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Microsporidia are unicellular fungi that are intracellular parasites of animals, including humans. They are both complex and simple, armed with a sophisticated infection apparatus and possessing the smallest eukaryotic nuclear genomes. The microsporidian Encephalitozoon cuniculi has a genome size of 2.9 Mb, which is smaller than many bacterial genomes. Genome reduction and compaction in size, content, and form has been interpreted as an adaptation to parasitism. One of the effects of genome size reduction concerns intron evolution — E. cuniculi has retained only a few extremely short spliceosomal introns. This thesis examines the splicing of introns in the spore stage. The introns were retained in spores, suggesting life-stage specific splicing and splicing inhibition. How the short introns are recognized was also examined. Unique splicing signal motifs were predicted, and were used to find additional introns. The intron density was doubled for this species, and I also obtained data that counter current views about intron evolution in compacted genomes with low intron densities. I also predict that E. cuniculi introns are recognized in a unique way by the spliceosome.

Microsporidia are unicellular fungi that are intracellular parasites of animals, including humans. They are both complex and simple, armed with a sophisticated infection apparatus and possessing the smallest eukaryotic nuclear genomes. The microsporidian Encephalitozoon cuniculi has a genome size of 2.9 Mb, which is smaller than many bacterial genomes. Genome reduction and compaction in size, content, and form has been interpreted as an adaptation to parasitism. One of the effects of genome size reduction concerns intron evolution — E. cuniculi has retained only a few extremely short spliceosomal introns. This thesis examines the splicing of introns in the spore stage. The introns were retained in spores, suggesting life-stage specific splicing and splicing inhibition. How the short introns are recognized was also examined. Unique splicing signal motifs were predicted, and were used to find additional introns. The intron density was doubled for this species, and I also obtained data that counter current views about intron evolution in compacted genomes with low intron densities. I also predict that E. cuniculi introns are recognized in a unique way by the spliceosome.

Microsporidia are unicellular fungi that are intracellular parasites of animals, including humans. They are both complex and simple, armed with a sophisticated infection apparatus and possessing the smallest eukaryotic nuclear genomes. The microsporidian Encephalitozoon cuniculi has a genome size of 2.9 Mb, which is smaller than many bacterial genomes. Genome reduction and compaction in size, content, and form has been interpreted as an adaptation to parasitism. One of the effects of genome size reduction concerns intron evolution — E. cuniculi has retained only a few extremely short spliceosomal introns. This thesis examines the splicing of introns in the spore stage. The introns were retained in spores, suggesting life-stage specific splicing and splicing inhibition. How the short introns are recognized was also examined. Unique splicing signal motifs were predicted, and were used to find additional introns. The intron density was doubled for this species, and I also obtained data that counter current views about intron evolution in compacted genomes with low intron densities. I also predict that E. cuniculi introns are recognized in a unique way by the spliceosome. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Saccharomyces cerevisiae, the best-known representative of the Saccharomycotina subphylum, is an intron-poor organism with introns in only 5 % of its protein-coding genes. The most popular model of intron evolution suggests that intron-poor eukaryotes, such as S. cerevisiae, have undergone extensive intron loss throughout their evolutionary history. Against this background of intron loss, the retention of specific introns in the S. cerevisiae genome might be attributable to an evolutionary advantage that they provide. Introns have been shown to exhibit ‘function’ in various ways: through recognition of their sequence by RNA binding proteins, the adoption of secondary structures after transcription, the mechanism of splicing itself, and noncoding RNA genes embedded within them. In order to understand how RNA structures contribute to intron function, we first performed a computational screen using 306 alignments of S. cerevisiae intron orthologs. We identified conserved RNA structures in 19 introns that act either in trans as independent intron-encoded ncRNA genes or in cis within the pre-mRNA. Our results showed that introns with conserved secondary structures are conserved in yeast and experimental validation revealed they are frequently maintained in the cells after splicing. Our results suggest that the intron in GLC7 contains a novel ncRNA that regulates expression of its host transcript under stress conditions. Secondly, we focused on the HAC1 intron, which is known to be spliced upon the unfolded protein response by an endoribonuclease IRE1. We showed that the conservation of known intron-defining RNA hairpins in HAC1 extends to Fungi and Metazoa. Concurrently, we identified with high confidence those species that have lost the mechanism of this unconventional splicing. Thirdly, we investigated rates and mechanisms of intron loss within the whole Saccharomycetaceae family in order to develop our findings on the conservation of introns with RNA structures within the context of yeast evolution on both the species and clade level. Computational intron prediction supplemented by RNAseq data from four yeast species demonstrated that both intron loss and conservation of intronic ncRNAs were prevalent in yeast species, and that these patterns have been shaped by whole genome duplication. Lastly, we hypothesise that intron loss in recent yeast evolutionary history has been promoted by double strand break repair machinery.

