DEAD-box RNA helicase Belle posttranscriptionally promotes gene expression in an ATPase activity-dependent manner

Abstract

Drosophila Belle (human ortholog DDX3) is a conserved DEAD-box RNA helicase implicated in regulating gene expression. However, the molecular mechanisms by which Belle/DDX3 regulates gene expression are poorly understood. Here we performed systematic mutational analysis to determine the contributions of conserved motifs within Belle to its in vivo function. We found that Belle RNA-binding and RNA-unwinding activities and intrinsically disordered regions (IDRs) are required for Belle in vivo function. Expression of Belle ATPase mutants that cannot bind, hydrolyze, or release ATP resulted in dominant toxic phenotypes. Mechanistically, we discovered that Belle up-regulates reporter protein level when tethered to reporter mRNA, without corresponding changes at the mRNA level, indicating that Belle promotes translation of mRNA that it binds. Belle ATPase activity and amino-terminal IDR were required for this translational promotion activity. We also found that ectopic ovary expression of dominant Belle ATPase mutants decreases levels of cyclin proteins, including Cyclin B, without corresponding changes in their mRNA levels. Finally, we found that Belle binds endogenous cyclin B mRNA. We propose that Belle promotes translation of specific target mRNAs, including cyclin B mRNA, in an ATPase activity-dependent manner.

The Drosophila RNA Helicase Belle (DDX3) Non-Autonomously Suppresses Germline Tumorigenesis Via Regulation of a Specific mRNA Set

“…We speculated that Belle binding within 5 -UTRs may facilitate translation initiation by unwinding secondary structures of mRNAs owing to its helicase activity, whereas Belle interaction with 3 -UTR may contribute to either activation or repression of translation, according to previously published data [28]. The peaks determined in the coding regions suggest that Belle may participate in the elongation steps of translation as well [26,28].…”

“…The RNA helicase Belle is known to be essential for fly viability, fertility, and germline stem cell maintenance [17,25,26]. However, the regulatory and mechanistic roles of Belle in spermatogenesis are still largely obscure.…”

“…This developmental disorder recapitulates the SCOS phenotype in humans with DBY gene deletions and also leads to severe male sterility [25,29]. Belle functions as an upstream regulator of expression of mitotic cyclins A and B both in the testes and ovaries [25,26]. However, regulatory and mechanistic actions of Belle in spermatogenesis are still largely understudied.…”

“…Belle protein is expressed ubiquitously and possesses extremely high functional pleiotropy in fly tissues, being involved in the ecdysone-triggered transcriptional cascade during metamorphosis [18]; in the small RNA-based silencing mechanisms [19,20]; in the Notch-induced differentiation of ovarian follicle cells and establishment of correct anterior-posterior polarity of the developing oocyte [21]; in the proper chromosome segregation during mitotic anaphase in S2 cells [22]; in the circadian rhythmicity in Drosophila neurons [23]; and in the cell cycle regulation in somatic tissues and the germline [24][25][26]. RNA helicase Belle is shown to regulate the translation of a set of specific mRNAs in an ATP-dependent manner [18,[26][27][28]. As such, Belle is found as a member of a large ribonucleoprotein complex which includes Smaug, Cup, Me31B, Trailer hitch, PABPC, eIF4E proteins, and the CCR4-Not complex interacting with nanos mRNA in early embryos.…”

“…This complex provides translational repression of the target mRNAs by shortening their poly(A)-tails [28]. Belle function is essential for male and female fertility [17,25,26]. Defects in cytokinesis during male meiosis and subsequent disorganization of spermatid bundles have been observed for hypomorph bel alleles [17].…”