Saccharomyces cerevisiae is an intron-poor organism with introns present in only 5% of its genes. The most prominent group of intron-containing genes are ribosomal protein (RP) genes. They are highly expressed and most of them are present as two paralogs. Parenteau et al. described the existence of intron- dependent intergenic regulatory circuits controlling expression ratios of RP paralogs. In this project, we did not confirm the regulation in 6 out of 7 tested regulatory circuits. We validated the regulation between RPL22 paralogs. We further showed that Rpl22 protein blocks the pre-mRNA splicing of both paralogs, with RPL22B paralog being more sensitive. Rpl22 protein binds to the stem-loop of RPL22B intron - disruption of the binding domain of Rpl22 proteins leads to loss of interaction. Moreover, the regulation seems to be working the same way in yeast Kluyveromyces lactis, which has only a single RPL22 copy. Overall, these results lead to better understanding of intergenic regulation, which adjusts the expression ratio between functionally different RPL22 paralogs. Key words introns, ribosomal protein genes, Rpl22, RPL22 paralogs, pre-mRNA splicing, Saccharomyces cerevisiae

Recentemente, tem sido demonstrado que a maioria dos RNAs transcritos em células humanas são RNAs não-codificadores de proteínas (ncRNAs) originados de íntrons ou regiões intergênicas. Em trabalhos anteriores realizados por nosso grupo, foram descritos longos ncRNAs transcritos de regiões intrônicas de genes codificadores e cuja expressão foi correlacionada ao grau de diferenciação de tumores de próstata, apontando para a relevância fisiológica desta classe de transcritos. Apesar de sua abundância, as propriedades, funções e regulação da grande maioria dos ncRNAs ainda não foram elucidadas. O objetivo do presente trabalho foi investigar a biossíntese de ncRNAs intrônicos em células humanas, primordialmente a contribuição da RNA Polimerase II (RNAP II), bem como aspectos de sua regulação. Primeiramente, o modelo de regulação da expressão gênica por hormônio andrógeno foi utilizado para avaliação da participação direta de um fator de transcrição de RNAP II, o Receptor de Andrógeno (AR), na modulação da transcrição de ncRNAs intrônicos. Utilizando-se a técnica de imunoprecipitação da cromatina, foi detectada a ligação do AR ao elemento de resposta a andrógeno (ARE) presente em um possível promotor de um transcrito intrônico antisenso (derivado do locus Myo5A), cuja expressão é aumentada em células da linhagem LNCaP tratadas com o hormônio. A ligação ao ARE foi induzida pelo tratamento, sugerindo que o efeito do andrógeno na expressão do ncRNA é mediado pelo AR. Em uma segunda abordagem, o efeito da inibição da transcrição por RNAP II com α-amanitina por 24 h em células LNCaP foi avaliado com o uso de microarranjos de oligonucleotídeos representando transcritos total ou parcialmente intrônicos, além de éxons de genes codificadores. A expressão de menos de 20 % dos transcritos intrônicos foi afetada, fração significativamente menor que a observada para os transcritos exônicos (40 %). Ainda que a maioria dos ncRNAs intrônicos diferencialmente expressos tenha sua abundância diminuída, interessantemente, 13 a 16 % foram aumentados, contrastando com aproximadamente 2 a 3 % de exônicos que aumentaram. Os resultados obtidos neste trabalho indicam que a RNAP II atua na transcrição de ncRNAs intrônicos, mas que uma fração considerável pode ser transcrita por outra RNA Polimerase. / It has been recently shown that the bulk of the transcription in human cells is comprised of non-protein-coding RNAs (or noncoding RNAs - ncRNAs) transcribed from introns and intergenic regions of the genome. Previous work from our group has demonstrated that expression of long intronic ncRNAs can be correlated to the degree of prostate tumor differentiation, underscoring the physiological relevance of these transcripts. However, the properties, functions, and regulation of this huge population of ncRNAs remain largely unknown. The present work aimed to investigate the biosynthesis of intronic ncRNAs and aspects of its regulation in human cells, focusing on the contribution of RNA Polymerase II (RNAP II). Initially, the model of regulation of gene expression by androgen hormone was used in order to evaluate the participation of the RNAP II transcription factor Androgen Receptor (AR) in the transcriptional regulation of intronic ncRNAs. Chromatin immunoprecipitation experiments revealed the binding of the AR in an androgen response element (ARE) present in a putative promoter driving the expression of an antisense intronic transcript in Myo5A locus in LNCaP cells. The interaction occurred in an androgen-inducible fashion, along with the up-regulation of the transcript, suggesting that hormone activation occurred in a direct manner mediated by the AR. In a different approach, the effect of RNAP II inhibition with α-amanitin for 24 h in LNCaP cells was analyzed using an oligoarray representing totally and partially intronic transcripts, as well as exons of proteincoding genes. The expression of less than 20 % of the intronic transcripts was affected by the treatment, contrasting to a significantly higher fraction observed for exonic messages (40 %). Moreover, most differentially expressed intronic transcripts were down-regulated, but strikingly 13 to 16 % were up-regulated in cells with blocked RNAP II, while this fraction for exonic transcripts was about 2 %. The results described here demonstrate that RNAP II in fact plays a role in intronic transcription in human cells, but also highlight that another transcriptional system may account for the biogenesis of a fraction of intronic ncRNAs.

