Assaying RNA chaperone activity in vivo using a novel RNA folding trap.

In the absence of proteins, RNAs often misfold in vitro due to alternative base pairings which result from the molecule being trapped in inactive conformations. We identify an in vivo folding trap in the T4 phage td gene, caused by nine base pairs between a sequence element in the upstream exon of the td gene and another at the 3' end of the intron. During translation, the ribosome resolves this interaction; consequently the intron folds correctly and splicing occurs. The introduction of a stop codon upstream of this base pairing prevents resolution of the inactive structure so that splicing cannot proceed. We have used this folding trap to probe for RNA binding proteins which, when overexpressed, either resolve the misfolded structure or impede its formation in vivo. We distinguish between proteins which recognize the intron structure and those which bind non-specifically and apparently ignore the intron. The first class, e.g. Neurospora crassa CYT-18, can rescue the exonic trap and intron mutants which cause a structural defect. However, known RNA chaperones such as Escherichia coli StpA and S12 and the HIV protein NCp7, only resolve the exonic trap without suppressing intron mutations. Thus, this structural trap enables detection of RNA chaperone activity in vivo.     

Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo.

The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA-protein interactions in HIV both in vivo and in vitro. Hitherto, inhibition of ribozyme catalysis has only been observed in vitro. We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo. All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro. Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA. In the absence of translation, two interactions between sequences in the upstream exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompetent conformations. Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B. Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA. Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation. This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E. coli 23 S rRNA and, thus, not translated. Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains. Missplicing is caused by an alternative structural element containing a cryptic 5' splice site, which serves as a substrate for the ribozyme. Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.     

Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro

We have altered the TACTAAC sequence in the yeast CYH2m gene intron to TACTACC. This mutation changes the nucleotide at the normal position of the branch in intron RNA lariats produced during pre-mRNA splicing, and it prevents splicing in vivo. In a yeast pre-mRNA splicing system, CYH2m pre-mRNA carrying the TACTACC mutation is not specifically cut or rearranged in any way. Substitution of an A for the first G of the CYH2m intron, converting the highly conserved GTATGT 5' splice site sequence to ATATGT, also blocks intron excision in vivo and in vitro: pre-mRNA carrying this mutation was still cut normally at the mutant 5' splice site in vitro, to give authentic exon 1 and an intron-exon 2 lariat RNA with an A-A 2'-5' phosphodiester linkage at the branch point. This lariat RNA is a dead-end product. The subsequent cleavage at the 3' splice site is therefore sensitive to the sequence of the 5' end of the intron attached at the branch point.     

The neurospora CYT-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core.

The Neurospora CYT-18 protein, a tyrosyl-tRNA synthetase, which functions in splicing group I introns in mitochondria, promotes splicing of mutants of the distantly related bacteriophage T4 td intron. In an in vivo assay, wild-type CYT-18 protein expressed in E. coli suppressed mutations in the td intron's catalytic core. CYT-18-suppressible mutations were also suppressed by high Mg2+ or spermidine in vitro, suggesting they affect intron structure. Both the N- and C-terminal domains of CYT-18 are required for efficient splicing, but CYT-18 with a large C-terminal truncation retains some activity. Our results indicate that CYT-18 interacts with conserved structural features of group I introns, and they provide direct evidence that a protein promotes splicing by stabilizing the catalytically active structure of the intron RNA.     

Influence of RNA structural stability on the RNA chaperone activity of the Escherichia coli protein StpA

Proteins with RNA chaperone activity are able to promote folding of RNA molecules by loosening their structure. This RNA unfolding activity is beneficial when resolving misfolded RNA conformations, but could be detrimental to RNAs with low thermodynamic stability. In order to test this idea, we constructed various RNAs with different structural stabilities derived from the thymidylate synthase (td) group I intron and measured the effect of StpA, an Escherichia coli protein with RNA chaperone activity, on their splicing activity in vivo and in vitro. While StpA promotes splicing of the wild-type td intron and of mutants with wild-type-like stability, splicing of mutants with a lower structural stability is reduced in the presence of StpA. In contrast, splicing of an intron mutant, which is not destabilized but which displays a reduced population of correctly folded RNAs, is promoted by StpA. The sensitivity of an RNA towards StpA correlates with its structural stability. By lowering the temperature to 25°C, a temperature at which the structure of these mutants becomes more stable, StpA is again able to stimulate splicing. These observations clearly suggest that the structural stability of an RNA determines whether the RNA chaperone activity of StpA is beneficial to folding.     

