DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis.

Area of Science:

• Molecular Biology
• Cell Biology
• Hematology

Background:

• DNA-dependent protein kinase (DNA-PK), composed of KU and DNA-PKcs, is a key factor in classical non-homologous end-joining (cNHEJ) DNA repair.
• KU's interaction with RNA is known, but its functional significance in mammals remains largely unexplored.
• Previous studies linked cNHEJ deficiency and TP53 loss to specific hematopoietic malignancies.

Purpose of the Study:

• To investigate the non-canonical functions of DNA-PK beyond its role in DNA repair.
• To elucidate the role of DNA-PK in ribosomal RNA (rRNA) biogenesis and hematopoiesis.
• To determine the impact of DNA-PKcs phosphorylation at specific sites on its cellular functions.

Main Methods:

• Utilized mouse models expressing kinase-dead DNA-PKcs, with and without TP53.
• Investigated the effect of blocking DNA-PKcs phosphorylation at T2609 and S2056 clusters.
• Examined DNA-PK localization, RNA binding, and co-purification with rRNA processing machinery.

Main Results:

• Kinase-dead DNA-PKcs, coupled with TP53 deficiency, led to myeloid disease, unlike the pro-B cell lymphoma seen in other cNHEJ/TP53 deficient mice.
• Inhibition of DNA-PKcs phosphorylation at T2609, but not S2056, caused KU-dependent defects in 18S rRNA processing, impaired protein synthesis, and bone marrow failure.
• DNA-PK binds to various cellular RNAs, including U3 snoRNA, and localizes to nucleoli in an rRNA-dependent manner, associating with the small subunit processome.

Conclusions:

• DNA-PK possesses critical RNA-dependent, cNHEJ-independent functions in ribosome biogenesis.
• The kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster are essential for these rRNA processing roles.
• These findings reveal a novel role for DNA-PK in maintaining hematopoietic stem cell function and preventing bone marrow failure.