The Effects of Pathogenic Dkc1 Mutations on Telomerase and Ribosome Biogenesis.
Authors: Gu Baiwei , Fan Jian-meng , Idol Rachel , Bessler Monica , Mason Philip J 
Published: October 2019

Abstract
Abstract 1163
Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome (BMF) associated with a predisposition to cancer. It is diagnosed by the triad of abnormal skin pigmentation, nail dystrophy and mucosal leukoplakia. X-linked DC, accounting for around one third of DC patients, is due to the mutations in the DKC1 gene which encodes the protein dyskerin. Dyskerin is a component of small nucleolar RNA particles (H/ACA snoRNP) active in the pseudouridylation of specific residues in nascent ribosomal RNA (rRNA). It also plays an important role in maintaining the telomere repeats at the ends of chromosomes by forming and stabilizing the telomerase complex. Patients with Dkc1 mutations show a wide variation of severity emphasizing the variable expression of the disease and the influence of other genetic and environmental factors. Most mutations are clustered, in the 3D structure, in a region important for RNA binding and few mutations are present in the pseudouridine synthesis activity domain. The relationship between the severity of DC and the position of the mutation in dyskerin is still unclear. It will be interesting to compare the pathogenic effects of Dkc1 mutations in different positions of dyskerin protein. Since the production of mutant lines of mouse is time consuming, expensive, and may be difficult, here we established a suitable in vitro system to express mutant dyskerin in MEF cells. We began with MEF cells containing a Dkc1 gene that is deleted in the presence of Cre. Mutated dyskerin introduced by a retroviral vector is expressed and wild type (WT) dyskerin is deleted by retrovirally introduced Cre-ERT2 and subsequent tamoxifen treatment, which activates the Cre-ERT2 protein. MEF cells are then single cell cloned and selected cells that grew well and contained for the mutant dyskerin gene but not the WT gene. We obtained cell lines with several mutations including I38T, H68Q, S121G and A353V as well as WT control. All of these mutations are recurrent ones and cause severe bone marrow failure in patients. Interestingly, we also obtained a D125A mutant cell line. This mutation has not been found in human patients but is an alteration of a crucial catalytic aspartic acid residue that is essential for pseudouridylation activity. All of these Flag-tagged dyskerin proteins showed a normal nucleolar sub-cellular distribution pattern. We chose the cells expressing similar levels of flag-tagged dyskerin to WT dyskerin to do further experiments. The exception to this was the D125A mutation because all the clones express tiny amounts of flag-tagged dyskerin. First, we measured the Dkc1 mRNA level of these mutant cells and found the expression of Dkc1 mRNA in these mutant cells is 3–8 fold higher than in WT cells (25 fold higher for D125A). This result indicates that the mutant dyskerin proteins in these cells are very unstable, so more mRNA has to be produced to maintain growth. Then, we studied the growth curves of these cells and found that all mutant cells showed more or less slower growth rate than WT. Notably, the D125A mutant cells showed extremely slow growth. We find that the expression of Terc is lower than WT in all mutant cells (10∼50% compared to WT) while telomerase activity was not significantly decreased except for the D125A mutant. By Q-FISH, we showed the telomere length of all mutant cells is shorter than WT control cells and the number of telomere free ends is increased in these mutant cells. These findings indicate that all Dkc1 mutations affect the telomerase function and cause rapidly shortening telomeres. In ribosome biogenesis we found that, although I38T, H68Q, S121G and A353V mutant cells did not show significant changes in the ribosomal profile and ribosomal processing, D125A mutant cell showed significantly delayed maturation of rRNA as well as dramatically decreased amounts of mature 80S ribosomes. Further investigation revealed that the D125A mutation abolished in vivo pseudouridylation of rRNA. Together with our previous results this study further indicates that compromised telomerase function is the major effect of the pathogenic Dkc1 mutations. We also show, surprisingly, that mammalian cells can grow and survive with no pseudouridine in their rRNA.

Disclosures:
Bessler: Alexion Pharmaceutical Inc: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; Taligen: Consultancy.