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PubMed1981

Expression of IgD may use both DNA rearrangement and RNA splicing mechanisms.

From a library of mouse sperm DNA, we have isolated two overlapping clones which contain the C(delta) gene. One of these clones also contains the C(mu) gene. The C(delta) gene is separated from the C(mu) membrane exons by approximately 2 kilobases (kb) of DAN. The C(delta) gene was identified by (a) hybridization to poly(A)(+)RNA prepared from the IgD-producing rat plasma cell tumor IR731, and (b) homology of a translated nucleotide sequence to the amino acid sequence of the human delta chain. The C(delta) gene spans 8 kb of DNA in the germ line. Plasmid subclones of the C(delta) gene were used as probes in Southern and RNA blot experiments. RNA blot analysis of cytoplasmic poly(A)(+)RNA from IR731 and a mu(+)delta(+) B-cell hybridoma revealed 1.6- and 2.7-kb delta mRNA species with different 3' ends, which presumably encode the secreted and membrane-bound forms, respectively, of the delta chain. Southern blot analysis of DNA from two mu(+)delta(+) lymphomas revealed that the C(delta) gene is in the germ-line configuration in each case. Restriction map analysis of C(mu) and C(delta) genomic clones isolated from a library of normal mu(+)delta(+) B-cell DNA also gave no evidence for DNA rearrangement in the region between the C(mu) and C(delta) genes. Taken together, these data suggest that IgD expression in mu(+)delta(+) B cells does not involve a V(H)-to-C(delta) DNA switch rearrangement. We propose that simultaneous expression of C(delta) and C(delta) with a single V(H) gene is mediated by two alternative routes of RNA processing of a primary nuclear transcript which contains the V(H), C(mu), and C(delta) genes. In contrast, analogous experiments with myeloma IR731 DNA revealed that the C(mu) gene has been deleted from the myeloma DNA and that the C(delta) gene has undergone DNA rearrangement, presumably including a switch recombination of the V(H) gene from the C(mu) to the C(delta) gene. These results indicate that two alternative mechanisms may be used in the expression of IgD molecules-RNA splicing in B cells and DNA rearrangement in plasma cells.

Animals↗
PubMed1979

SV40 recombinants carrying a functional RNA splice junction and polyadenylation site from the chromosomal mouse beta maj globin gene.

We have introduced a fragment of the chromosomal mouse beta major globin gene into SV40 and used the resultant hybrid virus to infect cultured monkey kidney cells. The mouse DNA fragment, which contains an intervening sequence and a poly(A) addition site, has been inserted in both possible orientations relative to the SV40 late region promoter. While the fragment is transcribed regardless of orientation, the RNA splice signal and poly(A) addition site are utilized only when the fragment is inserted in the "sense" orientation. Thus genomic mouse signals for both splicing and polyadenylation are recognized across species boundaries. Furthermore, since only an 18 bp segment was included on the 5' border of the intervening sequence, we can define a maximum length for this splice signal.

Animals↗
PubMed1996

RNA splice site utilization by simian immunodeficiency viruses derived from sooty mangabey monkeys.

Alternative splicing of the full-length, primary transcript into numerous subgenomic mRNAs is one way that lentiviral gene expression is regulated. Because the behaviors of different viral isolates might reflect in part differences in splicing, we Investigated the patterns of splice site utilization by simian immunodeficiency viruses (SIVs)-originally isolated from sooty mangabey monkeys (Cercocebus atys) We used reverse transcription-polymerase chain reaction (RT-PCR), molecular cloning, and DNA sequencing approaches to characterize SIVdeltaB670, a pathogenic and neurovirulent isolate, and SIVsmmH4, a related molecular clone. The majority of randomly selected SIVdeltaB670 and SIVsmmH4 partial cDNAs contained tat, rev, nef, and long terminal repeat (LTR) intron splice donor and acceptor sites positioned as expected based on the proviral sequence of SIVsmmH4. Nearly all (87%) of the partial cDNAs analyzed contained a spliced LTR intron. A greater number of partial cDNAs derived from SIVdeltaB670-infected cells contained putative alternatively spliced introns In comparison to SIVsmmH4, including two previously undocumented splice junctions involving the LTR intron splice donor. These data provide the first comprehensive analysis of splice site utilization by an isolate of SIV in comparison to a related molecular clone and the first characterization of SIVsmm splice site utilization.

