No description available.

No description available.

Mobile group II introns are retroelements that are found in prokaryotes, archaea, and the organelles of plants and fungi, but not in the nuclear genomes of eukaryotes. They consist of a catalytically active RNA and intron-encoded reverse transcriptase, which together promote site-specific integration into DNA sites in a mechanism called retrohoming. The group II intron Ll.LtrB has been developed into a programmable, DNA-targeting agent called "targetron", which is widely used in bacteria and an attractive technology for gene targeting in eukaryotes. However, group II intron genome targeting in human cells has not been equivocally shown. This dissertation focuses on the hypothesis that the low Mg2+-concentrations found in higher eukaryotes present a natural barrier to group II introns. First, I studied E. coli host proteins that aid group II intron retrohoming and found that synthesis of a second DNA-strand relies on host replication restart proteins. Next, I demonstrated that mutations in the distal stem of the catalytic core domain V (DV) improve Ll.LtrB retrohoming in a low Mg2+-concentration E. coli mutant and in biochemical assays. These results suggest that DV is involved in an RNA-folding step that becomes rate limiting at low Mg2+. Subsequently, I performed directed evolution of the intron RNA by injecting in vitro prepared mutant intron libraries into Xenopus laevis oocyte nuclei. The mutations were analyzed using Roche 454 sequencing to generate an intron fitness landscape, which revealed conserved positions and potentially beneficial mutations, enabling enhanced retrohoming in Xenopus oocytes. Finally, I used a hybrid Pol II/T7 Ll.LtrB eukaryotic expression system to show that high exogenous MgCl2 in the growth media enables retrohoming into plasmids and genomic DNA in human cells. In vivo directed evolution and mutation analyses using PacBio RS circular consensus sequencing indicated that only a few mutations may improve intron activity in human cells. This dissertation provides evidence that efficient group II intron retrohoming in human cells is limited by low Mg2+-concentrations and develops new approaches for overcoming this limitation to enable use of group II introns for gene targeting in higher organisms. / text

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The dynamic rearrangements of RNA and protein complexes and the fidelity of pre-mRNA splicing are governed by DExD/H-box ATPases. One of the spliceosomal ATPases, Brr2, is believed to facilitate conformational rearrangements during spliceosome activation and disassembly. It features an unusual architecture, with two consecutive helicase-cassettes, each comprising a helicase and a Sec63 domain. Only the N-terminal cassette exhibits catalytic activity. By contrast, the C-terminal half of Brr2 engages in protein interactions. Amongst interacting proteins are the Prp2 and Prp16 helicases. The work presented in this thesis aimed at studying and assigning functional relevance to the bipartite architecture of Brr2 and addressed the following questions: (1) What role does the catalytically inert C-terminal half play in Brr2 function, and why does it interact with other RNA helicases? (2) Which RNAs interact with the different parts of Brr2? (1) In a yeast two-hybrid screen novel brr2 mutant alleles were identified by virtue of abnormal protein interactions with Prp2 and Prp16. Phenotypic characterization showed that brr2 C-terminus mutants exhibit a splicing defect, demonstrating that an intact C-terminus is required for Brr2 function. ATPase/helicase deficient prp16 mutants suppress the interaction defect of brr2 alleles, possibly indicating an involvement of the Brr2 C-terminus in the regulation of interacting helicases. (2) Brr2-RNA interactions were identified by the CRAC approach (in vivo Crosslinking and analysis of cDNA). Physical separation of the N-terminal and C-terminal portions and their individual analyses indicate that only the N-terminus of Brr2 interacts with RNA. Brr2 cross-links in the U4 and U6 snRNAs suggest a step-wise dissociation of the U4/U6 duplex during catalytic activation of the spliceosome. Newly identified Brr2 cross-links in the U5 snRNA and in pre-mRNAs close to 3’ splice sites are supported by genetic analyses. A reduction of second step efficiency upon combining brr2 and U5 mutations suggests an involvement of Brr2 in the second step of splicing. An approach now described as CLASH (Cross-linking, Ligation and Sequencing of Hybrids) identified Brr2 associated chimeric sequencing reads. The inspection of chimeric U2-U2 sequences suggests a revised secondary structure for the U2 snRNA, which was confirmed by phylogenentic and mutational analyses. Taken together these findings underscore the functional distinction of the N- and C-terminal portions of Brr2 and add mechanistic relevance to its bipartite architecture. The catalytically active N-terminal helicase-cassette is required to establish RNA interactions and to provide helicase activity. Conversely, the C-terminal helicase-cassette functions solely as protein interaction domain, possibly exerting regulation on the activities of interacting helicases and Brr2 itself.

The spliceosome is a complex, dynamic ribonucleoprotein (RNP) complex that undergoes numerous conformational changes during its assembly, activation, catalysis and disassembly. Defects in spliceosome assembly are thought to trigger active dissociation of faulty splicing complexes. A yeast genetic screen was performed to identify components of the putative discard pathway. This study found that weak mutant alleles of SPP382 suppress defects in several proteins required for spliceosome activation (Prp38p, Prp8p and Prp19p) as well as substrate mutations (weak branch point mutants). This evolutionary conserved protein had been found in both yeast and mammalian splicing complexes. However, its role in splicing had not been elucidated. This study focused on understanding the cellular role of Spp382p in splicing and particularly in the discard pathway. Spp382p was found to be essential for normal splicing and for efficient intron turnover. Since Spp382p binds Prp43p and is required for intron release in vitro, spp382 mediated suppression of splicing factor mutations might reflect lowered Prp43p activity. In agreement with this, we find that prp43 mutants also act as suppressors. This leads us to propose a model in which defects in spliceosome assembly, like those caused by prp38-1, prompt Spp382p-mediated disassembly of the defective complex via Prp43p Bolstering this theory, we find that Spp382p is specifically recruited to defective complexes lacking the 5 exon cleavage intermediate and spp382 mutants suppress other splicing defects. I show by stringent proteomic and two-hybrid analyses that Spp382p interacts with Cwc23p, a DnaJ-like protein present in the spliceosome and co-purified the Prp43p-DExD/H-box protein. In this study, I also show that Cwc23p is itself essential for splicing and normal intron turnover. Enhanced expression of another protein, Sqs1p, structurally related to Spp382p and also found associated with Prp43p is inhibitory to both growth and splicing. Synthetic lethal and dosage suppression studies bolster a functional linkage between Spp382p, Cwc23p, Sqs1p and Prp43p and together, the data support the existence of a Spp382p -dependent spliceosome integrity (SPIN) complex acting to remove defective spliceosomes.

