O sistema NADPH oxidase humano é responsável pela geração de reativos intermediários do oxigênio e defeitos neste sistema resultam na Doença Granulomatosa Crônica (DGC). Nesta tese de doutorado, investigamos o efeito do IFN-g sobre eventos pós-transcricionais em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. Os dados obtidos sugerem que o uso do IFN-g in vitro interfere no processamento da mensagem causando aumento da expressão de transcritos do gene CYBB e NCF1 em células B-EBV de indivíduos sadios e pacientes DGC analisados. Observamos também que o IFN-g dimunui a expressão dos genes THOC4 NONO, SF3A1, SRRM1 e UPF3A e promove aumento de expressão de SRSF10, SNRPA1 e C2 em células B-EBV de paciente X-DGC secundária a defeitos de splicing. Identificamos que o IFN-g e o TNF-α aumentam a expressão das proteínas envolvidas no processo do splicing. Concluímos que o IFN-g aumenta a expressão de genes importantes para uma resposta eficiente do sistema imunológico, incluindo os do sistema NADPH oxidase, além de promover aumento da expressão de genes e de proteínas relacionados ao processo de splicing, que podem estar relacionados aos efeitos benéficos observados no uso do IFN-g em pacientes com DGC ligada ao X, ocasionada por defeitos de splicing. / The human phagocyte NADPH oxidase is responsible for the generation of reactive oxygen intermediates and defects in this system result in Chronic Granulomatous Disease (CGD). In this PhD Thesis, we investigated the effect of IFN-g on post-transcriptional events in normal individuals and patients with X-linked CGD, caused by splicing defects. The obtained data suggests that the use of IFN-g in vitro interferes in the message processing causing an increase of expression of CYBB and NCF1 gene transcripts in B-EBV cells of healthy individuals and analyzed CGD patients. We also observed that IFN-g decreases the expression of THOC4, NONO, SF3A1, SRRM1 and UPF3A, and increases the expression of SRSF10, SNRPA1 and C2 genes in cells from X-CGD patients, due to splicing defects. We identified that IFN-g and TNF-α induce expression of proteins involved in the splicing process. We conclude that IFN-g increases the expression of important genes for an effective immune response, including the NADPH oxidase system genes, and promotes augment of gene and protein expression related to the splicing process, which may be related to the beneficial effects related to the use of IFN-g in CGD patient caused by splicing defects.

O gene do Retardo Mental do X Frágil (FMR1) possui 17 éxons e seu transcrito primário pode sofrer splicing alternativo, havendo, entre outros eventos, possibilidade de exclusão ou inclusão do éxon 12. O produto da expressão do FMR1, a proteína do retardo mental do X frágil (FMRP), possui papéis importantes no sistema nervoso central, atuando como repressora da tradução de RNAm em espinhas dendríticas e controlando a síntese de proteínas envolvidas na função sináptica. Entre dois domínios centrais do tipo KH presentes na FMRP, o segundo (KH-2) é responsável pela interação da proteína aos polissomos. O domínio KH-2 é codificado pelos éxons 9 a 13 do FMR1 e possui a alça variável mais longa já observada entre proteínas humanas, que é codificada pelos éxons 11 e 12. A inclusão do éxon 12 no RNAm do FMR1 causa uma extensão em fase dessa alça variável do KH-2 da FMRP. Estas isoformas apresentam expressão significativa em neurônios cortico-cerebrais e cerebelares do rato, no primeiro mês pós-natal. Este trabalho baseia-se em resultados prévios do grupo de pesquisa, em que se identificaram sequências curtas no íntron 12 do FMR1, com potencial para agir como acentuadores de splicing. Baseando-nos na hipótese de que essas sequências constituem elementos transcritos que se ligam a fatores proteicos do núcleo celular, potencialmente reguladores do splicing do pré-RNAm do FMR1, realizamos ensaios de precipitação por afinidade com extratos nucleares de córtex cerebral de rato e transcritos do loco, biotinilados. Análises por espectrometria de massas revelaram enriquecimento de proteínas nucleares, contendo domínios de ligação a RNA, principalmente aquelas