Browsing by Subject "Exoribonucleases"

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Investigating the Role of DIS3L2 in Perlman Syndrome and Wilms Tumor
(2018-06-29) Hunter, Ryan Wayne; Amatruda, James F.; Mendell, Joshua T.; Abrams, John M.; Conrad, Nicholas
Wilms tumor, while the most common pediatric kidney tumor, has a poorly understood etiology. Several recent studies have uncovered a role for loss of let-7 in its pathogenesis. One crucial mechanism through which let-7 expression is controlled is via the activity of the RNA-binding protein LIN28, which binds the precursor of let-7 and mediates the addition of a series of uridines to the 3′ end. This oligouridylation marks pre-let-7 for degradation by the exoribonuclease DIS3L2. Loss-of-function mutations in DIS3L2 lead to the Perlman congenital overgrowth syndrome, characterized by high neonatal mortality and, interestingly, a strong predisposition to Wilms tumor. Furthermore, DIS3L2 has been found to be deleted or mutated in some cases of sporadic Wilms tumor. The importance of let-7 in Wilms tumorigenesis and a purported role for DIS3L2 in the LIN28-let-7 pathway have led to speculation that aberrant let-7 expression underlies Wilms tumor susceptibility in DIS3L2-deficient contexts. It is still unclear, though, how loss of DIS3L2 could lead to altered let-7 levels, as the uridylated pre-let-7 species that DIS3L2 degrades is believed to be a dead-end product that cannot be further processed by DICER into mature let-7. Thus far, regulation of mature let-7 levels by DIS3L2 has only been examined in a limited number of cell lines. To determine whether DIS3L2 regulation of let-7 differs in a broader set of cell types, we used genome-editing to knockout DIS3L2 in a wide-ranging panel of cell lines with differing levels of LIN28 expression. Consistent with prior reports, loss of DIS3L2 had no effect on mature let-7 expression in these cell lines. However, it remained possible that the regulation of let-7 by DIS3L2 differed in cell populations relevant to Wilms tumor pathogenesis in vivo. Thus, to examine DIS3L2 function in these contexts, we used CRISPR-Cas9 to generate mouse lines harboring either Dis3l2-null alleles or mutations commonly observed in Perlman syndrome. Interestingly, Dis3l2 mutants recapitulated some aspects of Perlman syndrome, including neonatal mortality and genitourinary abnormalities, but not overgrowth or Wilms tumor. Moreover, the phenotype that results from the most common Perlman mutation is the same as that seen in the Dis3l2 loss-of-function mouse model, suggesting that the DIS3L2 mutations reported in Perlman syndrome are indeed loss-of-function. Finally, we examined the molecular function of DIS3L2 in isolated primary nephron progenitor cells (NPCs), a likely cell of origin of Wilms tumors, from Dis3l2-null and wild-type embryos. Consistent with our findings in the cell lines, loss of DIS3L2 in NPCs does not affect let-7 expression or activity, yet leads to upregulation of Igf2, which is reported to be overexpressed in 70-80% of Wilms tumors. Therefore, Igf2 poses an attractive candidate for both overgrowth and oncogenesis associated with DIS3L2 loss.
The Roles of Small and Long Non-Coding RNAs in Regulating Gene Expression
(2014-01-23) Xue, Zhihong; McKnight, Steven L.; Liu, Yi; Liu, Qinghua; Conrad, Nicholas
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.