In this collection of papers I have summarized my investigations into the field of evolutionary genetics and more specifically into patterns of biodiversity and evolutionary processes. The lizards (and frogs) studied here share common features in that they are largely present in unique environments, which are also regions that are biologically understudied. Most of these taxa show high degrees of endemism, interesting natural history characteristics, and each group manifests distinctive adaptations of general evolutionary interest. My work in the genus Telmatobius has been a progressive approach that began in my MS program, and it first focused on alpha taxonomy, morphological variation, and species boundaries. This work led to new studies initiated and completed at BYU involving further taxonomic revision (Formas et al., 2003; Chapter 1), and then revisiting and re-evaluating species boundaries established earlier (with allozyme markers) and this time with population level molecular (mitochondrial DNA) markers (Chapter 2). Our results indicate that the striking differences in size, coloration and general appearance in the various Lake Titicaca morphotypes are not genetically based. Further, there is evidence that these morphotypes have evolved very rapidly after demographic bottlenecks eroded present genetic variability. Telmatobius frogs of Lake Titicaca are listed by the International (IUCN) as critically endangered. We support this classification and further suggest studies to explore open questions like the possibility of adaptation along ecological resource gradients. Lizards of the genus Microlophus are interesting but for different reasons, and studies of this group constitutes the bulk of my dissertation work. The genus includes both Galapagos insular species, and continental taxa distributed in a linear gradient along > 4000 km of the western coast of South America. In studying Microlophus I first tackled the unresolved phylogenetic relationships within the genus (Chapter 3) and then pay attention to phylogeographic aspects of the most speciose lizard radiation in the Galapagos Archipelago (Chapter 4). Chapter 3 is a single manuscript provisionally accepted in the journal Systematic Biology. This paper introduces the lizard genus Microlophus (“lava lizards”) as a study system, and includes a large nuclear data set accompanied by an equally large mitochondrial data set (7877 characters in total). This paper explicitly differentiates among sequence alignments of gene regions that vary in tempo and class of mutational events. We show that this recognition is important and we suggest ways to appropriately deal with the alignment of multi-locus non-coding DNA data sets. A secondary finding in this study is that mtDNA and nDNA topologies are discordant with each other but that both are strongly supported, and that the nuclear topology is concordant with species distribution patterns along coastal South America. We hypothesize that in this particular region of the tree, the nuclear genome recovers a topology that is closer to the species tree, and conflicts occur due to likely secondary contact of distantly related taxa, suggesting that unique taxonomic relationships in the mtDNA gene tree are the result of hybridization. This last point highlights the value of dense taxonomic and character sampling for teasing apart different aspects of evolutionary processes. Chapter 4 is a manuscript to be submitted to the journal Evolution; in this study we further investigate the most speciose radiation of Microlophus in the Galapagos, based on an unparalleled sampling of most islands and small islets in the Archipelago. We use mtDNA sequences to both test hypothesized between-island colonization routes, as well as the expectation that within-island phylogeographic structure should be greater on older islands. Our mtDNA gene tree is strongly supported and allows rejection of previous alternatives, and we propose a novel sequence of between-island colonization events. Our results also reject the idea of phylogeographic structure been related solely to island age. Instead, we provide evidence to suggest that active volcanism as a major player in the generation of genetic diversity in within-island environments, and this is further compounded by the seemingly stochastic nature of within-island long-distance colonization routes mediated by ocean currents. We suggest that the direction and intensity of these currents, as currently understood, are insufficient to generate a priori hypotheses of oceanic colonization routes and their influence on gene flow. We do show that the standard stepping-stone model of migration, where genetic interchange is only possible among neighboring localities, does not explain much of the within-island population genetic structure unraveled by this