In vivo selection of better self-splicing introns in Escherichia coli: the role of the P1 extension helix of the Tetrahymena intron

In vivo selection was used to improve the activity of the Tetrahymena pre-rRNA self-splicing intron in the context of heterologous exons. The intron was engineered into a kanamycin nucleotidyltransferase gene, with the pairing between intron bases and the 5' and 3' splice sites maintained. The initial construct failed to confer kanamycin resistance on Escherichia coli, although the pre-mRNA was active in splicing in vitro. Random mutation libraries were constructed to identify active intron variants in E. coli. All the active mutants sequenced contained mutations disrupting a base-paired region above the paired region P1 (referred to as the P1 extension region or P1ex) that involves the very 5' end of the intron. Subsequent site-directed mutagenesis confirmed that these P1ex mutations are responsible and sufficient to activate the intron splicing in E. coli. Thus, it appears that too strong of a secondary structure in the P1ex element can be inhibitory to splicing in vivo. In vitro splicing assays demonstrated that two P1ex mutant constructs splice six to eight times faster than the designed construct at 40 microM GTP concentration. The relative reaction rates of the mutant constructs compared to the original design are further increased at a lower GTP concentration. Possible mechanisms by which the disrupted P1ex structure could influence splicing rates are discussed. This study emphasizes the value of using libraries of random mutations to improve the activity of ribozymes in heterologous contexts in vivo.     

YdgT, the Hha paralogue in Escherichia coli, forms heteromeric complexes with H-NS and StpA

In enteric bacteria, proteins of the Hha/YmoA family play a role in the regulation of gene expression in response to environmental factors. Interaction of both Hha and YmoA with H-NS has been reported, and an Hha/H-NS complex has been shown to modulate expression in Escherichia coli of the haemolysin operon of plasmid pHly152. In addition to the hns gene, the chromosome of E. coli and other enteric bacteria also includes the stpA gene that encodes the StpA protein, an H-NS paralogue. We report here the identification of the Hha paralogue in E. coli, the YdgT protein. As Hha paralogue, YdgT appears to fulfil some of the functions reported for StpA as H-NS paralogue: YdgT is overexpressed in hha mutants and can compensate, at least partially, some of the hha-induced phenotypes. We also demonstrate that YdgT interacts both with H-NS and with StpA. Protein cross-linking studies showed that YdgT/H-NS heteromeric complexes are generated within the bacterial cell. The StpA protein, which is subjected to Lon-mediated turnover, was less stable in the absence of Hha or YdgT. Our findings suggest that Hha, YdgT and StpA may form complexes in vivo.     

Roles of the DNA binding proteins H-NS and StpA in homologous recombination and repair of bleomycin-induced damage in Escherichia coli

The DNA binding protein H-NS promotes homologous recombination in Escherichia coli, but the role of its paralog StpA in this process remains unclear. Here we show that an hns mutant, but not an stpA mutant, are marginally defective in conjugational recombination and is sensitive to the double-strand-break-inducing agent bleomycin. Interestingly, the hns stpA double mutant is severely defective in homologous recombination and more bleomycin-sensitive than is the hns or stpA single mutant, indicating that the stpA mutation synergistically enhances the defects of homologous recombination and the increased bleomycin-sensitivity in the hns mutant. In addition, the transduction analysis in the hns stpA double mutant indicated that the stpA mutation also enhances the defect of recombination in the hns mutant. These results suggest that H-NS plays an important role in both homologous recombination and repair of bleomycin-induced damage, while StpA can substitute the H-NS function. The recombination analysis of hns single, stpA single, and hns stpA double mutants in the recBC sbcA and recBC sbcBC backgrounds suggested that the reduction of the hns single or hns stpA double mutants may not be due to the defect in a particular recombination pathway, but may be due to the defect in a common process of the pathways. The model for the functions of H-NS and StpA in homologous recombination and double-strand break repair is discussed.     