More than 90% of human genes undergo a processing step called splicing, whereby non-coding introns are removed from initial transcripts and coding exons are ligated together to yield mature messenger RNA. Roughly 50% of human genetic diseases correspond to aberrant splicing. Splicing is catalyzed by an RNA/protein machine called the spliceosome. RNA components of the spliceosome are at least partly responsible for splicing catalysis. In addition, in vitro analyses implicate an essential and very highly conserved protein, Prp8, in orchestrating key steps in spliceosome assembly and possibly catalysis. Interestingly, mutant alleles of Prp8 are the cause of retinitis pigmentosa, an inherited form of retinal degeneration. A key goal is elucidation of the precise role of Prp8 in the spliceosome by high resolution structural analysis. The large size of Prp8 and its insolubility hinder progress in this regard. Instead, structural understanding of Prp8 can be gained by investigating domains in isolation; however there is only limited information as to what domain boundaries are and few hints about the functional relevance of putative domains. Here we have further defined the previously proposed domain IV in Prp8, and identified the domain IV core. Structural determination of the domain IV core reveals an RNase H fold, which could not be predicted based on primary sequence alone. RNase H recognizes A-form nucleic acid duplexes, which strongly suggests the domain IV core interacts with double-stranded RNA in the context of the spliceosome. Characterizing the binding preferences of the domain IV revealed the highest affinity is for a 4-helix junction structure adopted by the very RNAs at the spliceosome active site. Our characterization of the protein/RNA binding interface by complementary footprinting techniques currently provides the best model of how RNA interacts with an essential protein component at the heart of the spliceosome.

No description available.

Splicing is the process where pre-mRNA is converted to mRNA via two transesterification reactions. With this process unwanted sequences of nucleic acids, known as introns, are removed allowing only the coding nucleic acid sequences, exons, to remain. This process is catalysed by a dynamically assembled, highly complex macromolecular machine called the spliceosome, which is made up of five small nuclear ribonucleoproteins (snRNPs). To date, the spliceosome has defied conventional methods for conclusive characterisation, resulting in it being relatively poorly understood, although advances have been made.1, 2 Apart from being of interest due to the fact that splicing is an essential life process, it is also of interest medically. Disruption to the splicing process can produce incorrectly formed mRNA, which plays a part in many diseases.3 Small molecule inhibitors which bind to, and inhibit, the functions of individual proteins would “stall” the spliceosome,4 circumventing its dynamic nature. These inhibitors could also form the basis of new drugs, treating diseases which incorrectly formed mRNA can cause. Previously reported small molecule inhibitors have inhibited splicing at the early stages of spliceosome assembly.5-7 However, our target protein snu1148 belongs to the U5 snRNP, which is involved later on in the splicing cycle. Inhibition of Snu114 should, therefore, lead to accumulation of spliceosome complexes produced at later stages of the cycle. Homology studies of Snu114 indicated a strong correlation of amino acid sequences with ribosomal growth factors EF-2 and EF-G. This study allowed us to target Snu114 using known EF-2 and EF-G inhibitors, sordarin and fusidic acid, which were tested and found to have significant splicing inhibition activity. A series of derivatives of these parent compounds were then attempted in an effort to improve splicing inhibition activity and to analyse the structure-activity relationship of fusidic acid and sordarin as splicing inhibitors. The biosynthesis of sordarin proved to be difficult and only a few derivatives were synthesised, however an improvement was made to splicing inhibition activity by forming sordaricin 32. Various fusidic acid derivatives were successfully synthesised, leading to an analysis of the structure-activity relationship of fusidic acid as a splicing inhibitor. Most fusidic acid derivatives produced a lower splicing inhibition activity than fusidic acid. However, fusidic acid derivative 229 had an equivalent inhibition activity to that found for fusidic acid. This result leads us to believe that the C-3 hydroxyl moiety of fusidic acid would be an ideal area for modification in future studies.

Through a genome wide study of spliceosome recruitment in Saccharomyces cerevisiae, we were able to identify a set of protein-coding genes that despite the fact that they contain no introns and their mRNA is not known to be spliced; their loci were occupied by the spliceosome. Bioinformatic analysis revealed the existence of splicing signals on these genes. Detailed analysis of BDF2, a representative gene, revealed that the spliceosome negatively regulates its mRNA levels through an unconventional one-step splicing reaction that cleaves BDF2 mRNA and targets the cleavage products for degradation. In an effort to clarify the mechanism of spliceosome recruitment to BDF2 locus, we identified that Bdf1, the redundant to Bdf2 factor, is required for the recruitment of the spliceosome at BDF2 and the subsequent down-regulation of its mRNA levels. The above led us to propose a new role for the spliceosome in the regulation of gene expression. Finally, we investigated the generality of this regulatory mechanism is S. cerevisiae and identified a set of genes which can be differentially spliced and whose physiological expression could be potentially regulated by the spliceosome.