The Roles of Small and Long Non-Coding RNAs in Regulating Gene Expression




Xue, Zhihong

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Recent studies have revealed that a large proportion of a eukaryotic genome is transcribed into non-coding RNAs (ncRNAs). Based on size, these RNAs can be classified as small non-coding RNAs (sRNAs) and large non-coding RNAs (lncRNAs). The ncRNA regulatory networks control various levels of gene expression and play significant roles in diverse biological processes. Argonaute proteins, the core proteins in RNAi pathways, are required for the biogenesis of some sRNAs, including the PIWI-interacting RNAs and some microRNAs. How Argonautes mediate maturation of sRNAs independent of their slicer activity was not clear.The maturation of the Neurospora miRNA-like sRNA, milR-1, requires the Argonaute protein QDE-2, Dicer, and exonuclease QIP. Here, I reconstituted this Argonaute-dependent sRNA biogenesis pathway in vitro, and demonstrated that QDE-2 mediates milR-1 maturation by recruiting exosome and QIP, and by determining the size of milR-1. QIP first separates the QDE-2-bound duplex milR-1 precursor and then mediates 3’ to 5’ trimming and maturation of milR-1 precursor together with exosome using a hand-over mechanism. Our results establish a biochemical mechanism of an Argonaute-dependent sRNA biogenesis pathway and critical roles of exosome in sRNA processing. Natural antisense RNAs, which are mostly lncRNAs, are widely found in eukaryotic organisms and have been implicated in diverse physiological processes. The physiological importance of antisense RNAs and how they regulate sense RNAs are not clear. frequency (frq) encodes a core component of the Neurospora circadian oscillator. Here, I demonstrated that the simultaneous transcription of qrf, the long non-coding frq antisense RNA, represses frq transcription by inducing RNA polymerase II collision-triggered premature transcription termination and chromatin modifications. The expression of frq also inhibits the expression of qrf and surprisingly, drives the antiphasic rhythm of the qrf transcripts in the dark. The mutual inhibition of frq and qrf transcription forms a double negative feedback loop that is required for robust and sustained circadian rhythmicity. Our results establish antisense transcription as an essential feature in a eukaryotic circadian system and demonstrate the importance and mechanism of antisense RNA action. Together, the studies described in this dissertation shed light on the mechanisms of gene expression regulated by sRNAs and lncRNAs.

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