Secondary structural analysis of human lncRNAs

In the past decade, long noncoding RNAs (lncRNAs) have been increasingly recognized as important regulators of gene expression at various levels (1). The human genome encodes thousands of lncRNAs (2), and an increasing number of these lncRNAs have been associated with human
diseases (3). lncRNA structures are expected to play essential roles in gene regulatory functions, but our current understanding of them remains limited. Traditional methods for RNA structure determination each has its limitations:
biophysical approaches, such as NMR or crystallography, are not feasible for large RNAs which are relatively more flexible; traditional chemical probing methods often focus on small regions of single RNAs (4). To overcome these
constraints, we developed a novel method for high-throughput probing of RNA structure using massively parallel sequencing (Mod-seq (5)). Compared to traditional RNA structure probing methods, Mod-seq provides substantial
improvements in throughput, allowing rapid and simultaneous probing of the whole transcriptome (5, 6). My thesis work focused on using both experimental
methods and computational methods to study the structure of human lncRNAs. I first developed Mod-seeker, an automatic data analysis pipeline for Mod-seq
(5, 6). I then focused on studying the structure of lncRNA NEAT1, an essential component of mammalian nuclear paraspeckles (7, 8). Structure probing and comparative analyses suggest lack of evidence of covariant base-pairs in
NEAT1 across mammals. However, a conserved long-range interaction was observed that may contribute to NEAT1’s scaffolding function in paraspeckle
formation. The experiments described in this thesis suggest that lncRNAs can have conserved cellular functions without maintaining conserved secondary structures, even when they function as structural scaffolds. This work is one of
the first attempts to use both chemical probing and computational modelling to study the secondary structure of lncRNAs. The case study of NEAT1 lncRNA
structure helps us understand its function in paraspeckle formation and gives insights into the contributions of lncRNA structures towards their functions.