Amino Acid Sequence↗
PubMed1996

Conformational switches involved in orchestrating the successive steps of group I RNA splicing.

Group I introns possess a conserved guanosine residue at their 3' end, termed omegaG, that, in the case of the Tetrahymena pre-rRNA, is a major determinant of the second step of splicing. We examined the role of omegaG in self-splicing of the 249-residue group I intron of the Anabaena PCC7120 tRNAleu precursor. Contrary to observations with the Tetrahymena pre-rRNA intron, a mutation that places an adenosine residue at the omega position did not have a severe effect on the second step of splicing; neither 3' splice-site selection nor the rate of the second step was altered. The first step of splicing, however, was now readily reversed. This unexpected effect also resulted from a mutation that altered the nucleoside specificity of the intronic guanosine-binding site. The theme common to these mutations is that reversal of the first step of splicing results when there is not a strong interaction between the guanosine-binding site and the omega residue. This suggests that a major role of omegaG is to compete with the exogenous guanosine molecule added to the intron in the first step of splicing for the single guanosine-binding site of the intron. From these data, we are able to extend the mechanism for the self-splicing reaction of this intron by proposing two distinct conformational changes between the first and second steps of the splicing. The first of these is the exchange of the exogenous nucleoside for the omega nucleoside. This is the equilibrium that we can perturb by mutations at either the omega position or the guanosine-binding site. An additional conformational change then fully activates the intron for the second step of splicing.

Anabaena↗
PubMed1992

Solid-phase synthesis of branched oligoribonucleotides related to messenger RNA splicing intermediates.

The chemical synthesis of oligoribonucleotides containing vicinal (2'-5')- and (3'-5')-phosphodiester linkages is described. The solid-phase method, based on silyl-phosphoramidite chemistry, was applied to the synthesis of a series of branched RNA [(Xp)nA2' (pN)n3'(pN)n] related to the splicing intermediates derived from Saccharomyces cerevisiae rp51a pre-messenger RNA. The branched oligonucleotides have been thoroughly characterized by nucleoside and branched nucleotide composition analysis. Branched oligoribonucleotides will be useful in the study of messenger RNA splicing and in determining the biological role of RNA 'lariats' and 'forks' in vivo.

Base Sequence↗
PubMed2000

Alternative RNA splicing generates a glycosylphosphatidylinositol-anchored form of ceruloplasmin in mammalian brain.

Ceruloplasmin is a copper-containing ferroxidase that is essential for normal iron homeostasis. Whereas ceruloplasmin in plasma is produced and secreted by hepatocytes, in the brain a glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin is expressed on the surface of astrocytes. By using a cDNA cloning approach, we have now determined that the GPI-anchored form of ceruloplasmin is generated by alternative RNA splicing. The splicing occurs downstream of exon 18 and replaces the C-terminal 5 amino acids of the secreted form with an alternative 30 amino acids that signal GPI anchor addition. RNase protection analysis demonstrates that the GPI-anchored form is the major form in the brain, whereas the secreted form predominates in the liver. Individuals with aceruloplasminemia, a hereditary deficiency of ceruloplasmin, have severe iron deposition in a number of organs, including the brain where it results in neurodegeneration. Therefore, this novel GPI-anchored form of ceruloplasmin is likely to play an important role in iron metabolism in the central nervous system.

Alternative Splicing↗
PubMed1984

RNA splicing in neurospora mitochondria: self-splicing of a mitochondrial intron in vitro.

