Browsing by Subject "RNA splicing"

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Bacterial gene targeting using group II intron L1.LtrB splicing and retrohoming
(2008-05) Yao, Jun, 1974-; Lambowitz, Alan
The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer for reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base pairing of the intron RNA. Consequently, Ll.LtrB introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, I show that an Escherichia coli gene disruption library obtained using randomly inserted Ll.LtrB introns contains most viable E. coli gene disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. I also demonstrate that Ll.LtrB introns can be used for efficient gene targeting in a variety of Gram-negative and positive bacteria, including E. coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, Bacillus subtilis, and Staphylococcus aureus. Ll.LtrB introns expressed from a broad-host-range vector or an E. coli-S. aureus shuttle vector yielded targeted disruptions in a variety of test genes in these organisms at frequencies of 1-100% without selection. By using an Ll.LtrB intron that integrates in the sense orientation relative to target gene transcription and thus could be removed by RNA splicing, I disrupted the essential gene hsa in S. aureus. Because the splicing of the Ll.LtrB intron by the intron-encoded protein is temperature-sensitive, this method yields a conditional hsa disruptant that grows at 32oC, but not at 43oC. Finally, I developed high-throughput screens to identify E. coli genes that affect either the splicing or retrohoming of the Ll.LtrB intron. By using these screens, I identified fourteen mutants in a variety of genes that have decreased intron retrohoming efficiencies and additional mutants that have increased intron retrohoming efficiencies, in some cases apparently resulting from increased stability of the intron RNA.
Genetic mapping of nuclear suppressors of splicing-deficient chloroplast introns, and a novel rhodanese-domain protein required for chloroplast translation in Chlamydomonas
(2010-12) Luo, Liming, 1967-; Herrin, David L.; Roux, Stan J.; La Claire, John; Jansen, Robert K.; Browning, Karen
Although many group I (GI) introns can self-splice in vitro, their splicing is promoted by proteins in vivo. Only a few splicing factors that specifically promote GI intron splicing have been identified, however, none are from chloroplasts, which is the subject of this study. In previous work from our lab, a strategy was developed to identify splicing factors for chloroplast GI introns of Chlamydomonas by using suppressor genetics. A mutant with reduced splicing of the chloroplast 23S rRNA intron (Cr.LSU) was generated. Then, 3 nuclear suppressors (7120, 71N1 and 7151) with substantially restored splicing of Cr.LSU were isolated and partially characterized. However, the suppressor gene(s) were not identified. In this study, I have used genetic mapping to make a renewed attempt to isolate these genes. Using polymorphisms between the 137C strain that was used for suppressor isolation, and a new strain of C.reinhardtii (S1D2), the nuclear suppressor mutations in 7120 and 71N1 were mapped to a region on chromosome III that is essentially devoid of recombination. Based on the recombination maps, the suppressor gene in 7120 is located within a ~418-kb region from bp 2,473,064 to 2,891,232, whereas the suppressor in 71N1 is likely located within a ~236-kb subregion from bp 2,473,064 to 2,709,377. It is possible that these mutations are in the same gene; however, the maps could not be refined further due to the lack of recombination in this 418-kb region. I also attempted to compare the genomic sequence of the 7120 suppressor, which was obtained by next-generation sequencing, with the Chlamydomonas reference genome (JGI, v.4). Next-generation sequencing of 7120 revealed the existence of abundant repetitive sequences and transposable elements clustered in a ~40-kb subregion of the recombinationally suppressed 418-kb region on chromosome III. I suggest that the high frequency of repetitive sequences and transposable elements in this region may be the reason for the suppressed recombination. Searching for candidate genes in the mapped region led me to examine a novel protein that was predicted to have a putative chloroplast transit-peptide, and an RNA binding domain. Further bioinformatic analysis revealed a single rhodanese domain with an active-site cysteine. The protein was expressed in E.coli as the full-length and predicted mature forms, plus a small His-tag. The purified mature protein had rhodanese catalytic activity, based on the fact that it was able to transfer sulfur from thiosulfate to cyanide. Also, western blot analysis with a polyclonal antibody produced in rabbits showed that the cellular protein migrated on SDS gels close to the mature protein and faster than the full-length protein, indicative of an organelle-targeted protein. The antibody also showed that the cellular protein co-fractionated with chloroplasts. To gain insight into its in vivo function, the gene was knocked down using the tandem RNAi system (Rohr et al., 2004), which produced strains (5) with reductions of 31% to 76% in the mRNA level, and ~30% to ~60% in the protein level. These strains were sensitive to bright light, and had reduced rates of growth under all conditions, which are characteristics of chloroplast translation mutants. Thus, chloroplast protein synthesis was examined by radioisotope pulse-labeling in the presence of cycloheximide, which showed that the RNAi strains were broadly and negatively affected, and RT-PCR and northern blot revealed only normal chloroplast mRNA levels. These data have identified a new rhodanese-family enzyme that is required for chloroplast translation, which I have designated “CRLT”, for chloroplast rhodanese-like translation.
Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase with group I intron RNAs
(2002) Myers, Christopher Allan; Lambowitz, Alan
The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase, or CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. To investigate how CYT-18 stabilizes the active RNA structure, I used an Escherichia coli genetic assay with the phage T4 td intron to systematically test the ability of the CYT-18 protein to compensate for structural defects in the P7 region of the group I intron core. P7 is a long-range base-pairing interaction of the P3-P9 domain that forms the binding site for the splicing co-factor guanosine. My results show that CYT-18 can suppress numerous mutations that impair the self-splicing of the td intron, including mutations that disrupt base-pairing within the P7 region. CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site. Structure mapping experiments with some selected mutant introns showed that the P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain. Previous work suggested that CYT-18 recognizes a conserved tRNA-like structure of the group I intron catalytic core. I used directed hydroxyl radical cleavage assays to show that the nucleotide-binding fold and C-terminal domains of CYT-18 interact with the expected group I intron cognates of the aminoacyl-acceptor stem and D-anticodon arms, respectively. Further, three-dimensional graphic modeling, supported by biochemical data, shows that conserved regions of group I introns can be superimposed over interacting regions of the tRNA in a Thermus thermophilus TyrRS/tRNATyr cocrystal structure.
Nuclear genes that promote splicing of the chloroplast group-I 23S rRNA intron and an organelle intron database, FUGOID
(2002-08) Li, Fei; Herrin, David L.
Cr. LSU is a self-splicing Group I intron in the chloroplast 23S rRNA gene of the green alga Chlamydomonas reinhardtii. Trans-acting factors are likely required to promote the splicing of this intron in vivo; however, nothing is known of the trans requirements for Group I intron splicing in chloroplasts. I have used a genetic approach in this study of possible splicing factors for Cr. LSU. Single nucleotide substitutions were made in the core helices P4, P6, and P7, and in the metal-binding GAAA motif in the J4/5 region of the intron. In vitro assays showed that these substitutions had very strong effects on Cr.LSU self-splicing; however, splicing of all but the P6 mutations could be partially recovered by vi increasing the Mg2+ concentration, indicative of structural effects. The mutant constructs were transformed into chloroplasts to replace the wild-type intron. Surprisingly, only the P4 mutants became homoplasmic, indicating that the other mutations were lethal. The splicing-deficient P4 125A mutant, which exhibited slow growth and light sensitivity, was used to isolate suppressor strains that showed a substantial restoration of Cr.LSU splicing. Genetic analysis of the 7151, 7120 and 71N1 suppressors indicated that these mutations are in at least 2 nuclear genes. The 7151 suppressor, which defines the css1 (chloroplast splicing suppressor) gene, was shown to have no discernible phenotype with the wild-type intron, and to be dominant in vegetative diploids containing the mutant intron. These results indicate that the Cr.LSU intron is unusually sensitive to single base changes in the core, and, moreover, provide the first identification of plant genes that promote splicing of a group I intron in vivo. Toward the eventual cloning of the css1 gene, a library was constructed in a novel cosmid vector, SCBN, using genomic DNA from the 7151 suppressor mutant of C. reinhardtii. Part of the library was used to generate an indexed collection of 10,340 individual clones in 110 96-well microtiter plates. The library can be easily replicated (for DNA preparation) and has an average insert size of ~37 kb. Thus, it covers almost 4 genome equivalents. The library may allow the cloning of css1 by complementation of the P4 125A mutant. Finally, I have developed a web-based relational database, FUGOID (Functional Genomics of Organellar Introns Database) that collects and integrates vii viii various functional (and some structural) data on organellar (mitochondrial and chloroplast)introns. The main information provided by FUGOID includes intron sequence, subclass, resident ORF, self-splicing capability, host gene, protein factor(s) involved in splicing, mobility, insertion site, twintron, seminal references and taxonomic position of host organism. Users can access the database with any common web browser on a variety of operating systems. The main page of the database is available at http://wnt.cc.utexas.edu/~ifmr530/introndata/main.htm.
RNA/protein interactions during group II intron splicing and toward group II intron targeting in mammalian cells
(2005) Cui, Xiaoxia; Lambowitz, Alan
Group II introns are both catalytic RNAs and retrotransposable elements. Group II intron-encoded proteins (IEPs) have maturase activity, which promotes intron RNA splicing, and reverse transcriptase activity, which functions in intron mobility. Previous studies of the Lactococcus lactis Ll.LtrB intron suggested a model in which its IEP binds first to a high-affinity binding site in intron subdomain DIVa and then makes additional contacts with the conserved catalytic core to stabilize the active RNA structure. In the absence of DIVa, the IEP promotes residual splicing by binding directly to the catalytic core. I developed E. coli genetic assays to detect in vivo splicing of the Ll.LtrB intron and identify regions in the IEP essential for interacting with different parts of the intron RNA. Mutational and biochemical analysis combined with three-dimensional structural modeling support the hypothesis that the extended N-terminal finger region of LtrA is involved in high-affinity binding of DIVa, possibly forming a binding pocket in combination with parts of the thumb domain (domain X), while other regions of the RT and X domains are potentially involved in binding the catalytic core. The Ll.LtrB intron works very efficiently for gene targeting in bacteria, and it is desirable to target mammalian genomes, for which efficient means of manipulation are lacking. I developed an expression system to produce Ll.LtrB intron RNA and IEP in cultured human cells and found that the expressed intron splices in vivo. I also explored different methods for introducing RNPs into cells, including electroporation and injection, and was able to detect a chromosomal targeting event with RNP electroporation. With improvements in targeting efficiency, group II introns would be generally useful for functional genomics and gene therapy.
