Ultraviolet (UV) radiation causes DNA lesions that cause DNA mutations, cell cycle arrest, or apoptosis if they are not repaired. While many of the key players in these repair processes are known, factors that may be redundant or play less essential but important roles are less well characterized. MicroRNAs (miRNAs) are small noncoding RNAs that transcriptionally regulate many genes and have been implicated in repair of UV induced damage. One key finding being that depletion of genes involved in the maturation of miRNAs causes UV sensitivity. This led Phil Calses, a graduate student in the Taniguchi Lab, to explore the role of miRNA processing genes in repairing DNA damage, only in this instance miRNAs are a red herring. In a recent Cell Reports article Calses and colleagues found that a miRNA processing protein, DGCR8, has a second and independent function in the nucleotide excision repair pathway.
The first observation in this study was that UV irradiation resulted in not one but two versions of DGCR8 on an immunoblot, one of which disappeared when cell lysates were treated with a phosphatase. Using phosphoproteomic mass spectrometry, nine possible phosphorylation sites were identified within DGCR8. Serine 153 of DGCR8 proved to be the important site for UV-induced phosphorylation, when it was mutated to alanine the phosphorylation could no longer be detected by immunoblot. This finding was further validated using a phospho-specific antibody to S153. This antibody was used to demonstrate that phosphorylation was dependent on the dose of radiation, and peaked approximately one hour after UV exposure. Moreover, this phosphorylation event occurred in multiple cell types and in response to many forms of DNA damage including multiple UV wavelengths, hydrogen peroxide, and other chemical agents.
Calses and colleagues generated many mutant versions of DGCR8 to identify the regions of the protein that were essential for DNA damage repair. Using these constructs they demonstrated that Ser153 phosphorylation was absolutely required for DNA damage repair, while the C-terminus of the protein plays an important role in miRNA processing, it was not required for this activity. Moreover, if Ser153 was mutated to alanine, cells became sensitized to UV, indicating this as an important feature of the repair pathway.
This activity was further characterized by measuring the type of DNA damage DGCR8 may be repairing. UV irradiation causes at least two different types of DNA lesions called CPDs and 6-4PPs, both of these products are corrected by the nucleotide excision repair pathway. Both cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) were elevated in cells depleted of DGCR8 after UV exposure and if DGCR8 was mutated at Ser153 these lesions remained at elevated levels.
All of these experiments suggested that DGCR8 played two very different roles. The first is to bind with an RNase, Drosha to form a complex that ensures miRNA maturation. The second is DGCR8 is phosphorylated at Ser153 and stimulates nucleotide excision repair after DNA damage. Consistent with this, DGCR8 bound to Drosha and RNA polymerase II. While these functions are independent it is still unclear if there is any cross-talk between them, “We showed that DGCR8 has two independent functions (microRNA processing and UV-resistance), but we did not show that these two roles are mutually exclusive. We know that RNA synthesis is slowed/halted following UV irradiation (irrespective of DGCR8 S153 phosphorylation), though. And efficient recovery of RNA synthesis after UV exposure is dependent on S153 phosphorylation of DGCR8” said Dr. Toshi Taniguchi.
This exciting work identified a new role of DGCR8, but perhaps more importantly, it emphasized the value of following the biology – even when your initial hypothesis turns out to be incorrect. The understanding of human biology is always important to future scientific studies, and DGCR8 may play an important role in cancer biology as well, explained Dr. Taniguchi, “DGCR8 is mutated in a subset of Wilms tumors and mutations of DGCR8 can be found in many different types of cancers in the TCGA dataset”. The Taniguchi lab is excited to follow up this work by understanding the role of DGCR8 in animal models of DNA damage.
Funding for this research was provided by the Howard Hughes Medical Institute, National Institutes of Health, the Fanconi Anemia Research Fund, and Fred Hutch Obliteride.
Calses PC, Dhillon KK, Tucker N, Chi Y, Huang J-W, Kawasumi M, Nghiem P, Wang Y, Clurman BE, Jacquemont C, Gafken PR, Sugasawa K, Saijo M, Taniguchi T. 2017. DGCR8 Mediates Repair of UV-Induced DNA Damage Independently of RNA Processing. Cell Rep, 19(1), 162-174.