study. From a biological conservation perspective the study of patterns of recent evolutionary history in the Galapagos provides with a window to evolutionary processes that have shaped and continue to impact the generation of biodiversity in the Galapagos Archipelago. Islands have long been viewed as natural laboratories of evolutionary change, and thus all island isolates are or could be distinctly important components of the larger, archipelago-wide processes. We provide working hypotheses for some of the demographic processes that might be generating within- and between-island biodiversity in this clade of lizards; confirmation of these explanations with independent data will have management implications for conserving the unique patterns observed in the Galapagos biota, but also the processes that generated these patterns.

Mobile genetic elements are DNA sequences that move around to different positions within one genome or between different genomes. Mobile DNA elements were initially considered as selfish DNA sequences parasitizing the organism’s genome. However, this view has changed with the discovery of several mobile genetic elements which play important evolutionary and functional roles. Such understanding has led to a new connotation for these genetic elements such as drivers or natural molecular tools of genome evolution. Extensive research over the past several years has also led to the identification of several new mobile genetic elements including transposons, segregation distorters, heritable organisms, introns and inteins. Homing endonucleases (HEnases) are a group of rare cutting site-specific doublestranded DNA endonucleases encoded by open reading frames within introns, inteins or free standing genes in all the three forms of life including viruses. These enzymes confer mobility to themselves and their encoding sequences by a gene conversion event termed “homing”. During the homing process, the endonuclease inflicts a double-strand break at or near the homing site of the intein-/intron-less allele, which is subsequently repaired by the host DNA repair machinery resulting in the inheritance of intein/intron. The first homing endonuclease identified was the Saccharomyces cerevisiae mitochondrial genetic marker ‘ω’, which affects the polarity of recombination. This genetic marker, which was later shown to be a mobile group I intron, was present in the mitochondrial 21S rRNA gene and encodes a homing endonuclease. HEnases are distinguished for being able to recognise long DNA sequences (14-40 bp), and display disparate cleavage mechanisms. Unlike restriction endonucleases, these enzymes tolerate sequence polymorphism in their recognition region which provides a mechanism for increasing their genetic diversity. Substantial efforts are underway to explore the possibility of utilizing HEnases as tools for genome mapping, cloning of megabase DNA fragments and gene targeting. HEnases are divided into five sub-families on the basis of their conserved sequence and structural motifs: LAGLIDADG, GIY-YIG, H-N-H, His-Cys box and PD-(D/E)-XK families. Among these, LAGLIDADG family is the largest, most prevalent and well-studied class of HEnases. Homing enzymes that contain a single copy of LAGLIDADG motif per polypeptide chain, such as ICreI, I-MsoI and I-CeuI function as homodimers and recognize and cleave palindromic and pseudo-palindromic DNA sequences. On the other hand, HEnases that harbour two copies of LAGLIDADG motifs including I-AniI, PI-SceI and I-SceI act as monomers and recognize and cleave their DNA target sites with considerable asymmetry. Eubacterial RecA proteins are important for a number of cellular processes such as homologous recombination, DNA repair, restoration of stalled replication forks and SOS response. RecA protein and the process of homologous recombination, which is the main mechanism of genetic exchange, are evolutionarily conserved among a range of organisms. However, few mycobacterial species such as Mycobacterium tuberculosis and Mycobacterium leprae were found to be an exception as they harboured in-frame insertion of an intein-coding sequence in their recA genes. In these organisms, RecA is synthesized as a large precursor, which undergoes protein splicing resulting in the formation of an intein and functionally active RecA protein. The milieu in which RecA precursor undergoes splicing differs substantially between M. tuberculosis and M. leprae. M. leprae RecA precursor (79 kDa) undergoes splicing only in mycobacterial species, whereas M. tuberculosis RecA precursor (85 kDa) is spliced efficiently in Escherichia coli as well. Intriguingly, M. tuberculosis and M. leprae RecA inteins differ greatly in their size, primary sequence and location within the recA gene, thereby suggesting two independent origins during evolution. The