Snu40p and Snu66p are required for spliceosome activation at suboptimal temperatures
(2008-05) Roth, Andrew Adam; Stevens, Scott W.
In addressing the pre-mRNA substrate, the splicing machinery requires rearrangement of multiple RNA and protein components. The classical model of spliceosome formation begins with the U1 snRNA recognition of the 5" splice site and U2 snRNP interaction with the branch point. This process is followed by the engagement of a pre-assembled U4/U6·U5 tri-snRNP to form the A2-1 complex. The spliceosome is subsequently activated through a number of structural rearrangements. Among these is the unwinding of the U4/U6 intermolecular helix by the tri-snRNP component Brr2p. While numerous protein components of the tri-snRNP have been identified, the function of many of these remain unknown. The nonessential Snu66p (U4/U6·U5-110K in humans) stably associates only with the U4/U6·U5 tri-snRNP while the similarly nonessential Snu40p (U5-52K in humans) associates exclusively with the U5 snRNP. To understand why two non-essential pre-mRNA splicing factors have been so well conserved through great evolutionary distances, we examined their roles in the assembly and function of the tri-snRNP. Removal of SNU40 alone does not affect snRNP levels, however deletion of SNU66 results in reduced levels of tri-snRNP. The U4/U6·U5 snRNPs in [Delta]snu66 cells are resistant to the ATP-dependent U4/U6 unwinding by Brr2p, and profound U4/U6 accumulation occurs at reduced temperatures. Remarkably, subsequent removal of SNU40 in a [Delta]snu66 strain bypasses the tri-snRNP formation defect while unwinding of U4/U6 remains defective. Additional investigation revealed that Prp6p, another tri-snRNP protein, is destabilized from the complex. Based upon this data in total, I present a model in which Snu40p and Snu66p interact sequentially with Prp6p to maintain directionality for proper biogenesis of the tri-snRNP. Further, the U4/U6 unwinding defect of the double mutant should theoretically arrest the A2-1 spliceosome. Indeed, native gel analysis confirms the buildup of a large complex later determined to be A2-1. I have purified this complex, functionally tested its catalytic viability, and identified its components via mass spectrometry. This is the first full characterization of the A2-1 precatalytic spliceosome complex in Saccharomyces cerevisiae.
Structural studies of a group I intron splicing factor and a continuous three-dimensional DNA lattice
(2005) Paukstelis, Paul John; Lambowitz, Alan
The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing group I introns by stabilizing the catalytically active structure of the intron RNA. I determined 1.95 Å X-ray crystal structure of a C-terminally truncated CYT-18 protein (∆C423-669) that efficiently splices the Neurospora crassa ND1 intron and other group I introns. The structure shows that CYT-18’s nucleotide-binding fold and intermediate α-helical domain are essentially the same as those of closely related bacterial TyrRSs, except for an α-helical N-terminal extension (H0) and two small insertions (I and II) in the nucleotide-binding fold. The X-ray crystal structure in conjunction with site-directed hydroxyl radical cleavage data enabled the construction of a refined model of the CYT-18/group I intron RNA complex. The model shows that the group I intron RNA, like tRNATyr, binds across the surface of the two subunits of the homodimer, but interacts with the side opposite the aminoacylation active site. Though CYT-18 contains a tRNATyr binding site, and there are structural similarities between group I introns and tRNAs, these results demonstrate that CYT-18 adapted to function in intron splicing by acquiring a distinct binding site for group I introns. DNA has proved to be a versatile material for the rational design and assembly of nanometer scale objects. I solved the crystal structure of a continuous three-dimensional DNA lattice formed by the self-assembly of a DNA 13-mer to 2.1 Å resolution. The structure consists of stacked layers of parallel helices with adjacent layers linked through parallel-stranded base pairing. The hexagonal lattice geometry contains solvent channels large enough to allow 3’-linked guest molecules into the crystal. I have successfully used these parallel base pairs to design and produce crystals with greatly enlarged solvent channels. This lattice may have applications as a molecular scaffold for structure determination of guest molecules, as a molecular sieve, or in the assembly of molecular electronics. Predictable non-Watson-Crick base pairs may serve as a new tool in structural DNA nanotechnology.