Indiana University-Purdue University Indianapolis (IUPUI) / Alternative splicing is a regulatory mechanism that generates multiple mRNA transcripts from a single gene, allowing significant expansion in proteome diversity. Disruption of splicing mechanisms has a large impact on the transcriptome and is a significant driver of complex diseases by producing condition-specific transcripts. Recent studies have reported that mis-spliced RNA transcripts can be another major source of neoantigens directly associated with immune responses. Particularly, aberrant peptides derived from unspliced introns can be presented by the major histocompatibility complex (MHC) class I molecules on the cell surface and elicit immunogenicity. In this dissertation, we first developed an integrated computational pipeline for identifying IR-induced neoantigens (IR-neoAg) from RNA sequencing (RNA-Seq) data. Our workflow also included a random forest classifier for prioritizing the neoepitopes with the highest likelihood to induce a T cell response. Second, we analyzed IR neoantigen using RNA-Seq data for multiple myeloma patients from the MMRF study. Our results suggested that the IR-neoAg load could serve as a prognosis biomarker, and immunosuppression in the myeloma microenvironment might offset the increasing neoantigen load effect. Thirdly, we demonstrated that high IR-neoAg predicts better overall survival in TCGA pancreatic cancer patients. Moreover, our results indicated the IR-neoAg load might be useful in identifying pancreatic cancer patients who might benefit from immune checkpoint blockade (ICB) therapy. Finally, we explored the association of IR-induced neo-peptides with neurodegeneration disease pathology and susceptibility. In conclusion, we presented a state-of-art computational solution for identifying IR-neoAgs, which might aid neoantigen-based vaccine development and the prediction of patient immunotherapy responses. Our studies provide remarkable insights into the roles of alternative splicing in complex diseases by directly mediating immune responses. / 2023-08-16

Group II introns are highly structured RNAs that carry out self-splicing reactions. The multiple turnover version of one of these introns, termed the D135 ribozyme, is derived from the mitochondrial aI5γ intron of Saccharomyces cerevisiae and is widely studied as a model RNA for group II intron folding. An important current goal is to probe global changes during its folding with or without DEAD-box chaperone proteins. My initial experiments to study global compaction using small angle X-ray scattering (SAXS) of D135 reveal rapid initial compaction. Unexpectedly, slower increases in Rg value and forward scattering were observed and shown to result from dimerization of the ribozyme. Dimerization was also observed with native electrophoretic mobility shift assays. Here, I have characterized the dimerization process at various conditions. Dimerization requires Mg2+, with similar concentration dependence as tertiary folding, and the dimer is efficiently disrupted by the ATP-dependent activity of DEAD-box proteins. Dimerization does not affect ribozyme catalysis, as both the monomer and the dimer are shown to be fully active. Further experiments showed that dimerization results from duplex formation by an artificial 3’ tail that has extensive self-complementarity, as the deletion of this tail ablates dimerization. Constructs lacking this artificial 3’ tail are likely to simplify further study of the folding process of this ribozyme. / text

