Visualizing Rare Watson-Crick-Like Tautomeric and Anionic Mismatches in DNA and RNA

The central dogma of molecular biology relies on the correct Watson-Crick (WC) geometry of canonical deoxyribonucleic acid (DNA) dG•dC and dA•dT base pairs to replicate and transcribe genetic information with speed and an astonishing level of fidelity. In addition, the Watson-Crick geometry of canonical ribonucleic acid (RNA) rG•rC and rA•rU base pairs is highly conserved to ensure that proteins are translated with high fidelity. However, numerous other potential nucleobase tautomeric and ionic configurations are possible that can give rise to entirely new pairing modes between the nucleotide bases. Very early on, James Watson and Francis Crick recognized their importance and in 1953 postulated that if bases adopted one of their less energetically disfavored tautomeric forms (and later ionic forms) during replication it could lead to the formation of a mismatch with a Watson-Crick-like geometry and could give rise to “natural mutations.”

Since this time numerous studies have provided evidence in support of this hypothesis and have expanded upon it; computational studies have addressed the energetic feasibilities of different nucleobases’ tautomeric and ionic forms in siico; crystallographic studies have trapped different mismatches with WC-like geometries in polymerase or ribosome active sites. However, no direct evidence has been given for (i) the direct existence of these WC-like mismatches in canonical DNA duplex, RNA duplexes, or non-coding RNAs; (ii) which, if any, tautomeric or ionic form stabilizes the WC-like geometry. This thesis utilizes nuclear magnetic resonance (NMR) spectroscopy and rotating frame relaxation dispersion (R1ρ RD) in combination with density functional theory (DFT), biochemical assays, and targeted chemical perturbations to show that (i) dG•dT mismatches in DNA duplexes, as well as rG•rU mismatches RNA duplexes and non-coding RNAs, transiently adopt a WC-like geometry that is stabilized by (ii) an interconnected network of rapidly interconverting rare tautomers and anionic bases. These results support Watson and Crick’s tautomer hypothesis, but additionally support subsequent hypotheses invoking anionic mismatches and ultimately tie them together. This dissertation shows that a common mismatch can adopt a Watson-Crick-like geometry globally, in both DNA and RNA, and whose geometry is stabilized by a kinetically linked network of rare tautomeric and anionic bases. The studies herein also provide compelling evidence for their involvement in spontaneous replication and translation errors.

Posttranscriptional Regulation of Embryonic Neurogenesis by the Exon Junction Complex

The six-layered neuron structure in the cerebral cortex is the foundation for human mental abilities. In the developing cerebral cortex, neural stem cells undergo proliferation and differentiate into intermediate progenitors and neurons, a process known as embryonic neurogenesis. Disrupted embryonic neurogenesis is the root cause of a wide range of neurodevelopmental disorders, including microcephaly and intellectual disabilities. Multiple layers of regulatory networks have been identified and extensively studied over the past decades to understand this complex but extremely crucial process of brain development. In recent years, post-transcriptional RNA regulation through RNA binding proteins has emerged as a critical regulatory nexus in embryonic neurogenesis. The exon junction complex (EJC) is a highly conserved RNA binding complex composed of four core proteins, Magoh, Rbm8a, Eif4a3, and Casc3. The EJC plays a major role in regulating RNA splicing, nuclear export, subcellular localization, translation, and nonsense mediated RNA decay. Human genetic studies have associated individual EJC components with various developmental disorders. We showed previously that haploinsufficiency of Magoh causes microcephaly and disrupted neural stem cell differentiation in mouse. However, it is unclear if other EJC core components are also required for embryonic neurogenesis. More importantly, the molecular mechanism through which the EJC regulates embryonic neurogenesis remains largely unknown. Here, we demonstrated with genetically modified mouse models that both Rbm8a and Eif4a3 are required for proper embryonic neurogenesis and the formation of a normal brain. Using transcriptome and proteomic analysis, we showed that the EJC posttranscriptionally regulates genes involved in the p53 pathway, splicing and translation regulation, as well as ribosomal biogenesis. This is the first in vivo evidence suggesting that the etiology of EJC associated neurodevelopmental diseases can be ribosomopathies. We also showed that, different from other EJC core components, depletion of Casc3 only led to mild neurogenesis defects in the mouse model. However, our data suggested that Casc3 is required for embryo viability, development progression, and is potentially a regulator of cardiac development. Together, data presented in this thesis suggests that the EJC is crucial for embryonic neurogenesis and that the EJC and its peripheral factors may regulate development in a tissue-specific manner.

