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At least 19 recordsLinked to original sources
PubMed2006

Design of expression vectors for RNA interference based on miRNAs and RNA splicing.

RNA interference (RNAi) mediates sequence-specific post-transcriptional gene silencing in many eukaryotes and is used for reverse genetic studies and therapeutics. RNAi is triggered by double-stranded small interfering RNAs (siRNAs), which can be processed from small hairpin RNAs generated from an expression vector. In some recently described vectors, the siRNAs are expressed from synthetic stem-loop precursors of microRNAs (miRNAs) driven by polymerase II promoters. We have designed new RNAi vectors, designated pSM155 and pSM30, that take into consideration miRNA processing and RNA splicing by placing the miRNA-based artificial miRNA expression cassettes inside of synthetic introns. Like the original miRNA vectors, we show that the pSM155 and pSM30 constructs efficiently down-regulate expression of firefly luciferase and an endogenous gene, phospholipase D2. Moreover, the expression of a coexpressed fluorescent marker is substantially improved by this new design. Another improvement of these new vectors is incorporation of two inverted BsmBI sites placed internal to the arms of the new miRNA-based vectors, so oligos used for cloning are shorter and the cost is reduced. These RNAi vectors thus provide new tools for gene suppression.

Base Sequence↗
PubMedDate not supplied

Specific interaction between the self-splicing RNA of Tetrahymena and its guanosine substrate: implications for biological catalysis by RNA.

Splicing of the ribosomal RNA precursor of Tetrahymena has previously been shown to require no protein in vitro; the cleavage-ligation activity is intrinsic to the RNA molecule. Analysis of the reaction kinetics with guanosine, which is a substrate in the reaction, and with several guanosine analogues suggests that guanosine binds to a specific site on the pre-rRNA. It appears that the RNA, like an enzyme, binds its substrate to promote the rate and specificity of a biological reaction.

Animals↗
PubMed2001

RNA ligands generated against complex nuclear targets indicate a role for U1 snRNP in co-ordinating transcription and RNA splicing.

RNA ligands were generated against various gene products present in HeLa nuclear extract. Functional profiling was performed to identify RNA ligands that modulate RNA polymerase II (pol II)-mediated transcription. Unexpectedly, four of the eight inhibitor ligands identified by this screen contained an 11-nucleotide sequence identical to the 5'-splice site of eukaryotic pre-mRNAs. Such ligands were shown to impede pre-initiation complex assembly on a cytomegalovirus promoter. In addition, U1 small nuclear ribonucleoprotein particles (snRNP) and pol II had been co-immunoprecipitated in the absence of transcription. These results suggest a role for U1 snRNP in co-ordinating transcription and RNA splicing.

Base Sequence↗
PubMed1992

Spliced RNA of woodchuck hepatitis virus.

Polymerase chain reaction was used to investigate RNA splicing in liver of woodchucks infected with woodchuck hepatitis virus (WHV). Two spliced species were detected, and the splice junctions were sequenced. The larger spliced RNA has an intron of 1300 nucleotides, and the smaller spliced sequence shows an additional downstream intron of 1104 nucleotides. We did not detect singly spliced sequences from which the smaller intron alone was removed. Control experiments showed that spliced sequences are present in both RNA and DNA in infected liver, showing that the viral reverse transcriptase can use spliced RNA as template. Spliced sequences were detected also in virion DNA prepared from serum. The upstream intron produces a reading frame that fuses the core to the polymerase polypeptide, while the downstream intron causes an inframe deletion in the polymerase open reading frame. Whereas the splicing patterns in WHV are superficially similar to those reported recently in hepatitis B virus, we detected no obvious homology in the coding capacity of spliced RNAs from these two viruses.

Animals↗
PubMed2002

Multiple RNA splicing and the presence of cryptic RNA splice donor and acceptor sites may contribute to low expression levels and poor immunogenicity of potential DNA vaccines containing the env gene of equine infectious anemia virus (EIAV).

