Defense Date

12-21-2020

Graduation Date

Spring 5-2021

Availability

One-year Embargo

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

Committee Chair

Mihaela-Rita Mihailescu

Committee Member

Jeffrey D. Evanseck

Committee Member

Michael Cascio

Committee Member

Danith H. Ly

Abstract

Paraspeckles are a relatively newly identified subnuclear body whose function is still largely unknown. They are membraneless organelles found within mammalian nuclei originally identified as being comprised of different paraspeckle proteins (PSPs), and later proven to also contain RNA. This study investigates the long non-coding RNA nuclear enriched abundant transcript 1 (NEAT1), which has been found to be indispensable in the formation of paraspeckles, with each containing up to 50 NEAT1 molecules. NEAT1 has two isoforms, the shorter NEAT1_1, and the longer NEAT1_2. The focus of this work will be on the NEAT1_2, which acts as a scaffold in the formation of the paraspeckles. Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons and several studies indicate NEAT1 upregulation in ALS, presumably due to an increase in paraspeckle formation in motor neurons of ALS patients. We identified five regions within NEAT1_2 that have the potential to form secondary structure G quadruplexes (GQs), with one of the GQs being localized 20 nucleotides away from a triple helix (TH) secondary structure previously identified at its 3’ terminus, formed by the binding of an adenine (A)-rich tail to two uracil (U)-rich motifs. The proximity of the TH and GQ at the 3’ end of the RNA leads to the hypothesis that these two structures will affect one another, potentially affecting the stability of NEAT1_2 Here, we used various biophysical techniques to characterize the GQ structure present near the 3’ end of NEAT1_2 and determine how the presence or absence of the GQ affects the formation of the terminal TH. We and others have shown that the potential GQ region nearest the TH terminus, “GQ5” does indeed form a GQ. We have also shown that this GQ is parallel and intramolecular. Various binding studies reveal that when the GQ is unable to form, the TH is also unable to form, supporting our hypothesis that the formation of the TH is dependent upon the GQ structure. Understanding the secondary structure of NEAT1_2 can help in the overall understanding of paraspeckle structure and function, how NEAT1 and paraspeckles may work together in the progression of some diseases, and lead to potential treatment of neurodegenerative diseases like ALS.

Language

English

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