“Newter”-ing the Nicodemite: Reception of John Calvin’s Quatre sermons (1552) in Sixteenth-Century England

This dissertation examines the publication history of a single work: John Calvin’s 1552 Quatre sermons de M. Jehan Calvin traictans des matières fort utiles pour nostre temps, avec briefve exposition du Pseaume lxxxvii. Overlooked for both its contribution to Calvin’s wider corpus and its surprising popularity in English translation, successive editions of Quatre sermons display how Calvin’s argument against the behavior of so-called “Nicodemites” was adapted to various purposes unrelated to refuting religious dissimulation. The present study contributes to research in Calvin’s anti-Nicodemism by highlighting the fruitfulness of focusing on a discrete work and its reception. Borrowing a term (“Newter”) from John Field’s 1579 translation of Quatre sermons, this study’s title adumbrates its argument. English translators capitalized on the intrinsic malleability of a nameless and faceless opponent, the Nicodemite, and the adaptability of Quatre sermons’ genre as a collection of sermons to reshape—or, if you will, disfigure—both Calvin’s original foes and his case against them to advance various new agenda. Yet they were not the first to use the reformer’s sermons this way. They could have learned this from Calvin himself.

My examination of Quatre sermons opens by setting the work in the context of Calvin’s other writings and his political situation (Introduction, chapters one and two). Calvin’s unrelenting literary assault on French Nicodemism over three decades has long been recognized for its consistency and negativity. Yet scholars have tended to neglect how Calvin’s polemic against religious dissimulation could exhibit significant flexibility according to the needs of his context. Whereas Calvin’s preface promises simply to revisit his previous argument against participation in the Mass, his approach to Nicodemism in Quatre sermons seems adapted to accomplish goals beyond decrying false worship, offering a carefully-crafted apology for Calvin’s pastoral authority directed at his political situation. Repeatedly emphasizing God’s purpose to bless his children through the ministry of a rightly-ordered church, Quatre sermons marks a shift in Calvin’s anti-Nicodemite rhetoric away from purely negative critique, stressing instead God’s provision of spiritual nurture via political exile. Read in light of Calvin’s 1552 context, two audiences emerge: sermons ostensibly targeting believers in France who hid their faith also appear especially designed to silence Calvin’s foes in Geneva.

The remainder of the study examines the reception of Quatre sermons in the rapidly shifting religious and social contexts of Marian and Elizabethan England, where it appeared in more unique editions than any of Calvin’s writings besides the Institutio and the reformer’s 1542/45 Genevan Catechism. Calvin’s anti-Nicodemism has not been examined for its distinct contribution to the overall English reception of his thought. Five English versions of Quatre sermons appeared between 1553 and 1584—four of these under a Protestant queen, a situation quite different from the French context Calvin addressed. After situating Calvin’s position within the currents of Tudor Protestant anti-Nicodemism (chapter three), I place each of the five translations in its particular context, investigating prefaces, appendices, marginalia, and translation methods to discover how and why individuals used Quatre sermons (chapters four to six). Like Calvin in 1552, those who brought Quatre sermons to English readers were not primarily concerned with Nicodemism. Rather, the malleability of Calvin’s Nicodemite as polemical opponent and the flexibility of Quatre sermons as a sequence of discrete, interrelated parts made it popular with those eager to press Calvin into the service of a variety of diverse goals he could not have imagined, including turning his anti-Nicodemism against fellow members of the English church.