Decoding lncRNAs : mapping the structures, functions, and evolution of the roX long noncoding RNAs in Drosophila dosage compensation
- The last decade of genomic research has revealed that animal genomes are replete with thousands of long noncoding RNA (lncRNA) genes. Though they lack the capacity to encode proteins, lncRNAs are numerous, diverse, and engaged in important biological phenomena, such as regulating chromatin state, maintaining genome stability, and coordinating chromosome conformation. Many are implicated in organismal development, cell state determination, and disease etiology -- yet the functions of the vast majority of lncRNAs are completely unknown (see lncRNA review in Chapter 1). A key to decoding the genome lies in closing this gap in our knowledge of lncRNAs. Advancements in the field require the development of RNA-centric techniques for interrogating lncRNA functions and mechanisms. In addition to contributing to our understanding of genomic complexity, the burgeoning lncRNA field represents a veritable Gold Rush of new biological "parts" -- encoded in RNA. Indeed, RNA is a robust engineering substrate and has been used to build such synthetic biological devices as small molecule sensors, genome editors, and reaction scaffolds. This dissertation describes my exploration of how lncRNAs work (Chapter 2), how their functions are organized (Chapter 3), and how they evolve (Chapter 4). As a model system for studying lncRNAs, my work focuses primarily on the roX lncRNAs in Drosophila dosage compensation. Dosage compensation is the epigenetic equalization of gene expression from sex chromosomes, which male flies accomplish by doubling expression from their single X chromosome. Dosage compensation relies on the roX lncRNAs, which coordinate the assembly of the dosage compensation complex and target it to the X chromosome. The two roX lncRNAs (roX1 and roX2) are essential to male viability, yet are genetically redundant despite their drastically different sizes and sequences. How can these two seemingly different lncRNAs share a common function? What are their molecular roles in dosage compensation? Which RNA elements encode these functions? How have the roX RNAs, the X chromosome, and the dosage compensation complex evolved together? My research described herein addresses these and other questions. First, I determined that the functional subunit of the roX lncRNAs is a repetitive structural motif. In this study, I integrated results from transcriptome-wide RNA--protein interaction profiling, RNA secondary structure mapping, and genetics. These analyses pinpointed short repeated sequence elements that fold into tandem, double-stranded RNA stem-loop structures; these structures in turn act as combinatorial docking sites for the dosage compensation complex proteins. The functional redundancy of roX1 and roX2 derives from their sharing such structural features. This research highlights the focal nature of functional elements encoded within vast noncoding RNA space, and provides further evidence that lncRNA function can be encoded at the level of secondary structure (see Chapter 2 and Appendix 1). Following this work, I examined the functional and structural organization of the roX lncRNAs. To do this, I developed a technique for dissecting lncRNA domains in situ, called domain-specific chromatin isolation by RNA purification (dChIRP). In applying this new method, I discovered that roX1 contains six RNA domains with specific spatial topologies and distinct functions. This study provided exemplary proof of the hypothesis that lncRNAs are organized into modular domains with distinct functions (see Chapter 3 and Appendix 2). The evolutionary origins and trajectories of lncRNAs are poorly understood, primarily because lncRNAs exhibit notoriously poor sequence conservation and evolve rapidly. In a recent study, I investigated the phenomenon of lncRNA evolution and developed a systematic method for identifying lncRNA homologs in distantly related species. This strategy discovered 66 roX lncRNA orthologs in ~40 divergent fly species. Phylogenetic comparisons of these roX orthologs revealed that lncRNA structures, domains, and functions are conserved despite rapidly evolving primary sequence. I traced complex evolutionary histories in the lives of these lncRNAs, including gene duplication and decay, rapid loss or gain of functional elements, and deep common ancestry of structural domains. Furthermore, I mapped the roX lncRNAs' genomic binding sites in four distantly related species, thus representing the first ever metagenomic analysis of lncRNA--genome interactions. This analysis revealed that roX-bound sites evolve rapidly in proximity, can originate from pre-existing splicing signals, and are constrained by even spacing along the X chromosome (see Chapter 4). My most recent work focuses on extended this study to more distantly related flies towards understanding the evolutionary origins of lncRNAs involved in dosage compensation, and the identification of other such chromatin-associated lncRNAs (Appendix 3). My work has explored how the roX lncRNAs work, the organization of their functions into structural domains, and their co-evolution with the dosage compensation complex and X chromosome. This work provides a framework for studying lncRNAs through the lenses of genetics, biochemistry, structure, and evolution, a toolkit for characterizing lncRNA functions, and the motivation for applying these strategies to other lncRNAs of interest.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Quinn, Jeffrey Jerome
|Stanford University, Department of Bioengineering.
|Endy, Andrew D
|Smolke, Christina D
|Endy, Andrew D
|Smolke, Christina D
|Statement of responsibility
|Jeffrey Jerome Quinn.
|Submitted to the Department of Bioengineering.
|Thesis (Ph.D.)--Stanford University, 2016.
- © 2016 by Jeffrey Jerome Quinn
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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