Human DDX21 binds and unwinds RNA guanine quadruplexes

Abstract

Guanine quadruplexes (G4s) are an important structure of nucleic acids (DNA and RNA) with roles in several cellular processes. RNA G4s require specialized unwinding enzymes, of which only two have been previously identified. We describe the results of a simple and specific mass spectrometry guided method used to screen HEK293T cell lysate for G4 binding proteins. From these results, we validated the RNA helicase protein DDX21. DDX21 is an established RNA helicase, but has not yet been validated as a G4 binding protein. Through biochemical techniques, we confirm that DDX21-quadruplex RNA interactions are direct and mediated via a site of interaction at the C-terminus of the protein. Furthermore, through monitoring changes in nuclease sensitivity we show that DDX21 can unwind RNA G4. Finally, as proof of principle, we demonstrate the ability of DDX21 to suppress the expression of a protein with G4s in the 3΄ UTR of its mRNA.

An RNA guanine quadruplex regulated pathway to TRAIL-sensitization by DDX21

“…Recently we discovered DDX21 to be an RNA rG4 binding protein that has the ability to destabilize rG4s 14 . Until now studies have focused on model RNA systems and there are no rG4s that DDX21 is known to interact with endogenously.…”

“…Our data underline the potential for the integration of rG4 therapeutics with an existing cancer treatment interest. Current research suggests that DDX21 has a unique specificity for certain rG4s that may be dependent on loop sequence 14,42 . Further work aims to identify the structure of the TV2 rG4 that DDX21 is recognizing, deciphering the specificity of DDX21 may potentially allow for the design of small molecules that specifically block its action.…”

“…To look for potential G4 targets of DDX21 we wanted to compare cell populations expressing a wildtype and a mutant (M4) DDX21 with impaired G4 binding. The M4 mutant contains a PRGQR to YEGIQ mutation in the C-terminal repeat region of DDX21 that has been previously shown to disrupt rG4 binding and unwinding activities 14 . Differences between the wildtype DDX21 recovery (WT) and M4 DDX21 recovery experiments should be due to differences in DDX21 G4 interactions.…”

“…Abnormal levels of DDX21 protein has been observed in colorectal 10 and breast cancers 11 (where it has been correlated with disease free survival, possibly mediated by regulating c-Jun activity and rRNA biogenesis 12,13 ) as well as in schizophrenic patients, making it a potential therapeutic target. Recently, we have shown that DDX21 can bind and unwind guanine-quadruplexes (rG4s) and affect the translation of a reporter construct with a rG4 in its 5' UTR 14 . This activity is mediated by an evolutionarily conserved region repeat region (F/PRGQR) in its C-terminus that preferentially interacts with the backbone of RNA rG4s 15 .…”

Diverse Roles of DEAD/DEAH-Box Helicases in Innate Immunity and Diseases

“…DDX21, also known as nucleolar RNA helicase II, is an ATP-independent helicase associated with many nuclear and nucleolar events, such as ribosomal RNA biogenesis [158] or unwinding of R-loops [159] and RNA guanine quadruplexes [160]. DDX21 also interacts with mitotic regulator PP1 [161] and oncoprotein DEK [162].…”