occurrence of inteins in the obligate mycobacterial pathogens M. tuberculosis, M. leprae and M. microti, initially suggested that RecA inteins might play a role in pathogenesis or virulence, however this was found to be not the case due to the subsequent identification of these intervening sequences in several non pathogenic mycobacterial strains. Sequence comparison of RecA inteins suggested that they belong to the LAGLIDADG class of homing endonucleases. Accordingly, we have shown earlier that M. tuberculosis RecA intein (PI-MtuI), is a novel LAGLIDADG homing endonuclease, which displays dual target specificity in the presence of alternative cofactors in an ATP-dependent manner. The genome of M. leprae, a gram positive bacillus reveals that in contrast to the genomes of other mycobacterial species, it has undergone extensive deletions and decay and thereby represents an extreme case of reductive evolution. In such a scenario of massive gene decay and function loss in the leprosy bacillus, and dissimilarities in size and primary structures among mycobacterial RecA inteins, it was of interest to examine whether M. leprae recA intervening sequence can encode a catalytically active homing endonuclease. To this end, the intervening sequence corresponding to M. leprae recA intein was PCR amplified, cloned, overexpressed and purified to homogeneity using IMPACT protocol. The identity of the purified RecA intein was ascertained by sequencing 9 amino acid residues at the N-terminal end and Western blot analysis using anti-PI-MleI antibodies. Purified enzyme was found to be devoid of any contaminating exonuclease. Protein crosslinking experiments using glutaraldehyde suggested that PI-MleI exists in solution as a monomer, consistent with double-motif LAGLIDADG enzymes. To test whether the purified PI-MleI can bind to the DNA and display any DNA-binding specificity, we carried out electrophoretic mobility shift assays with both single-stranded and double-stranded cognate DNA. The enzyme displayed robust binding to cognate doublestranded DNA, compared to the cognate single-stranded DNA. DNA binding was further found to be sequence independent though the presence of the cognate sequence was required for maximal binding. The stability and specificity of PI-MleI-cognate DNA complexes were further examined by salt titration and competition experiments, which indicated high stability and specificity. After establishing the stable binding of recombinant PI-MleI to the cognate duplex DNA, we next investigated its endonuclease activity on the cognate plasmid pMLR containing the intein-less recA allele, in the absence or presence of divalent cations. The cleavage was monitored by the conversion of supercoiled pMLR to nicked circular as well as linear duplex DNA. PI-MleI exhibited both single-stranded nicking and double-stranded DNA cleavage activity. PI- MleI exhibits endonuclease activity both in the presence of Mg2+ or Mn2+ through a two step reaction. PI-MleI mediated cleavage though was found to be divalent cation dependent however was nucleotide cofactor independent, unlike PI-MtuI, which cleaves the cognate DNA substrate in the presence of ATP. PI-MleI endonuclease activity was assayed under different conditions and found to display a broad divalent cation, pH and temperature dependence. The kinetic experiments revealed slow turnover rate of PI-MleI suggesting its weak endonuclease activity in contrast to robust cleavage activity displayed by several other known LAGLIDADG homing endonucleases. An intriguing observation emerged from the cleavage site mapping of PI-MleI at singlenucleotide resolution. PI-MleI displayed a staggered double- strand break in the homing site by nicking in the left flanking sequence 44 to 47 bp and in the right flanking sequence 16 to 25 bp, away from the intein insertion site. Similar cleavage patterns have been earlier observed for few GIY-YIG homing endonucleases. To gain further mechanistic insights into the PI-MleI mediated catalysis, we examined the binding of PI-MleI to the cognate DNA by DNase I and (OP)2 Cu footprinting experiments. Both the footprinting approaches revealed interaction of PI-MleI with a region upstream and downstream of its own insertion site, conferring protection to 16 nucleotide residues on the upper and 12 nucleotide residues on the lower strand, respectively. The asymmetric footprints have been earlier observed for some members of LAGLIDADG-type homing endonucleases wherein protection on the complementary strands was found to be out of register by 2 to 3 nucleotides, respectively. In case of PI-MleI, however the footprint formed on the complementary strands of the homing site is non-overlapping, indicating the asymmetric mode of interaction of the enzyme. Surprisingly, PI-MleI footprint was not evident at the cleavage