relacionadas à regulação e processamento de pré-RNAm, sobretudo o splicing / Fragile X Mental Retardation 1 gene (FMR1) comprises 17 exons. Its primary transcript is subject to alternative splicing, allowing for the possibility of exon 12 inclusion or skipping, among other events. The product of FMR1 gene expression, fragile X mental retardation protein (FMRP), has important roles in the central nervous system, acting as a translational repressor in dendritic spines, thus controlling the synthesis of proteins involved in synaptic function. FMRP has two central KH domains. One of them (KH-2) is responsible for its interaction with polysomes. The KH-2 domain is encoded by FMR1 exons 9 to 13. It contains the longest variable loop ever observed among human KH-containing proteins, which is encoded by FMR1 exons 11 and 12. Exon-12 inclusion in FMR1 mRNA causes an in-frame extension of FMRP KH-2 domain variable loop. These isoforms appear significantly expressed in cortico-cerebral and cerebellar neurons of the rat in the first month after birth. We have previously identified short sequences within FMR1 intron 12 that may potentially act as splicing enhancers. Our study is based on the hypothesis that those sequences when transcribed should bind to nuclear protein factors that may function as FMR1 exon 12 pre-mRNA splicing regulators. To initiate an experimental approach to test that hypothesis, we conducted affinity precipitation assays with rat cerebral cortex nuclear extracts and biotinylated transcripts. Mass spectrometry analyses disclosed proteins that have been described to be enriched in the cell nucleus, contain RNA-binding domains, and be functionally related to pre-mRNA processing, notably splicing

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Carboxylesterases (CES) are enzymes that metabolize a wide variety of compounds including esters, thioesters, carbamates, and amides. In humans there are three known carboxylesterase genes CES1, CES2, and CES3. Irinotecan (CPT-11) and capecitabine are important chemotherapeutic prodrugs that are used for the treatment of colorectal cancer. Of the three CES isoenzymes, CES2 has the highest catalytic efficiency for irinotecan activation. There is large inter-individual variation in response to treatment with irinotecan. Life-threatening late-onset diarrhea has been reported in approximately 13% of patients receiving irinotecan. Several studies have reported single nucleotide polymorphisms (SNPs) for the CES2 gene. However, there has been no consensus on the effect of different CES2 SNPs and their relationship to CES2 RNA expression or irinotecan hydrolase activity. Three CES2 mRNA transcripts of approximately 2kb,3kb, and 4kb have been identified by multi-tissue northern analysis. The expressed sequence tag (EST) database indicates that CES2 undergoes several splicing events that could generate up to six potential proteins. Four of the proteins CES2, CES2458-473, CES2+64, CES21-93 were studied to characterize their expression and activity. Multi-tissue northern analysis revealed that CES2+64 corresponds to the 4kb and 3kb transcripts while CES21-93 is located only in the 4 kb transcript. CES2458-473 is an inactive splice variant that accounts for approximately 6% of the CES2 transcripts in normal and tumor colon tissue. There is large inter-individual variation in CES2 expression in both tumor and normal colon samples. Characterization of CES2+64 identified the protein as normal CES2 indicating that the signal peptide is recognized in spite of the additional 64 amino acids at the N-terminus. Sub-cellular localization studies revealed that CES2 and CES2+64 localize to the ER, and CES21-93 localizes to the cytoplasm. To date CES2 SNP data has not provided any explanation for the high inter-individual variability in response to irinotecan treatment. Multi-tissue northern blots indicate that CES2 is expressed in a tissue specific manner. We have identified the CES2 variants which correspond to each mRNA transcript. This information will be critical to defining the role of CES2 variants in the different tissues.

No description available.