We have used Neurospora nuclear mutant cyt-18-1, which accumulates a number of unspliced mitochondrial precursor RNAs, to identify rapidly mitochondrial introns that are self-splicing in vitro. Incubation of deproteinized whole mitochondrial RNA from the mutant with 32P-GTP resulted in strong labeling of a 1.3 kb RNA, subsequently identified as cytochrome b (cob) intron 1, and weaker labeling of additional RNAs. Self-splicing of cob intron 1, including precise cleavage and ligation, was confirmed using an in vitro transcript synthesized from the SP6 promoter. The in vitro splicing reaction was shown to be analogous to that for the Tetrahymena nuclear rRNA intron. Since splicing of cob intron 1 is inhibited in a recessive nuclear mutant, we infer that this essentially RNA-catalyzed splicing reaction must be facilitated by a protein in vivo.

Base Sequence↗
PubMed1992

The role of the tnv protein and tnv RNA splicing signals in replication of HIV-1 IIIB isolates.

The requirement for tnv, a tat-env-rev fusion protein expressed by the IIIB strain of HIV-1, was tested. The expression of tnv was prevented by altering the 5' splice site that flanks the central coding exon of tnv. Mutants that carry such an altered 5' splice site replicate normally in an established T-cell line and in peripheral blood lymphocytes, demonstrating that tnv has no effect on virus replication. However, two mutants that carry an alteration in the 3' splice site of the same exon are replication defective. The 3' splice site mutations result in significant reduction in the expression of the 16-kDa tat protein and induce the expression of large amounts of a 19-kDa rev-related protein that initiates within the central coding exon of tnv. S1 nuclease analysis reveals that splicing to the central tnv exon occurs with substantially increased efficiency via the use of an alternate 3' splice site six nucleotides 3' from the mutated site. The effect of the 3' splice site mutations on viral protein expression and replication are fully reversed by a second site mutation that eliminates the alternate splice site.

Base Sequence↗
PubMed1998

An RNA splicing enhancer-like sequence is a component of a splicing inhibitor element from Rous sarcoma virus.

The accumulation in infected cells of large amounts of unspliced viral RNA for use as mRNA and genomic RNA is a hallmark of retrovirus replication. The negative regulator of splicing (NRS) is a long cis-acting RNA element in Rous sarcoma virus that contributes to unspliced RNA accumulation through splicing inhibition. One of two critical sequences located in the NRS 3' region resembles a minor class 5' splice site and is required for U11 small nuclear ribonucleoprotein (snRNP) binding to the NRS. The second is a purine-rich region in the 5' half that interacts with the splicing factor SF2/ASF. In this study we investigated the possibility that this purine-rich region provides an RNA splicing enhancer function required for splicing inhibition. In vitro, the NRS acted as a potent, orientation-dependent enhancer of Drosophila doublesex pre-mRNA splicing, and enhancer activity mapped to the purine-rich domain. Analysis of a number of site-directed and deletion mutants indicated that enhancer activity was diffusely located throughout a 60-nucleotide area but only the activity associated with a short region previously shown to bind SF2/ASF correlated with efficient splicing inhibition. The significance of the enhancer activity to splicing inhibition was demonstrated by using chimeras in which two authentic enhancers (ASLV and FP) were substituted for the native NRS purine region. In each case, splicing inhibition in transfected cells was restored to levels approaching that observed for the NRS. The observation that a nonfunctional version of the FP enhancer (FPD) that does not bind SF2/ASF also fails to block splicing when paired with the NRS 3' region supports the notion that SF2/ASF binding to the NRS is relevant, but other SR proteins may substitute if an appropriate binding site is supplied. Our results are consistent with a role for the purine region in facilitated snRNP binding to the NRS via SF2/ASF.

Animals↗
PubMed2002

The alpha(1A) subunits of rat brain calcium channels are developmentally regulated by alternative RNA splicing.