sites and this could be due to the unstable binding of the intein at these regions. To decipher the interaction of PI-MleI at the cleavage sites and to ascertain if these interactions have any functional implications in terms of alterations in base-pairing positioning or strand separation to mediate DNA catalysis, we probed the structure of PI-MleI-DNA complexes with KMnO4. KMnO4 treatment of PI-MleI-cognate DNA complexes revealed the presence of hypersensitive T residues on both the strands at the cleavage sites, but showed no such reactive T residues within the PI-MleI-binding regions. Also, hyper-sensitive T residues were not seen at or near the intein-insertion site or in the region between binding and cleavage sites suggesting that PI-MleI upon binding its cognate DNA induces distortions selectively at the cleavage region. To validate these findings and to test whether such alterations occurred on all substrate DNA molecules or on a small sub-population of target molecules, we used a more sensitive 2-aminopurine fluorescence approach. To this end, six cognate duplex DNA molecules each containing 2-aminopurine (2-AP) at different positions such as at the insertion site, in the DNAbinding region, at or near to the cleavage sites were synthesized to monitor helical distortions in the target DNA. The 2-AP containing cognate DNA duplexes were incubated with increasing concentrations of PI-MleI in the assay buffer and monitored the changes in 2-AP fluorescence intensity in the spectral region from 330 to 450 nm. Out of the 2-AP placed at several positions within the cognate substrate, only the 2-aminopurines at the cleavage site showed enhanced fluorescence with PI-MleI addition, consistent with the hyper-sensitivity of T residues during KMnO4 probing. The findings suggest that DNA distortion might assist PI-MleI in widening the minor groove at the cleavage site and make the scissile phosphates accessible to the enzyme active site similar to what has been seen with other LAGLIDADG homing enzymes. These observations suggest that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage sites and generates two staggered double-strand breaks. Together, these finding indicate the modular structure of PI-MleI having separate domains for DNA target recognition and cleavage and a bipartite structure of its homing site. After demonstrating the endonuclease activity of PI-MleI, we next examined the active site residues of PI-MleI involved in double-stranded DNA cleavage, which would further provide insights into its catalytic mechanism. Previously, sequence alignment analyses of LAGLIDADG enzymes carried out using different alignment programs identified the presence of 115VLGSLMGDGP123 sequence as DOD motif I (Block C) and 185LQRAVYLGDG194 or 210VLAIWYMDDG219C sequences as catalytic DOD motif II (Block E) in M. leprae RecA intein (PI-MleI). The bioinformatics analyses though on one hand identified the catalytic motifs in PI-MleI, on the other hand led to conflicting data in regard to the identity and the specific position of the catalytic DOD motif II within the PI-MleI polypeptide. We therefore, performed site-directed mutagenesis of key residues in these catalytic motifs and examined their effect on PI-MleI mediated catalysis. A wealth of mutagenesis and structural data, which exists concerning HEnases, suggests that catalytic centers carry essential aspartate residues, one in each of the LAGLIDADG motifs Accordingly, we chose to mutate conserved aspartates that have been previously implicated in catalysis. By site-directed mutagenesis, we constructed five mutant proteins, in which Asp122 was mutated to alanine, cysteine and threonine; whereas Asp193 and Asp218 were mutated to alanine. The identity of each mutant was ascertained by determining the complete nucleotide sequence of the mutant gene. Mutant proteins were further purified to >95% homogeneity using the purification strategy developed for wild type PI-MleI and were found to be devoid of any contaminating exonuclease. To study the effect of mutations in PI-MleI active site residues on its DNA-binding affinity, we examined the binding characteristics of the wild type PI-MleI and its aspartate variants with the intein-less recA substrate and the stability of protein-DNA complexes. All the mutants displayed similar binding affinity to the cognate DNA as that of the wild type PI-MleI, as judged by the comparison of their binding constants (Kd) which were found to be of the same order. Comparison of salt titration isotherms of wild type PI-MleI and its aspartate variants further revealed the similar salt titration midpoint for most of the mutants as that of wild type enzyme suggesting similar protein-DNA complexes stability. Although these results indicate the occurrence of stable complexes between PI-MleI variants and