[ES] La introducción de la secuenciación de ARN a nivel de célula única (scRNA-seq) en el ámbito de la transcriptómica ha redefinido nuestro entendimiento de la diversidad celular, arrojando luz sobre los mecanismos subyacentes a la heterogeneidad tisular. No obstante, al inicio de esta tesis, las limitaciones de a esta tecnología obstaculizaban su aplicación en el estudio de procesos complejos, entre ellos el splicing alternativo. A pesar de ello, los patrones de splicing a nivel celular planteaban incógnitas que esta tecnología tenía el potencial de resolver: ¿es posible observar, a nivel celular, la misma diversidad de isoformas que se detecta mediante RNA-seq a nivel de tejido? ¿Qué función desempeñan las isoformas alternativas en la constitución de la identidad celular? El objetivo de esta tesis es desbloquear el potencial del scRNA-seq para el análisis de isoformas, abordando sus dificultades técnicas y analíticas mediante el desarrollo de nuevas metodologías computacionales. Para lograrlo, se trazó una hoja de ruta con tres objetivos. Primero, se establecieron cuatro requisitos para el estudio de las isoformas mediante scRNA-seq, llevando a cabo una revisión de la literatura existente para evaluar su cumplimiento. Tras completar este marco con simulaciones computacionales, se identificaron las debilidades y fortalezas de los métodos de scRNA-seq y las herramientas computacionales disponibles. Durante la segunda etapa de la investigación, estos conocimientos se utilizaron para diseñar un protocolo óptimo de procesamiento de datos de scRNA-seq. En concreto, se integraron datos de lecturas largas a nivel de tejido con datos de scRNA-seq para garantizar una identificación adecuada de las isoformas así como su cuantificación a nivel celular. Este proceso permitió ampliar las estrategias computacionales disponibles para la reconstrucción de transcriptomas a partir de lecturas largas, mejoras que fueron implementadas en SQANTI3, software de referencia en transcriptómica. Por último, los datos procesados se utilizaron para desarrollar un nuevo método de análisis de co-expresión de isoformas a fin de desentrañar redes de regulación del splicing alternativo implicadas en la constitución de la identidad celular. Dada la elevada variabilidad de los datos de scRNA-seq, este método se basa en la utilización de una estrategia de correlación basada en percentiles que atenúa el ruido técnico y permite la identificación de grupos de isoformas co-expresadas. Una vez configurada la red de co-expresión, se introdujo una nueva estrategia de análisis para la detección de patrones de co-utilización de isoformas que suceden de forma independiente a la expresión a nivel de gen, denominada co-Differential Isoform Usage. Este enfoque facilita la identificación de una capa de regulación de la identidad celular atribuible únicamente a mecanismos post-transcripcionales. Para una interpretación biológica más profunda, se aplicó una estrategia de anotación computacional de motivos y dominios funcionales en las isoformas definidas con lecturas largas, revelando las propiedades biológicas de las isoformas involucradas en la red de co-expresión. Estas investigaciones culminan en el lanzamiento de acorde, un paquete de R que encapsula las diferentes metodologías desarrolladas en esta tesis, potenciando la reproducibilidad de sus resultados y proporcionando una nueva herramienta para explorar la biología de las isoformas alternativas a nivel de célula única. En resumen, esta tesis describe una serie de esfuerzos destinados a desbloquear el potencial de los datos de scRNA-seq para avanzar en la comprensión del splicing alternativo. Desde un contexto de escasez de herramientas y conocimiento previo, se han desarrollado soluciones de análisis innovadoras que permiten la aplicación de scRNA-seq al estudio de las isoformas alternativas, proporcionando recursos innovadores para profundizar en la regulación post-transcripcional y la función celular. / [CA] La introducció de la seqüenciació d'ARN a escala de cèl·lula única (scRNA-seq) en l'àmbit de la transcriptòmica ha redefinit el nostre enteniment de la diversitat cel·lular, projectant llum sobre els mecanismes subjacents a l'heterogeneïtat tissular. Malgrat les limitacions inicials d'aquesta tecnologia, especialment en el context de processos complexos com l'splicing alternatiu, els patrons d'splicing a escala cel·lular plantejaven incògnites amb potencial de resolució: és possible observar, a escala cel·lular, la mateixa diversitat d'isoformes que es detecta mitjançant RNA-seq en teixits? Quina funció tenen les isoformes alternatives en la constitució de la identitat cel·lular? L'objectiu d'aquesta tesi és desbloquejar el potencial del scRNA-seq per a l'anàlisi d'isoformes alternatives, abordant les seues dificultats tècniques i analítiques amb noves metodologies computacionals. Per a això, es va traçar una ruta amb tres objectius. Primerament, es van establir quatre requisits per a l'estudi de les isoformes mitjançant scRNA-seq, amb una revisió de la literatura existent per avaluar-ne el compliment. Després de completar aquest marc amb simulacions computacionals, es