The env gene is an excellent candidate for inclusion in any DNA-based vaccine approach against equine infectious anemia virus (EIAV). Unfortunately, this gene is subjected to mutational pressure in E. coli resulting in the introduction of stop codons at the 5' terminus unless it is molecularly cloned using very-low-copy-number plasmid vectors. To overcome this problem, a mammalian expression vector was constructed based on the low-copy-number pLG338-30 plasmid. This permitted the production of full-length EIAV env gene clones (plcnCMVenv) from which low-level expression of the viral surface unit glycoprotein (gp90) was detected following transfection into COS-1 cells. Although this suggested the nuclear export of complete env mRNA moieties at least two additional polypeptides of 29 and 20kDa (probably Rev) were produced by alternative splicing events as demonstrated by the fact that their synthesis was prevented by mutational inactivation of EIAV env splice donor 3 (SD3) site. The plcnCMVenv did not stimulate immune responses in mice or in horses, whereas an env construct containing an inactivated SD3 site (plcnCMVDeltaSD3) did induce weak humoral responses against gp90 in mice. This poor immunogenicty in vivo was probably not related to the inherent antigenicity of the proteins encoded by these constructs but to some fundamental properties of EIAV env gene expression. Attempts to modify one of these properties by mutational inactivation of known viral RNA splice sites resulted in activation of previously unidentified cryptic SD and slice acceptor sites.

Amino Acid Sequence↗
PubMed1991

DNA template effect on RNA splicing: two copies of the same gene in the same nucleus are processed differently.

Many cellular and viral genes are parts of complex transcription units containing multiple splicing choices. During the course of an adenoviral replicative cycle, different spliced versions of a single gene predominate, depending on the stage of infection. This is true for several adenoviral genes. In this paper we show for the viral E1B transcription unit that splice site usage regulates this process. The change in alternative splicing in this system does not depend on the sequence of the transcribed genes. Non-adenoviral genes, such as the SV40 early region and the polyoma early region, which normally show little or no regulation of spliced RNA product formation, become regulated for mRNA production after insertion into the adenoviral genome. Additional studies show that E1B splicing regulation in adenovirus is a cis effect. Staggered infections using two discernable viral genomes resulted in a situation where both early and late genomes exist in the same nucleus. Neither genome was able to impose its regulated splicing pattern on the other, indicating that the cue for the switch in viral gene splicing is not directly dependent on global changes in trans-acting splicing factors. This suggests a model where the signal for changes in RNA processing for the E1B gene is linked to the state of the DNA template or its localization within nuclear subcompartments.

Adenoviridae↗
PubMed1996

Solution structure of the donor site of a trans-splicing RNA.

BACKGROUND: RNA splicing is both ubiquitous and essential for the maturation of precursor mRNA molecules in eukaryotes. The process of trans-splicing involves the transfer of a short spliced leader (SL) RNA sequence to a consensus acceptor site on a separate pre-mRNA transcript. In Caenorhabditis elegans, a majority of pre-mRNA transcripts receive the 22-nucleotide SL from the SL1 RNA. Very little is known about the various roles that RNA structures play in the complex conformational rearrangements and reactions involved in premRNA splicing. RESULTS: We have determined the solution structure of a domain of the first stem loop of the SL1 RNA of C. elegans, using homonuclear and heteronuclear NMR techniques; this domain contains the splice-donor site and a nine-nucleotide hairpin loop. In solution, the SL1 RNA fragment adopts a stem-loop structure: nucleotides in the stem region form a classical A-type helix while nucleotides in the hairpin loop specify a novel conformation that includes a helix, that extends for the first three residues; a syn guanosine nucleotide at the turn region; and an extrahelical adenine that defines a pocket with nucleotides at the base of the loop. CONCLUSION: The proximity of this pocket to the splice donor site, combined with the observation that the nucleotides in this motif are conserved among all nematode SL RNAs, suggests that this pocket may provide a recognition site for a protein or RNA molecule in the trans-splicing process.

Animals↗
PubMed1987

Self-splicing RNA and an RNA enzyme in Tetrahymena.

The RNA molecules transcribed from many eukaryotic genes are interrupted by intervening sequences, which are removed by a process called RNA splicing. One structurally related group of intervening sequences, the group I intervening sequences, are found in a variety of microorganisms. Some of these, including the group I intervening sequence from the ribosomal RNA precursor of Tetrahymena thermophila, have been shown to mediate their own splicing in an RNA-catalyzed reaction. Following its excision from the ribosomal RNA precursor, the Tetrahymena intervening sequence acts as an enzyme, cutting and rejoining RNA substrates.

