Date of Award
Doctor of Philosophy (PhD)
Chemistry & Biochemistry
Thomas R. Cech
A key aspect of cellular function is the proper assembly and utilization of ribonucleoproteins (RNPs). Defects in the formation of RNPs lead to "RNP hypo-assembly diseases", which can be caused by RNA degradation out-competing RNP assembly. Examples of such human diseases include Dyskeratosis Congenita (DC) and Spinal Muscular Atrophy (SMA).
In order to test the hypothesis that specific RNA quality control pathways were responsible for degradation of RNAs in these diseases, I used yeast and mammalian cell lines as model systems to investigate two different diseases, SMA and DC. In SMA, Sm-site mutations in yeast U1 snRNA led to their rapid degradation by two different RNA decay pathways: 3’ to 5’ decay in the nucleus by Trf4/Rrp6, and 5’ to 3’ decay in the cytoplasm by Dcp2/Xrn1. Cytoplasmic degradation of snRNAs when the Sm site is mutated is conserved in human cells. The cytoplasmic decay patway is also responsible for the degradation of snRNAs when SMN levels are reduced, as is the case in SMA. Importantly, inhibition of snRNA decapping through DCP2 knockdown rescued splicing defects observed for some mRNAs when SMN is limiting. These results suggest that inhibition of snRNA decay could rescue some phenotypes of SMA.
For the investigation of human telomerase RNA (hTR) quality control pathways in DC, disease causing mutations were introduced in hTR, along with the depletion of dyskerin in human cells. These models established that hTR is degraded by PAPD5/EXOSC10 in the nucleus, and DCP2/XRN1 in the cytoplasm. Inhibition of hTR decay rescued both the sub-cellular localization of hTR, as well as telomerase activity in human cells. Additionally, PARN, which is a 3’ to 5’ exonuclease, stabilized hTR through deadenylation of its 3’ end. PARN mutations also lead to a severe form of DC. I have identified other possible substrates of PARN in human cells, which suggest that PARN deadenylates a number of stable non coding RNAs in human cells, but influences the stability of a select few. This potentially explains why PARN mutations cause a more severe form of DC than mutations in telomerase components.
Shukla, Siddharth, "Investigation of RNA Quality Control Pathways in RNP Hypo-Assembly Diseases" (2016). Chemistry & Biochemistry Graduate Theses & Dissertations. 199.