubMed1995

Aspects of splice site selection in constitutive and alternative pre-mRNA splicing.

RNA splicing is an indispensable step for expression of many eukaryotic genes. Combinations of 5' and 3' splice sites should be correctly selected in both constitutive and alternative splicing. Recent studies have revealed mechanisms of alternative splicing in some systems, in which specific regulators play vital roles in splice site selection. On the other hand, essential splicing factors such as SR proteins modulate splice site usage of general machinery. Specific regulators and splicing factors such as SR proteins have some common structural features. With these related components, a similar machinery of splice site selection is involved in constitutive and alternative splicing.

Alternative Splicing↗
PubMed2006

Utilization of host SR protein kinases and RNA-splicing machinery during viral replication.

Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine-arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. In studies reported here, we use the HIV genome as a model. We show that HIV expression decreases overall SR protein/activity. However, we also show that HIV expression is significantly increased (20-fold) when one of the SR proteins, SRp75 is phosphorylated by SR protein kinase (SRPK)2. Thus, inhibitors of SRPK2 and perhaps of functionally related kinases, such as SRPK1, could be useful antiviral agents. Here, we develop this hypothesis and show that HIV expression down-regulates SR proteins in Flp-In293 cells, resulting in only low-level HIV expression in these cells. However, increasing SRPK2 function up-regulates HIV expression. In addition, we introduce SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. Although an isonicotinamide compound, SPRIN340 (or its derivatives) remain to be optimized for better specificity and lower cytotoxicity, we show here that SRPIN340 suppresses propagation of Sindbis virus in plaque assay and variably suppresses HIV production. Thus, we show that SRPK, a well known kinase in the cellular RNA-processing machinery, is used by at least some viruses for propagation and hence suggest that SRPIN340 or its derivatives may be useful for curbing viral diseases.

Cell Line↗
PubMed1993

Feline immunodeficiency virus gene expression: analysis of the RNA splicing pattern and the monocistronic rev mRNA.

The transcription pattern of the feline immunodeficiency virus (FIV) genome in a feline CD4+ cell line was examined. In addition to the genomic RNA (9.2 kb), at least five FIV-specific transcripts [5.2, 4.4 (doublet), 1.7 and 1.4 kb] were detected by using subgenomic restriction enzyme fragments of an FIV molecular clone or FIV-specific oligonucleotides as probes. Among these transcripts, the 9.2, 5.2 and 4.4 (doublet) kb mRNAs were not expressed in the cytoplasm of cells transfected with a rev- mutant. To determine the location of splice junctions in the FIV genome, we used PCR to amplify and clone cDNAs corresponding to the viral mRNAs from infected cells. The region between pol and env was found to contain at least two splice donor and three splice acceptor sites. Two splice acceptor sites were detected in the 3' region of env. By hybridization analysis and sequencing of cDNA clones, it was revealed that the medium sized mRNAs are derived from a single splice event, with different splice acceptor sites, and that the two smaller transcripts are doubly or triply spliced mRNAs. Our results demonstrate a complex pattern of alternative splicing of FIV mRNAs. Furthermore, we identified monocistronic rev mRNA species that employ a unique splice acceptor site.

Amino Acid Sequence↗
PubMed1983

RNA splicing in Neurospora mitochondria. The large rRNA intron contains a noncoded, 5'-terminal guanosine residue.

The 35 S percursor of the Neurospora mitochondrial large rRNA contains a 2.3-kilobase intron located towards its 3' end. The intron RNA is excised in a single cleavage-ligation reaction and is detectable in mitochondria by Northern hybridization experiments. We now show (i) that the free intron RNA is a full-length linear molecule, and (ii) that it, like the Tetrahymena nuclear rRNA intron, contains an extra, noncoded guanosine residue at its 5' end. The latter finding suggests that the Neurospora mitochondrial large rRNA may be spliced via a phosphoester transfer mechanism similar to that proposed for the "self-splicing" Tetrahymena intron.

Base Sequence↗