Animals↗
PubMed1991

Control of adenovirus alternative RNA splicing: effect of viral DNA replication on RNA splice site choice.

The primary transcripts of most adenovirus transcription units are processed into multiple, alternatively spliced mRNAs. The relative concentrations of such differentially processed mRNAs changes during the infectious cycle. The factors that control this temporal shift in mRNA abundance have not yet been characterized. In the experiments presented here we have examined mRNA synthesis from three viral transcription units: two early regions E1a and E1b, and late region L1. We show that viral DNA replication plays a key role in the control of cytoplasmic mRNA expression from these regions. In the absence of efficient late protein synthesis, viral DNA replication was sufficient to induce a substantial fraction of the E1a, E1b and L1 transcripts to shift from the early to the late pattern of mRNA structure. The shift was not complete under the conditions used, suggesting that viral proteins, although not essential for the process, play an important regulatory role. The requirement for late viral protein synthesis differed between the three transcription units examined. This dependence was most pronounced for correct L1 mRNA production. Viral DNA replication was sufficient to trigger a significant shift in L1 alternative 3' splice site selection. However, in the absence of late translation the L1 pre-mRNA was aberrantly spliced.

Adenovirus Early Proteins↗
PubMed2005

RNA splicing promotes translation and RNA surveillance.

Aberrant mRNAs harboring premature termination codons (PTCs or nonsense codons) are degraded by the nonsense-mediated mRNA decay (NMD) pathway. mRNAs transcribed from genes that naturally acquire PTCs during lymphocyte development are strongly downregulated by PTCs. Here we show that a signal essential for this robust mRNA downregulatory response is efficient RNA splicing. Strong mRNA downregulation can be conferred on a poor NMD substrate by either strengthening its splicing signals or removing its weak introns. Efficient splicing also strongly promotes translation, providing a molecular explanation for enhanced NMD and suggesting that efficient splicing may have evolved to enhance both protein production and RNA surveillance. Our results suggest simple approaches for increasing protein expression from expression vectors and treating human genetic diseases caused by nonsense and frameshift mutations.

Base Sequence↗
PubMed1990

Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns.

Sex-specific alternative RNA splicing of the doublesex (dsx) pre-mRNA results in sex-specific polypeptides that regulate both male and female somatic sexual differentiation in Drosophila melanogaster. We have molecularly characterized a class of dsx mutations that act in cis to disrupt the regulation of dsx RNA processing, causing the dsx pre-mRNA to be spliced in the male-specific pattern regardless of the chromosomal sex of the fly. These dsx mutations are associated with rearrangements in the female-specific exon just 3' to the female-specific splice acceptor. The mutations do not affect the female-specific splice sites or intron that are identical to wild-type sequences. These results indicate that sequences in the female-specific exon are important for the regulation of sex-specific RNA splicing, perhaps by acting as sites of interaction with trans-acting regulators. Furthermore, the data suggest that female-specific regulation of dsx RNA processing occurs by promoting the usage of the female splice acceptor site, rather than by repressing the usage of the alternative male-specific splice acceptor.

Amino Acid Sequence↗
PubMed1981

Nucleotide sequence complementarity between adenovirus 2-coded VA RNA and host cell pre-mRNA. A possible regulatory mechanism of cellular RNA splicing by VA RNA.

Using a computer program, complementary of nucleotide sequences was assessed between adenovirus 2-coded VA RNA and presumptive cellular and viral 'pre-mRNAs'. In this paper, the possibility is considered that the splicing of cellular 'pre-mRNA' can be regulated in such a way that the formation of the proper intramolecular double-stranded hairpin structures, key elements for RNA splicing, is prevented by the binding of VA RNAI or RNAII to the nucleotide sequences around the exon-intron and intron-exon joint sites of cellular 'pre-mRNA' molecules. Complementarity assessment showed that VA RNAI can bind to the joint sites in such a way as to form an omega shape at two separate regions around the joint sites of cellular 'pre-mRNA'. Whereas VA RNAI is not capable of binding to viral hexon 'pre-mRNA' in the same manner as it does to cellular 'pre-mRNA', the binding may occur in a different way. Such differential binding is discussed in relation to the post-transcriptional sequence selection which takes place during the late phase of adenovirus infection.

