RNA structures regulating ribosomal protein biosynthesis

Thesis advisor: Michelle M. MeyerMost commonly known for being the blueprint for proteins, RNA also plays vital roles in gene regulation. Non-coding RNAs, functional RNA molecules that are not translated into proteins, are potential regulatory agents in bacteria. Ribosomal autogenous regulatory elements are short transcribed sequences between the promoter and a protein coding region that regulate expression of their associated gene(s), though they are not themselves translated. These sequences form RNA secondary structures that can regulate at either the transcriptional or translational level. These riboregulators have been well characterized in gram-negative bacteria such as Escherichia coli, but in gram-positive bacteria far less is known regarding how r-proteins are regulated. My main goal has been to find riboregulators of r-protein synthesis in Bacilli and determine their consensus structures and phylogenetic distributions. I have utilized the RNA homology search program Infernal, coupled with our high-capacity genomic context visualization tool, to identify homologues of ribosomal-protein autogenous regulatory RNAs found in Bacilli. The alignments produced from this work determine consensus secondary structures and phylogenetic distribution of these regulator RNAs that provide new insight into the structure and function of these RNAs.Thesis (MS) — Boston College, 2015.Submitted to: Boston College. Graduate School of Arts and Sciences.Discipline: Biology

Directing human embryonic stem cell differentiation by non-viral delivery of siRNA in 3D culture

Human embryonic stem cells (hESCs) hold great potential as a resource for regenerative medicine. Before achieving therapeutic relevancy, methods must be developed to control stem cell differentiation. It is clear that stem cells can respond to genetic signals, such as those imparted by nucleic acids, to promote lineage-specific differentiation. Here we have developed an efficient system for delivering siRNA to hESCs in a 3D culture matrix using lipid-like materials. We show that non-viral siRNA delivery in a 3D scaffolds can efficiently knockdown 90% of GFP expression in GFP-hESCs. We further show that this system can be used as a platform for directing hESC differentiation. Through siRNA silencing of the KDR receptor gene, we achieve concurrent downregulation (60–90%) in genes representative of the endoderm germ layer and significant upregulation of genes representative of the mesoderm germ layer (27–90 fold). This demonstrates that siRNA can direct stem cell differentiation by blocking genes representative of one germ layer and also provides a particularly powerful means to isolate the endoderm germ layer from the mesoderm and ectoderm. This ability to inhibit endoderm germ layer differentiation could allow for improved control over hESC differentiation to desired cell types.National Institutes of Health (U.S.) (Grant EB000244)National Institutes of Health (U.S.) (Grant DE016561)Alnylam Pharmaceuticals (Firm