A self-splicing RNA excises an intron lariat

We have investigated the in vitro self-splicing of a class II mitochondrial intron. A model pre-mRNA containing intron 5 gamma of the oxi 3 gene of yeast mitochondrial DNA undergoes an efficient intramolecular rearrangement reaction in vitro. This reaction proceeds under conditions distinct from those optimal for self-splicing of class I introns, such as the Tetrahymena nuclear rRNA intron. Intron 5 gamma is excised as a nonlinear RNA indistinguishable from the in vivo excised intron product by gel electrophoresis and primer extension analysis. Studies of the in vitro excised intron product strongly indicate that it is a branched RNA with a circular component joined by a linkage other than a 3'-5' phosphodiester. Two other products, the spliced exons and the broken form of the lariat, were also characterized. These results show that the class II intron products are similar to those of nuclear pre-mRNA splicing.     

Group II intron splicing in Escherichia coli: phenotypes of cis-acting mutations resemble splicing defects observed in organelle RNA processing.

The mitochondrial group IIB intron rI1, from the green algae Scenedesmus obliquus ' LSUrRNA gene, has been introduced into the lacZ gene encoding beta-galacto-sidase. After DNA-mediated transformation of the recombinant lacZ gene into Escherichia coli, we observed correct splicing of the chimeric precursor RNA in vivo. In contrast to autocatalytic in vitro self-splicing, intron processing in vivo is independent of the growth temperature, suggesting that in E.coli, trans -acting factors are involved in group II intron splicing. Such a system would seem suitable as a model for analyzing intron processing in a prokaryotic host. In order to study further the effect of cis -mutations on intron splicing, different rI1 mutants were analyzed (with respect to their splicing activity) in E.coli. Although the phenotypes of these E. coli intron splicing mutants were identical to those which can be observed during organellar splicing of rI1, they are different to those observed in in vitro self-splicing experiments. Therefore, in both organelles and prokaryotes, it is likely that either similar splicing factors or trans -acting factors exhibiting similar functions are involved in splicing. We speculate that ubiquitous trans -acting factors, via recent horizontal transfer, have contributed to the spread of group II introns.     

The Cbp2 protein stimulates the splicing of the omega intron of yeast mitochondria.

The Cbp2 protein is encoded in the nucleus and is required for the splicing of the terminal intron of the mitochondrial COB gene in Saccharomyces cerevisiae . Using a yeast strain that lacks this intron but contains a related group I intron in the precursor of the large ribosomal RNA, we have determined that Cbp2 protein is also required for the normal accumulation of 21S ribosomal RNA in vivo . Such strains bearing a deletion of the CBP2 gene adapt slowly to growth in glycerol/ethanol media implying a defect in derepression. At physiologic concentrations of magnesium, Cbp2 stimulates the splicing of the ribosomal RNA intron in vitro . Nevertheless, Cbp2 is not essential for splicing of this intron in mitochondria nor is it required in vitro at magnesium concentrations >5 mM. A similar intron exists in the large ribosomal RNA (LSU) gene of Saccharomyces douglasii . This intron does need Cbp2 for catalytic activity in physiologic magnesium. Similarities between the LSU introns and COB intron 5 suggest that Cbp2 may recognize conserved elements of the these two introns, and protein-induced UV crosslinks occur in similar sites in the substrate and catalytic domains of the RNA precursors.     

Multiple self-splicing introns in bacteriophage T4: evidence from autocatalytic GTP labeling of RNA in vitro

RNA from T4-infected cells yielded multiple end-labeled species when incubated with alpha-32P-GTP under self-splicing conditions. One of these corresponds to the previously identified intron from the td gene of T4, while others appear to represent additional group I introns in T4. Two loci distinct from the td gene were found to hybridize to a mixed alpha-32P-GTP-labeled T4 RNA probe. These mapped in or near the unlinked genes nrdB and nrdC. A fragment from the nrdB region that contains the intron has been cloned and shown to generate characteristic group I splice products with RNA synthesized in vivo and in vitro. Multiple introns, and the prospect that these occur within several genes in the same metabolic pathway, suggest a possible regulatory role for splicing in T4.     

Identification of the sequences responsible for the splicing phenotype of the regulatory intron of the L1 ribosomal protein gene of Xenopus laevis.