Base Sequence↗
PubMed1988

Regulation of Rous sarcoma virus RNA splicing and stability.

Only a fraction of retroviral primary transcripts are spliced to subgenomic mRNAs; the unspliced transcripts are transported to the cytoplasm for packaging into virions and for translation of the gag and pol genes. We identified cis-acting sequences within the gag gene of Rous sarcoma virus (RSV) which negatively regulate splicing in vivo. Mutations were generated downstream of the splice donor (base 397) in the intron of a proviral clone of RSV. Deletion of bases 708 to 800 or 874 to 987 resulted in a large increase in the level of spliced RSV RNA relative to unspliced RSV RNA. This negative regulator of splicing (nrs) also inhibited splicing of a heterologous splice donor and acceptor pair when inserted into the intron. The nrs element did not affect the level of spliced RNA by increasing the rate of transport of the unspliced RNA to the cytoplasm but interfered more directly with splicing. To investigate the possible role of gag proteins in splicing, we studied constructs carrying frameshift mutations in the gag gene. While these mutations, which caused premature termination of gag translation, did not affect the level of spliced RSV RNA, they resulted in a large decrease in the accumulation of unspliced RNA in the cytoplasm.

Avian Sarcoma Viruses↗
PubMed2006

Role of viral splicing elements and cellular RNA binding proteins in regulation of HIV-1 alternative RNA splicing.

In HIV-1 infected cells, over 40 different mRNA species are produced by alternative splicing of the single HIV-1 primary RNA transcript. In addition, approximately half of the HIV-1 primary RNA transcripts are not spliced and are exported to the cytoplasm where they serve as mRNA and as genomic RNA. In this article, we will review current knowledge of the mechanisms by which the HIV-1 alternative splicing is regulated. Several negatively and positively-acting cis-acting elements have been detected within the viral genome that repress or facilitate viral RNA splicing by binding to cellular proteins. These include exonic splicing silencers (ESS) and an intronic splicing silencer (ISS) that are selectively bound either by members of the hnRNP A/B family (hnRNPs A1, A1(B), A2, and B1) or by hnRNP H. Exonic splicing enhancers (ESE) are also present within the HIV-1 genome and are selectively bound by members of the SR protein family. ESS and ISS repression mediated by hnRNP A/B proteins occurs at early steps of splicing, prior to formation of pre-spliceosome complexes. Current models propose that ESS elements promote cooperative binding of hnRNP A/B proteins to the exon and prevent efficient binding of essential cellular splicing factors to the 3' splice site. SR proteins bound to ESE elements that are juxtaposed or overlapping ESS elements may counteract this inhibition. We will review data indicating the importance of the HIV-1 splicing elements and their cognate binding proteins for efficient virus replication. Differences in cis-acting splicing elements between the group M (major) and group O (outlier) HIV-1 strains will also be discussed. Finally we will review evidence suggesting the possibility that there may be changes in regulation of HIV-1 alternative splicing in infected human T cells, human macrophages and rodent cells.

Alternative Splicing↗
PubMed1999

The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation.

The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.

Carrier Proteins↗
PubMed1988

Sex-lethal, a Drosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins.

The switch gene, Sex-lethal (Sxl), controls sexual development and dosage compensation. It must be active in females and inactive in males throughout development. Analysis of Sxl cDNAs shows that this on/off regulation may be explained by differential RNA splicing; only female transcripts appear to encode functional products, whereas all male transcripts contain an exon that truncates the open reading frame. The functional female product shows sequence similarities with ribonucleoproteins, suggesting that it is an RNA binding protein. Thus, we propose that Sxl encodes a factor that interacts with both its own pre-mRNA (accounting for positive autoregulation) and that of downstream genes to confer female-specific splicing. In this way, a single, simple mechanism could account for both the maintenance and expression of the sexually determined state.

Amino Acid Sequence↗