target DNA, to further define the DNA-binding properties of each mutant protein, wild-type PI-MleI and its variants were assayed by DNase I footprinting. All the mutants (D122A, D122C, D122T, D193A and D218A) showed an asymmetric footprint and protection of ~16 nucleotide residues on the upper and 12 nucleotide residues on the lower strand, respectively, near the intein-insertion site similar to the wild type PI-MleI. Together, these observations suggest that the aspartate substitutions in the catalytic motifs do not alter DNA recognition specificity of PI-MleI or its variants, and may not play a direct role in protein-DNA interactions, again implicating the existence of a modular structure of PI-MleI with distinct DNA-binding and catalytic domains. Wild-type PI-MleI although binds near the intein insertion site, but however was found to induce helical distortions only at the cleavage sites. To explore, if aspartate substitutions have any effect on the structural modifications in target DNA sequence, we carried out 2-aminopurine fluorescence with wild type PI-MleI and its variants. In agreement with the wild type enzyme, all the mutants showed increase in fluorescence with target DNA containing 2-AP only at the cleavage sites, but not at the binding sites. However, quantitative measurements of fluorescence change suggested that D122A and D193A mutants show nearly two-fold decrease in the magnitudes of spectral change at the cleavage site compared to wild type and other variants suggesting their involvement in the helical distortion process. To study the effect of Asp substitutions on the catalytic activity of PI-MleI, we performed cleavage assays using cognate plasmid pMLR DNA, with increasing concentrations of wild-type PI-MleI, or its variants and measured the double-stranded cleavage activity. Whereas, D122A and D193A mutants were completely inactive in double-stranded DNA cleavage under the conditions of the cleavage assay, D218A showed DNA cleavage activity comparable to that of the wild type PI-MleI. Similarly, D122T showed decrease in doublestranded DNA cleavage activity. Interestingly, D122C variant showed ~2-fold enhanced DNA cleavage, compared to the wild-type enzyme.Together, these findings provide compelling evidence to conclude that 115VLGSLMGDGP123 and 185LQRAVYLGDG194 motifs (Blocks C and E, respectively), but not 210VLAIWYMDDG219 motif (Block E), and that residues Asp122 and Asp193 play a direct role with respect to the catalytic mechanism of PI-MleI. In summary, these results suggest that the structural and mechanistic aspects of PI-MleI catalysis are distinct from other well-characterized LAGLIDADG-type homing endonucleases and thus provide further insights into understanding the function and evolution of LAGLIDADG homing enzymes.

Lacking experimental data, protein function is often predicted from evolutionary and protein structure theory. Under the 'domain grammar' hypothesis the function of a protein follows from the domains it encodes. Under the 'orthology conjecture', orthologs, related through species formation, are expected to be more functionally similar than paralogs, which are homologs in the same or different species descended from a gene duplication event. However, these assumptions have not thus far been systematically evaluated. To test the 'domain grammar' hypothesis, we built models for predicting function from the domain combinations present in a protein, and demonstrated that multi-domain combinations imply functions that the individual domains do not. We also developed a novel gene-tree based method for reconstructing the evolutionary histories of domain architectures, to search for cases of architectures that have arisen multiple times in parallel, and found this to be more common than previously reported. To test the 'orthology conjecture', we first benchmarked methods for homology inference under the obfuscating influence of low-complexity regions, in order to improve the InParanoid orthology inference algorithm. InParanoid was then used to test the relative conservation of functionally relevant properties between orthologs and paralogs at various evolutionary distances, including intron positions, domain architectures, and Gene Ontology functional annotations. We found an increased conservation of domain architectures in orthologs relative to paralogs, in support of the 'orthology conjecture' and the 'domain grammar' hypotheses acting in tandem. However, equivalent analysis of Gene Ontology functional conservation yielded spurious results, which may be an artifact of species-specific annotation biases in functional annotation databases. I discuss possible ways of circumventing this bias so the 'orthology conjecture' can be tested more conclusively. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 6: Epub ahead of print.

