Text

theses

In eukaryotic cells, mRNA degradation plays an important role in the control of gene expression and is therefore highly regulated. The scavenger decapping enzyme DcpS is a multifunctional protein that plays a critical role in mRNA degradation. We first sought to identify DcpS target genes in mammalian cells using a cell permeable DcpS inhibitor compound, RG3039, which was initially developed for therapeutic treatment of Spinal Muscular Atrophy (SMA). Microarray analysis following DcpS decapping inhibition by RG3039 revealed the steady state levels of 222 RNAs were altered. Of a subset selected for validation by qRT-PCR, two non-coding transcripts dependent on DcpS decapping activity, were identified and referred to as DcpS Responsive Noncoding Transcript (DRNT) 1 and 2 respectively. Only the increase in DRNT1 transcript was accompanied with an increase of its RNA stability and this increase was dependent on both DcpS and Xrn1. Our data indicate that DcpS is a transcript-restricted modulator of RNA stability in mammalian cells and the RG3039 quinazoline compound is pleotropic, influence gene expression in both an apparent DcpS dependent and independent manner. A surprising development was uncovered in a collaborative study where two distinct mutations in the DcpS gene (c.636+1G>A, DcpSIns15 and c.947C>T, DcpST316M) were identified as the underlying cause of autosomal recessive intellectual disability within a consanguineous family. Both of the mutations were confirmed to disrupt DcpS decapping activity in vitro and/or in vivo, indicating that the decapping activity of DcpS is critical for normal neurological development. Consistent with a role for DcpS in neuronal cells, our studies with the DcpSIns15 variant uncovered a link between this variant DcpS and Spinal Muscular Atrophy (SMA). Exogenous expression of DcpSIns15 in SMA patient fibroblast cells increased SMN2 mRNA and corresponding SMN protein levels. Our findings suggest that strategies to shift wild type DcpS splicing patterns to partially yield the variant DcpS Ins15 splicing pattern may be beneficial for SMA therapeutics.

ETD_6602

Ph.D.

Includes bibliographical references

by Mi Zhou

doi:10.7282/T300044J

ETD doctoral

The author owns the copyright to this work.