van identificar les debilitats i fortaleses dels mètodes de scRNA-seq i de les eines computacionals disponibles. Durant la segona etapa de la investigació, aquests coneixements es van utilitzar per dissenyar un protocol òptim de processament de dades de scRNA-seq. En concret, es van integrar dades de lectures llargues a escala de teixit amb dades de scRNA-seq per a garantir una identificació adequada de les isoformes així com la seua quantificació a escala cel·lular. Aquest procés va permetre ampliar les estratègies computacionals disponibles per a la reconstrucció de transcriptomes a partir de lectures llargues, millores que van ser implementades en SQANTI3, un programari de referència en transcriptòmica. Finalment, les dades processades es van fer servir per a desenvolupar un nou mètode d'anàlisi de coexpressió d'isoformes amb l'objectiu de desentranyar xarxes de regulació de l'splicing alternatiu implicades en la constitució de la identitat cel·lular. Donada l'elevada variabilitat de les dades de scRNA-seq, aquest mètode es basa en la utilització d'una estratègia de correlació basada en percentils que minimitza el soroll tècnic i permet la identificació de grups d'isoformes coexpressades. Un cop configurada la xarxa de coexpressió, es va introduir una nova estratègia d'anàlisi per a la detecció de patrons de co-utilització d'isoformes que succeeixen de forma independent a l'expressió del seu gen, denominada co-Differential Isoform Usage. Aquest enfocament facilita la identificació d'una capa de regulació de la identitat cel·lular atribuïble únicament a mecanismes post-transcripcionals. Per a una interpretació biològica més profunda, es va aplicar una estratègia d'anotació computacional de motius i dominis funcionals en les isoformes definides amb lectures llargues, revelant les propietats biològiques de les isoformes involucrades en la xarxa de coexpressió. Aquestes investigacions culminen en el llançament d'acorde, un paquet de R que encapsula les diferents metodologies desenvolupades en aquesta tesi, potenciant la reproducibilitat dels seus resultats i proporcionant una nova eina per a explorar la biologia de les isoformes alternatives a escala de cèl·lula única. En resum, aquesta tesi descriu una sèrie d'esforços destinats a desbloquejar el potencial de les dades de scRNA-seq per a avançar en la comprensió de l'splicing alternatiu. Des d'un context de manca d'eines i coneixement previ, s'han desenvolupat solucions d'anàlisi innovadores que permeten l'aplicació de scRNA-seq a l'estudi de les isoformes alternatives, proporcionant recursos innovadors per a aprofundir en la regulació post-transcripcional i la funció cel·lular. / [EN] In the world of transcriptomics, the emergence of single-cell RNA sequencing (scRNA-seq) ignited a revolution in our understanding of cellular diversity, unraveling novel mechanisms in tissue heterogeneity, development and disease. However, when this thesis began, using scRNA-seq to understand Alternative Splicing (AS) was a challenging frontier due the inherent limitations of the technology. In spite of this research gap, pertinent questions persisted regarding cell-level AS patterns, particularly concerning the recapitulation of isoform diversity observed in bulk RNA-seq data at the cellular level and the roles played by cell and cell type-specific isoforms. The work conducted in the present thesis aims to harness the potential of scRNA-seq for alternative isoform analysis, outlining technical and analytical challenges and designing computational methods to overcome them. To achieve this, we established a roadmap with three main aims. First, we set requirements for studying isoforms using scRNA-seq and conducted an extensive review of existing research, interrogating whether these requirements were met. Combining this acquired knowledge with several computational simulations allowed us to delineate the strengths and pitfalls of available data generation methods and computational tools. During the second research stage, this insight was used to design a suitable data processing pipeline, in which we jointly employed bulk long-read and short-read scRNA-seq sequenced from full-length cDNAs to ensure adequate isoform reconstruction as well as sensitive cell-level isoform quantification. Additionally, we refined available transcriptome curation strategies, introducing them as innovative modules in the transcriptome quality control software SQANTI3. Lastly, we harnessed single-cell isoform expression data and the rich biological diversity inherent in scRNA-seq, encompassing various cell types, in the design of a novel isoform co-expression analysis method. Percentile correlations effectively mitigated single-cell noise, unveiling clusters of co-expressed isoforms and exposing a layer of regulation in cellular identity that operated independently of gene expression. We additionally introduced co-Differential Isoform Usage (coDIU) analysis, enhancing our ability to interpret isoform cluster networks. This endeavour, combined with the computational annotation of functional sites and domains in the long read-defined isoform models, unearthed a distinctive functional