Thesis (M.S.)--Miami University, Dept. of Zoology, 2005. / Title from first page of PDF document. Document formatted into pages; contains [1], ix, 75 p. : ill. Includes bibliographical references (p. 69-75).

Recentemente, tem sido demonstrado que a maioria dos RNAs transcritos em células humanas são RNAs não-codificadores de proteínas (ncRNAs) originados de íntrons ou regiões intergênicas. Em trabalhos anteriores realizados por nosso grupo, foram descritos longos ncRNAs transcritos de regiões intrônicas de genes codificadores e cuja expressão foi correlacionada ao grau de diferenciação de tumores de próstata, apontando para a relevância fisiológica desta classe de transcritos. Apesar de sua abundância, as propriedades, funções e regulação da grande maioria dos ncRNAs ainda não foram elucidadas. O objetivo do presente trabalho foi investigar a biossíntese de ncRNAs intrônicos em células humanas, primordialmente a contribuição da RNA Polimerase II (RNAP II), bem como aspectos de sua regulação. Primeiramente, o modelo de regulação da expressão gênica por hormônio andrógeno foi utilizado para avaliação da participação direta de um fator de transcrição de RNAP II, o Receptor de Andrógeno (AR), na modulação da transcrição de ncRNAs intrônicos. Utilizando-se a técnica de imunoprecipitação da cromatina, foi detectada a ligação do AR ao elemento de resposta a andrógeno (ARE) presente em um possível promotor de um transcrito intrônico antisenso (derivado do locus Myo5A), cuja expressão é aumentada em células da linhagem LNCaP tratadas com o hormônio. A ligação ao ARE foi induzida pelo tratamento, sugerindo que o efeito do andrógeno na expressão do ncRNA é mediado pelo AR. Em uma segunda abordagem, o efeito da inibição da transcrição por RNAP II com α-amanitina por 24 h em células LNCaP foi avaliado com o uso de microarranjos de oligonucleotídeos representando transcritos total ou parcialmente intrônicos, além de éxons de genes codificadores. A expressão de menos de 20 % dos transcritos intrônicos foi afetada, fração significativamente menor que a observada para os transcritos exônicos (40 %). Ainda que a maioria dos ncRNAs intrônicos diferencialmente expressos tenha sua abundância diminuída, interessantemente, 13 a 16 % foram aumentados, contrastando com aproximadamente 2 a 3 % de exônicos que aumentaram. Os resultados obtidos neste trabalho indicam que a RNAP II atua na transcrição de ncRNAs intrônicos, mas que uma fração considerável pode ser transcrita por outra RNA Polimerase. / It has been recently shown that the bulk of the transcription in human cells is comprised of non-protein-coding RNAs (or noncoding RNAs - ncRNAs) transcribed from introns and intergenic regions of the genome. Previous work from our group has demonstrated that expression of long intronic ncRNAs can be correlated to the degree of prostate tumor differentiation, underscoring the physiological relevance of these transcripts. However, the properties, functions, and regulation of this huge population of ncRNAs remain largely unknown. The present work aimed to investigate the biosynthesis of intronic ncRNAs and aspects of its regulation in human cells, focusing on the contribution of RNA Polymerase II (RNAP II). Initially, the model of regulation of gene expression by androgen hormone was used in order to evaluate the participation of the RNAP II transcription factor Androgen Receptor (AR) in the transcriptional regulation of intronic ncRNAs. Chromatin immunoprecipitation experiments revealed the binding of the AR in an androgen response element (ARE) present in a putative promoter driving the expression of an antisense intronic transcript in Myo5A locus in LNCaP cells. The interaction occurred in an androgen-inducible fashion, along with the up-regulation of the transcript, suggesting that hormone activation occurred in a direct manner mediated by the AR. In a different approach, the effect of RNAP II inhibition with α-amanitin for 24 h in LNCaP cells was analyzed using an oligoarray representing totally and partially intronic transcripts, as well as exons of proteincoding genes. The expression of less than 20 % of the intronic transcripts was affected by the treatment, contrasting to a significantly higher fraction observed for exonic messages (40 %). Moreover, most differentially expressed intronic transcripts were down-regulated, but strikingly 13 to 16 % were up-regulated in cells with blocked RNAP II, while this fraction for exonic transcripts was about 2 %. The results described here demonstrate that RNAP II in fact plays a role in intronic transcription in human cells, but also highlight that another transcriptional system may account for the biogenesis of a fraction of intronic ncRNAs.