Despite decades of effort by molecular systematists, the trees of life of eukaryotic organisms still remain partly unresolved or in conflict with each other. An ever increasing number of fully-sequenced genomes of various eukaryotes allows to consider gene and species phylogenies at genome-scale. However, such phylogenomics-based approaches also revealed that more taxa and more and more gene sequences are not the ultimate solution to fully resolve these conflicts, and that there is a need for sequence-independent phylogenetic meta-characters that are derived from genome sequences. Spliceosomal introns are characteristic features of eukaryotic nuclear genomes. The relatively rare changes of spliceosomal intron positions have already been used as genome-level markers, both for the estimation of intron evolution and phylogenies, however with variable success. In this thesis, a specific subset of these changes is introduced and established as a novel phylogenetic marker, termed near intron pair (NIP). These characters are inferred from homologous genes that contain mutually-exclusive intron presences at pairs of coding sequence (CDS) positions in close proximity. The idea that NIPs are powerful characters is based on the assumption that both very small exons and multiple intron gains at the same position are rare. To obtain sufficient numbers of NIP character data from genomic and alignment data sets in a consistent and flexible way, the implementation of a computational pipeline was a main goal of this work. Starting from orthologous (or more general: homologous) gene datasets comprising genomic sequences and corresponding CDS transcript annotations, the multiple alignment generation is an integral part of this pipeline. The alignment can be calculated at the amino acid level utilizing external tools (e.g. transAlign) and results in a codon alignment via back-translation. Guided by the multiple alignment, the positionally homologous intron positions should become apparent when mapped individually for each transcript. The pipeline proceeds at this stage to output portions of the intron-annotated alignment that contain at least one candidate of a NIP character. In a subsequent pipeline script, these collected so-called NIP region files are finally converted to binary state characters representing valid NIPs in dependence of quality filter constraints concerning, e.g., the amino acid alignment conservation around intron loci and splice sites, to name a few. The computational pipeline tools provide the researcher to elaborate on NIP character matrices that can be used for tree inference, e.g., using the maximum parsimony approach. In a first NIP-based application, the phylogenetic position of major orders of holometabolic insects (more specifically: the Coleoptera-Hymenoptera-Mecopterida trifurcation) was evaluated in a cladistic sense. As already suggested during a study on the eIF2gamma gene based on two NIP cases (Krauss et al. 2005), the genome-scale evaluation supported Hymenoptera as sister group to an assemblage of Coleoptera and Mecopterida, in agreement with other studies, but contradicting the previously established view. As part of the genome paper describing a new species of twisted-wing parasites (Strepsiptera), the NIP method was employed to help to resolve the phylogenetic position of them within (holometabolic) insects. Together with analyses of sequence patterns and a further meta-character, it revealed twisted-wing parasites as being the closest relatives of the mega-diverse beetles. NIP-based reconstructions of the metazoan tree covering a broad selection of representative animal species also identified some weaknesses of the NIP approach that may suffer e.g. from alignment/ortholog prediction artifacts (depending on the depth of range of taxa) and systematic biases (long branch attraction artifacts, due to unequal evolutionary rates of intron gain/loss and the use of the maximum parsimony method). In a further study, the identification of NIPs within the recently diverged genus Drosophila could be utilized to characterize recent intron gain events that apparently involved several cases of intron sliding and tandem exon duplication, albeit the mechanisms of gain for the majority of cases could not be elucidated. Finally, the NIP marker could be established as a novel phylogenetic marker, in particular dedicated to complementarily explore the wealth of genome data for phylogenetic purposes and to address open questions of intron evolution.

Group II introns are a class of mobile ribozymes found in bacteria and eukaryotic organelles that self-splice from precursor RNAs. The resulting lariat intron RNA can then insert into new genomic DNA sites through a reverse splicing reaction. Collectively, this process of intron mobility is termed “retrohoming.” Mobile group II introns encode a reverse transcriptase (RT) that stabilizes the catalytically active form of the intron RNA for both the forward and reverse splicing reactions and also converts the integrated intron RNA into DNA. This work aims to elucidate the structure of bacterial group II intron-encoded RTs and ultimately determine how they function in intron mobility. Although efforts to crystallize group II introns RTs have been unsuccessful, small angle X-ray scattering studies in conjunction with homology modeling have provided new insights into the structure and function of these enzymes. / text

A subset of mitochondrial group II introns of flowering plants has, over evolutionary time, lost characteristic features and employs unconventional splicing pathways. Given the potential impact of cold treatment on RNA folding, as well as on enzymatic activity and import of nuclear-encoded splicing machinery, I have examined the physical excised forms of aberrant introns from wheat embryos subjected to 4oC. My findings suggest a shift in biochemistry with cold treatment to novel splicing pathways that generate heterogeneous in vivo circularized forms for nad1 intron 2, nad2 intron 1 and the cox2 intron, in contrast to predominantly linear excised intron forms at room temperature. Interestingly, the highly degenerate nad1 intron 1, which due to DNA rearrangement has been broken into two halves that interact for splicing in trans, is excised exclusively by first-step hydrolysis at room temperature and under cold treatment. In this case, splicing culminates in two distinct linear half introns that appears correlated with an unusual 5’ terminal insert. This represents the first in vivo demonstration of hydrolytic trans-splicing. Based on northern analysis, cold treatment was further associated with reduced splicing efficiency for all introns surveyed. Moreover, study of precursor transcripts of the nad1a-intron 1a locus suggests the efficiency of end-maturation, including processing of the cotranscribed tRNA-Pro gene, is also reduced in the cold. My findings demonstrate a temperature-sensitivity of transcript maturation, particularly for RNA splicing, providing new insight into the impact of cold growth conditions on plant mitochondrial gene expression.