signature in coDIU genes. This research effort materialized in the release of acorde, an R package that encapsulates all analyses functionalities developed throughout this thesis, providing a reproducible means for the scientific community to further explore the depths of alternative isoform biology within single-cell transcriptomics. This thesis describes a complex journey aimed at unlocking the potential of scRNA-seq data for investigating AS and isoforms: from a landscape marked by the scarcity of tools and guidelines, towards the development of novel analysis solutions and the acquisition of valuable biological insight. In a swiftly evolving field, our methodological contributions constitute a significant leap forward in the application of scRNA-seq to the study of alternative isoform expression, providing innovative resources for delving deeper into the intricacies of post-transcriptional regulation and cellular function through the lens of single-cell transcriptomics. / The research project was funded by the BIO2015-71658 and BES-2016-076994 grants awarded by the Spanish Ministry of Science and Innovation / Arzalluz Luque, Á. (2024). Understanding Isoform Expression and Alternative Splicing Biology through Single-Cell RNAseq [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203888

Doctor of Philosophy / Department of Physics / Kristan L. Corwin / Optical frequency references have been widely used in applications such as navigation, remote sensing, and telecommunication industry. For stable frequency references in the near-infrared (NIR), lasers can be locked to narrow absorption features in gases such as acetylene. Currently, most Near NIR references are realized in free space setups. In this thesis, a low-loss hollow-core optical fiber with a diameter of sub millimeters is integrated into the reference setup to provide long interaction lengths between the filling gas and the laser field, also facilitate the optical interaction with low power levels. To make portable NIR reference, gas can be sealed inside the hollow-core fiber, by creating a photonic microcell. This work has demonstrated all-fiber optical frequency references in the Near IR by fabricating and integrating gas sealed photonic microcells in the reference setup. Also, a thoughtful study regarding the lineshape of the fiber-based reference has been accomplished. According the proper modeling of a shift due to lineshape, a correction was applied to our previous absolute frequency measurement of an NIR optical frequency reference. Furthermore, effects of the hollow-core fibers, including mode-dependence frequency shift related to surface modes are explored. In addition, angle splicing techniques, which will improve the performance of the fiber-based frequency reference have been created. Low transmission and return loss angle splices of photonic bandgap fiber, single mode PCF, and large core kagome to SMF-28 are developed and those fibers are demonstrated to be promising for photonic microcell based optical frequency references. Finally, a potentially portable optical metrology system is demonstrated by stabilizing a fiber-laser based frequency comb to an acetylene-filled optical fiber frequency reference. Further work is necessary to fabricate an all-fiber portable optical metrology system with high optical transmission and low molecular contamination.

Résumé : INTRODUCTION : Les protéines doivent se retrouver dans le bon compartiment cellulaire afin de pouvoir exercer leurs fonctions. Une fois synthétisées dans le cytoplasme, les protéines se déplacent à travers les différents compartiments cellulaires, guidés par des signaux de ciblage protéique. Des modifications post-traductionnelles jouent un rôle dans la régulation et le contrôle dynamique de la localisation des protéines. Des exemples dans la littérature indiquent que cette régulation existe également au niveau pré-traductionnel. Ce projet cherche à investiguer cette régulation pré-traductionnelle sur les motifs d’import et d’export nucléaires choisi comme signaux modèles. A l’aide de la bio-informatique, nous caractérisons cette régulation pré-traductionnelle, pour ensuite analyser de façon quantitative le niveau du contrôle de l’inclusion de ces motifs dans les transcrits en considérant des données de RNA-seq. MÉTHODE : Des données du transcriptome humain ont été regroupées dans une base de données locale MySQL. Une curation extensive de bases de données publiques a permis d’identifier les motifs d’import et d’export nucléaires, validés expérimentalement. Des scripts dans les langages Python et PHP ont été créés afin d’interroger la base de données et d’implémenter les algorithmes. Par la suite des ensembles de données de RNA-seq ont été analysés pour quantifier le niveau d’inclusion de ces motifs. RÉSULTATS : La majorité de ces motifs varie pour les gènes dont seulement certaines isoformes contiennent ce motif. Nous avons pu déterminer la distribution de la position des motifs chez l’humain, et caractériser quatre différents types de régulation pré-traductionnelles pour ces motifs. Ces catégories sont : les sites d’initiation et de terminaison différentiels