No description available.

Saccharomyces cerevisiae is an intron-poor organism with introns present in only 5% of its genes. The most prominent group of intron-containing genes are ribosomal protein (RP) genes. They are highly expressed and most of them are present as two paralogs. Parenteau et al. described the existence of intron- dependent intergenic regulatory circuits controlling expression ratios of RP paralogs. In this project, we did not confirm the regulation in 6 out of 7 tested regulatory circuits. We validated the regulation between RPL22 paralogs. We further showed that Rpl22 protein blocks the pre-mRNA splicing of both paralogs, with RPL22B paralog being more sensitive. Rpl22 protein binds to the stem-loop of RPL22B intron - disruption of the binding domain of Rpl22 proteins leads to loss of interaction. Moreover, the regulation seems to be working the same way in yeast Kluyveromyces lactis, which has only a single RPL22 copy. Overall, these results lead to better understanding of intergenic regulation, which adjusts the expression ratio between functionally different RPL22 paralogs. Key words introns, ribosomal protein genes, Rpl22, RPL22 paralogs, pre-mRNA splicing, Saccharomyces cerevisiae

The small ribosomal subunit gene of the Chaetomium thermophilum DSM 1495 is invaded by a nested intron at position mS1247, which is composed of a group I intron encoding a LAGLIDADG open reading frame interrupted by an internal group II intron. The first objective was to examine if splicing of the internal intron could reconstitute the coding regions and facilitate the expression of an active homing endonuclease. Using in vitro transcription assays, the group II intron was shown to self-splice only under high salt concentration. Both in vitro endonuclease and cleavage mapping assays suggested that the nested intron encodes an active homing endonuclease which cleaves near the intron insertion site. This composite arrangement hinted that the group II intron could be regulatory with regards to the expression of the homing endonuclease. Constructs were generated where the codon-optimized open reading frame was interrupted with group IIA1 or IIB introns. The concentration of the magnesium in the media sufficient for splicing was determined by the Reverse Transcriptase-Polymerase Chain Reaction analyses from the bacterial cells grown under various magnesium concentrations. Further, the in vivo endonuclease assay showed that magnesium chloride stimulated the expression of a functional protein but the addition of cobalt chloride to the growth media antagonized the expression. This study showed that the homing endonuclease expression in Escherichia coli can be regulated by manipulating the splicing efficiency of the group II introns which may have implications in genome engineering as potential ‘on/off switch’ for temporal regulation of homing endonuclease expression . Another objective was to characterize native homing endonucleases, cytb.i3ORF and I-OmiI encoded within fungal mitochondrial DNAs, which were difficult to express and purify. For these, an alternative approach was used where two compatible plasmids, HEase.pET28b (+)-kanamycin and substrate.pUC57-chloramphenicol, based on the antibiotic markers were maintained in Escherichia coli BL21 (DE3). The in vivo endonuclease assays demonstrated that these homing endonucleases were able to cleave the substrate plasmids when expressed, leading to the loss of the antibiotic markers and thereby providing an indirect approach to screen for potential active homing endonucleases before one invests effort into optimizing protein overexpression and purification strategies. / October 2016