Calcium influx through voltage-gated calcium channels governs important aspects of CNS development. Multiple alternative splicings of the pore-forming alpha(1) subunits have been evidenced in adult brain but little information about their expression during ontogenesis is presently available. The aim of this study was to focus on the expression of three rat voltage-gated calcium channel alpha(1A) splice variants (alpha(1A-a), alpha(1A-b) and alpha(1A-EFe)) during brain ontogenesis in vivo. Using a reverse transcription-polymerase chain reaction strategy, we found that the three isoforms have different timings of development throughout the brain: alpha(1A-b) is expressed from embryonic to the adult stage, alpha(1A--EFe) is restricted to the embryonic period whereas alpha(1A-a) is expressed only postnatally. In situ hybridization indicated that alpha(1A-a) and alpha(1A-b) isoforms develop with different regional and cellular patterns. In hippocampus and cerebellum, alpha(1A-b) represented the predominant isoform at all developmental stages. Taken together, these data reveal that alternative RNA splicing may modulate the alpha(1A) calcium channel properties during development.

Alternative Splicing↗
PubMed2001

Strong RNA splicing enhancers identified by a modified method of cycled selection interact with SR protein.

A modified method of cycled selection was used to characterize splicing enhancers for exon inclusion from a pool of beta-globin-based three exon/two intron pre-mRNAs with a variable number of random nucleotides incorporated in the internal exon. The pre-mRNAs generated by this method contained random sequences ranging from 0 to 18 nucleotides in length. This method was used to isolate particular splicing enhancer motifs from a previously enriched pool of extremely diverse enhancers. After four cycles of selection for mRNA containing the internal exon, a distinct enhancer motif (GACGAC...CAGCAG) was highly enriched. This motif served as strong splicing enhancers in a heterogeneous exon. We have shown here that the selected enhancer motif promotes exon inclusion through specific interaction with SRp30. We have also shown that although present in many of our selected splicing enhancers conforming to this motif, a typical purine-rich enhancer sequence is dispensable for either enhancer activity or binding with SRp30.

Amino Acid Motifs↗
PubMed1986

Expression of the mitochondrial split gene coding for cytochrome oxidase subunit I in S. cerevisiae: RNA splicing pathway.

We have studied the splicing pathway leading to the synthesis of cytochrome oxidase subunit I (COX I) mRNA, by analysing the transcription pattern of several oxi3- splicing deficient mutants located in the first four introns of the gene. The four introns contain long open reading frames (ORFs) in phase with the upstream exons. All the mutations block the excision of the mutated intervening sequence (IVS) from the pre-mRNA, and accumulate characteristic novel polypeptides of sizes which could correspond to the translation products of the intron's ORF. Most of the mutations do not affect the splicing of the following intervening sequences; only in the case of mutations in the aI1 intron is a polar effect observed on the splicing of the second intron, aI2. Our results indicate that the splicing of these two intervening sequences which both belong to the class II of introns described by Michel et al. (1982), is controlled by the activity of the maturases encoded by their respective ORFs and that the translation of the aI2 maturase depends on the previous excision of aI1 IVS. (Moreover, the aI1 maturase, which accumulates in some mutants, can efficiently splice aI2 IVS when the translation of the latter's proper maturase cannot occur).

Electron Transport Complex IV↗
PubMed1983

T-antigen expression by polyoma mutants with modified RNA splicing.

Polyoma virus mutants were constructed that could not express all the three T-antigens. The mutagenesis was directed to the two 5' splice sites utilized in the maturation of early RNA. The mutant bc1051 had a base change at the splice site of large T-antigen mRNA, and the mutants dl1061 and dl1062 had deletions at the corresponding splice point of small and middle T-antigen mRNA. The site was removed in mutant dl1061 and altered by fusion to upstream sequences in mutant dl1062. Analysis of viral RNA showed that dl1061 and dl1062 formed only large T-antigen mRNA, whereas bc1051 did not produce this RNA-species. However, the biological properties of dl1062 suggested that it also produced mRNA directing the synthesis of a small T-antigen-related polypeptide, at least in low amounts. Only mutant bc1051 could induce transformation of rat cells. In mouse 3T3 cells dl1062 multiplied to a limited extent, while bc1051 and dl1061 failed to produce virus. However, dl1061 DNA was synthesized at a low rate which could be increased to normal levels by co-transfection with mutant bc1051. This result suggests that polyoma small and middle T-antigen have a previously unrecognized function in the early phase of the infection process, or in viral DNA-synthesis.

