The Roles of RNA Polymerase I and III Subunits Polr1c and Polr1d in Craniofacial Development and in Zebrafish Models of Treacher Collins Syndrome

Abstract

Ribosome biogenesis is a global process required for growth and proliferation of all cells, yet perturbation of ribosome biogenesis during human development often leads to tissue-specific defects termed ribosomopathies. Transcription of the ribosomal RNAs (rRNAs) by RNA polymerases (Pol) I and III, is considered a rate limiting step of ribosome biogenesis and mutations in the genes coding for RNA Pol I and III subunits, POLR1C and POLR1D cause Treacher Collins syndrome, a rare congenital craniofacial disorder. Our understanding of the functions of individual RNA polymerase subunits, however, remains poor. We discovered that polr1c and polr1d are dynamically expressed during zebrafish embryonic development, particularly in craniofacial tissues. Consistent with this pattern of activity, polr1c and polr1d homozygous mutant zebrafish exhibit cartilage hypoplasia and cranioskeletal anomalies characteristic of humans with Treacher Collins syndrome. Mechanistically, we discovered that polr1c and polr1d loss-of-function results in deficient ribosome biogenesis, Tp53-dependent neuroepithelial cell death and a deficiency of migrating neural crest cells, which are the primary progenitors of the craniofacial skeleton. More importantly, we show that genetic inhibition of tp53 can suppress neuroepithelial cell death and ameliorate the skeletal anomalies in polr1c and polr1d mutants, providing a potential avenue to prevent the pathogenesis of Treacher Collins syndrome. Our work therefore has uncovered tissue-specific roles for polr1c and polr1d in rRNA transcription, ribosome biogenesis, and neural crest and craniofacial development during embryogenesis. Furthermore, we have established polr1c and polr1d mutant zebrafish as models of Treacher Collins syndrome together with a unifying mechanism underlying its pathogenesis and possible prevention.

Tissue-selective effects of nucleolar stress and rDNA damage in developmental disorders

“…1j and Extended Data Fig. 2c–e), a phenotype consistent with both TCS and published zebrafish phenotypes 8–10 . Notably, at higher morpholino doses, overall growth defects were evident in tcof1 morphants (Extended Data Fig.…”

“…Thus, development of craniofacial structures is hypersensitive to the loss of Ddx21 or its RNA helicase activity. These results were corroborated in zebrafish embryos injected with morpholinos blocking translation of ddx21 , where observed craniofacial phenotypes resembled those reported in the polr1d −/− and polr1c −/− models of TCS 8–10 , and were rescued by the co-injection of human DDX21 mRNA (Extended Data Fig. 2g–j).…”

“…For cartilage staining with alcian blue, embryos were collected 5 days post-fertilization. Alcian blue staining was performed as described 10 . For haemoglobin staining with o-dianisidine and immunofluorescence, embryos were collected 24 h post-fertilization.…”

“…Because TCS phenotypes were shown to result from defects in cNCCs 9,10,12,13 , we investigated whether Tcof1 loss cell-autonomously affects DDX21 functions in cNCCs. We used CRISPR-Cas9 genome editing to generate Tcof +/− and Tcof −/− mouse embryonic stem (ES) cells, which showed no appreciable defect in Ddx21 nucleolar localization (Fig.…”

POLR1D promotes colorectal cancer progression and predicts poor prognosis of patients

“…Immunofluorescence assay also showed that the intensity of β‐catenin was much stronger after POLR1D was up‐regulated in RKO cells, while was much weaker after POLR1D was down‐regulated in LOVO cells (Figure B). Furthermore, according to Kristin E. Noack Watt, polr1d −/− mutant embryos exhibited much higher level of tp53 compared to control ones, we decided to examined whether POLR1D could alter p53 protein level in CRC cells. As shown in Figure C, POLR1D overexpression decreased the p53 and its downstream gene p21 expression levels, while POLR1D knockdown displayed the opposite effect, indicating POLR1D inactivated p53 signaling in CRC cells.…”