de transcription /traduction, l’épissage (d’introns/d’exons) et le décalage du cadre de lecture. L’index d’inclusion du motif (MII) varie pour les gènes à travers différents tissus d’un même ensemble de données et varie également dans des tissus cancéreux. Certains gènes produisent des isoformes dont le MII est tissu-spécifique. CONCLUSION : La régulation pré-traductionnelle de l’inclusion de motifs de ciblage d’import et d’export nucléaires joue un rôle important sur la localisation de la protéine résultante. Les données de RNA-seq ont abouti à une analyse quantitative sur ces motifs dans différents tissus normaux et cancéreux. / Abstract : INTRODUCTION: Proteins have to be in the right compartment in order to perform their functions. Once synthesized in the cytosol, proteins are guided by sorting signals as they move through different subcellular compartments. Post-translational modifications play a role in the regulation and dynamic control of protein localization. Some examples in the literature show that this regulation also exist at the pre-translational level. The purpose of this research is to investigate this regulation on nuclear import and export motifs, chosen as model signals. Using bioinformatics, we characterize this pre-translational regulation then using RNA-seq data, perform a quantitative analysis on the level of motif inclusion among transcripts. METHODS: Data of the human transcriptome was put in MySQL in house. For this study, we used experimental data. Extensive manual curation was performed on nuclear import and export motifs that were experimentally validated and obtained from public databases. In order to interrogate the database and implement the algorithms, scripts in Python and PHP were used. RNA-seq data were used and analyzed in order to quantify the level of inclusion of these motifs. RESULTS: The majority of these motifs are only present in a subset of the coding isoforms of the genes. The position distribution of these motifs in the human proteome was determined. We characterized four different types of pre-translational regulation for alternative motifs. The categories a re: differential initiation and termination sites of transcription/translation, splicing (of introns/exons) and frameshift mutation. Genes have a motif inclusion index (MII) that varies among different types of tissues within the same dataset and varies as well in cancer tissues. Some genes produce isoforms with MII that are tissue-specific. CONCLUSION: Pre-translational regulation plays an important role in the inclusion of nuclear import and export motifs and the localization of proteins containing them. Quantitative analyses showing the behaviour of the motifs in different types of normal and cancer tissues were performed with RNA-seq data.

No description available.

Group I introns are catalytic RNAs with the ability to splice out of RNA transcripts, often without the aid of proteins. These self-splicing introns have been reengineered to create ribozymes with the ability to catalyze reactions. One such ribozyme, derived from a Pneumocystis carinii group I intron, has been engineered to sequence specifically remove a targeted segment from within an RNA substrate, which is called the trans excision-splicing reaction.The two catalytic steps of the trans excision-splicing reaction occur at positions on the substrate known as the 5' and 3' splice sites. Strict sequence requirements at these sites could potentially limit the target choices for the trans excision-splicing ribozyme, so the sixteen possible base pair combinations at the 5' splice site and the four possible nucleotides at the 3' splice site were tested for reactivity. All base pair combinations at the 5' splice site allow the first reaction step (5' hydrolysis) to occur and several combinations allow the second step to occur, resulting in trans excision-splicing product formation. Moreover, we found that non-Watson-Crick base pairs are important for 5' splice site recognition and prevent product degradation via hydrolysis at other sequence positions. The sequence requirement at the 3' splice site is absolute, as guanosine alone produced complete product.To date, the experiments with the trans excision-splicing ribozyme have been conducted in vitro. The further development of this ribozyme as a biochemical tool and as a potential therapeutic agent requires in vivo reactivity. Thus, a prokaryotic system was designed and tested to assess the catalytic potential of the trans excision-splicing ribozyme. We show that the ribozyme successfully excised a single, targeted nucleotide from a mutated green fluorescent protein transcript in Escherichia coli. On average, 12% correction was observed as measured by fluorescence and approximately 1.2% correction was confirmed through sequence analysis of isolated transcripts.We have used these studies to further characterize trans excision-splicing ribozymes in vitro and to pave the way for future development of this ribozymereaction in vivo. These results increase our understanding of this ribozyme and advance this reaction as a biochemical tool with potential therapeutic applications.