Antigens, Viral, Tumor↗
PubMed1992

RNA splicing in lower eukaryotes.

Recently, cis-acting elements and trans-acting RNA and protein factors necessary for splicing nuclear pre-mRNAs, group II introns or group III introns, have been discovered, and new roles for the splicing factors have been elucidated. Parallels among the pathways for splicing these different classes of introns have been identified.

Animals↗
PubMed2004

Topoisomerase I and II inhibitors control caspase-2 pre-messenger RNA splicing in human cells.

We have recently shown that the topoisomerase II inhibitor, etoposide (VP16), could trigger caspase-2 pre-mRNA splicing in human leukemic cell lines. This leads to increased inclusion of exon 9, which is specifically inserted into the short caspase-2S isoform mRNA and absent from the long caspase-2L isoform mRNA. One of the consequences of this alternative splicing is a decrease in the total amount of the mature form of caspase-2L mRNA and protein. In this study, we analyzed the effects of several representative molecules of various classes of cytotoxic agents on caspase-2 pre-mRNA splicing in both U937 leukemic cells and in HeLa cervix carcinoma cells. Very strikingly, both topoisomerase I (camptothecin and homocamptothecin derivatives) and II (VP16, amsacrine, doxorubicin, mitoxantrone) inhibitors induced exon 9 inclusion. DNA intercalating glycosyl indolocarbazole derivatives as well as DNA alkylating agents, such as cisplatin and melphalan, antimetabolites like 5-fluorouracil, and mitotic spindle poisons like vinblastine had no effect. Therefore, both classes of DNA topoisomerases can control pre-mRNA splicing of the caspase-2 transcript. In addition, the splicing reaction brought about by camptothecin was hampered in human CEM/C2 and in murine P388-45R leukemic deficient in topoisomerase I activity. Conversely, VP16 did not trigger caspase-2 alternative splicing in human HL60/MX2 leukemic cells harboring a mutant topoisomerase II. Minigene transfection analysis revealed that topoisomerase inhibitors did not change the splicing profile when cis-acting elements in intron-9, reported to control exon 9 inclusion independently of drug treatment, were removed. Rather, our experiments suggest that exon 9 inclusion induced by topoisomerase inhibitors reflects the activity exerted by topoisomerase I or II on proteins that control splicing reactions, or their direct involvement in pre-mRNA splicing.

Alternative Splicing↗
PubMed2001

Soluble and transmembrane isoforms of novel interleukin-17 receptor-like protein by RNA splicing and expression in prostate cancer.

Members of the interleukin-17 cytokine family are present in a variety of tissues (1-3), although the founding member, interleukin-17, is expressed exclusively in T cells and B cells (4-8). The cloning and characterization of a novel single-pass transmembrane protein with limited homology to the interleukin-17 receptor is reported. High mRNA levels were detected in prostate, cartilage, kidney, liver, heart, and muscle, whereas transcripts were barely detected in thymus and leukocytes. At least 11 RNA splice variants were found, transcribed from 19 exons on human chromosome 3p25.3-3p24.1. Differential exon usage was found in different tissues by quantitative reverse transcriptase-PCR. Predicted proteins range from 186 to 720 amino acids. Soluble secreted proteins lacking transmembrane and intracellular domains are predicted from several splice isoforms and may function as extracellular antagonists to cytokine signaling by functioning as soluble decoy receptors. Using antibodies directed at the cytoplasmic and extracellular domains of this protein, we investigated its localization and found that it was expressed in a variety of normal human tissues including prostate and in prostate cancer.

Alternative Splicing↗