Splicing of the regulated third intron of the L1 ribosomal protein gene of Xenopus laevis has been studied in vivo by oocyte microinjection of wild-type and mutant SP6 precursor RNAs and in vitro in the heterologous HeLa nuclear extract. We show that two different phenomena combine to produce the peculiar splicing phenotype of this intron. One, which can be defined constitutive, shows the same features in the two systems and leads to the accumulation of spliced mRNA, but in very small amounts. The low efficiency of splicing is due to the presence of a noncanonical 5' splice site which acts in conjunction with sequences present in the 3' portion of the intron. The second leads to the massive conversion of the pre-mRNA into site specific truncated molecules. This has the effect of decreasing the concentration of the pre-mRNA available for splicing. We show that this aberrant cleavage activity occurs only in the in vivo oocyte system and depends on the presence of an intact U1 RNA.     

Molecular genetics of group I introns: RNA structures and protein factors required for splicing--a review

In vivo and in vitro genetic techniques have been widely used to investigate the structure-function relationships and requirements for splicing of group-I introns. Analyses of group-I introns from extremely diverse genetic systems, including fungal mitochondria, protozoan nuclei, and bacteriophages, have yielded results which are complementary and highly consistent. In vivo genetic studies of fungal mitochondrial systems have served to identify cis-acting sequences within mitochondrial introns, and trans-acting protein products of mitochondrial and nuclear genes which are important for splicing, and to show that some mitochondrial introns are mobile genetic elements. In vitro genetic studies of the self-splicing intron within the Tetrahymena thermophila nuclear large ribosomal RNA precursor (Tetrahymena LSU intron) have been used to examine essential and nonessential RNA sequences and structures in RNA-catalyzed splicing. In vivo and in vitro genetic analysis of the intron within the bacteriophage T4 td gene has permitted the detailed examination of mutant phenotypes by analyzing splicing in vivo and self-splicing in vitro. The genetic studies combined with phylogenetic analysis of intron structure based on comparative nucleotide sequence data [Cech 73 (1988) 259-271] and with biochemical data obtained from in vitro splicing experiments have resulted in significant advances in understanding the biology and chemistry of group-I introns.     

Accurate pre-mRNA splicing using a nuclear extract from Bombyx mori fat body

A cell-free pre-mRNA splicing system was developed from the nuclear extract of Bombyx mori fat bodies. The synthetic pre-mRNA from the cytoplasmic actin gene of B. mori (BmA3 ) was used as a substrate for in vitro splicing. The pre-mRNA was accurately spliced in the nuclear extract prepared from the fat body. Sequencing of the amplicon by RT-PCR revealed that the spliced site in vitro was identical to the site in vivo. We found that a part of the input capped pre-mRNA was accurately spliced after 2h of incubation. In addition, the RT-PCR analysis precisely mapped the branch point to an adenine located at 30 nucleotides before the 3' end of the first intron in BmA3. This system was a first example that differentiated tissue in insects yielded active extracts for in vitro splicing.     

Normal and mutant human β-globin pre-mRNAs are faithfully and efficiently spliced in vitro

Human β-globin mRNA precursors (pre-mRNAs) synthesized in vitro from a bacteriophage SP6 promoter/β-globin gene fusion are accurately and efficiently spliced when added to a HeLa cell nuclear extract. Under optimal conditions, the first intervening sequence (IVS 1) is removed by splicing in up to 90% of the input. pre-mRNA. Splicing requires ATP and in its absence the pre-mRNA is neither spliced nor cleaved at splice junctions. Splicing does not require that the pre-mRNA contain a correct 5′ or 3′ end, a 3′ poly A tail, or a 5′-terminal cap structure. However, capping of the pre-mRNA significantly affects the specificity of in vitro processing. In the absence of a cap approximately 30%–40% of the pre-mRNA is accurately spliced, and a number of aberrantly cleaved RNAs are also detected. In contrast, capped pre-mRNAs are spliced more efficiently and produce fewer aberrant RNA species. The specificity of splice-site selection in vitro was tested by analyzing pre-mRNAs that contain β-thalassemia splicing mutations in IVS 1. Remarkably, these mutations cause the same abnormal splicing events in vitro and in vivo. The ability to synthesize mutant pre-mRNAs and study their splicing in a faithful in vitro system provides a powerful approach to determine the mechanisms of RNA splice-site selection.