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.

Keywords: ATPase; DEAD-box; Drosophila; RNA-binding; helicase; posttranscriptional gene regulation.

FIGURE 1.

Belle constructs and transgenic expression strategy and results showing that transgenic Belle ATPase mutants under belle promoter causes dominant toxicity. (A) Domain structure of Drosophila Belle. Belle has a DDX core with a DEAD motif and a HELICc domain, flanked by amino- and carboxy-terminal IDRs. (B) Description of Belle mutants used in this study. (C–F) Transgenic expression strategy used in this study. (C) Control flies have endogenous Belle. (D) Expression of transgenic Belle under endogenous Belle promoter (belle-Belle). (E) Eye-specific transgenic Belle expression achieved by using UAST-Belle and eye-specific Gal4 drivers (ey-Gal4 > Belle and longGMR-Gal4 > Belle). (F) Ovary-specific transgenic Belle expression achieved by using UASP-Belle and ovary-specific Gal4 drivers (MTD-Gal4 > Belle, tj-Gal4 > Belle, and MAT67-Gal4 > Belle). (G) Viability of belle::Belle transgenic flies in the genetic backgrounds of belle^+/+, belle^null, and belle^hyp1.

FIGURE 2.

RNA-binding, RNA-unwinding, and IDRs of Belle are important for normal eye development and ectopic expression of Belle ATPase mutants has dominant toxic effects in eyes. (A) Summary of eye phenotypes of control and Belle transgenic flies. belle::Belle transgenic expression in the absence of endogenous Belle in eyes was achieved using flies wIR; ey-Gal4, UAS-FLP/belle-Belle; FRT82B, belle⁴⁷¹¹⁰/FRT82B, GMR-hid (left column). Eye-specific transgenic Belle expression in the absence of endogenous Belle in eyes was achieved using flies wIR; ey-Gal4, UAS-FLP/UAST-3xFlag-Belle; FRT82B, belle⁴⁷¹¹⁰/FRT82B, GMR-hid (middle column). Eye-specific transgenic Belle expression in the presence of endogenous Belle in eyes was achieved using flies wIR; UAST-3xFlag-Belle/CyO; longGMR-Gal4/TM3, Sb (right column). Control flies, which express endogenous Belle but no transgenic Belle, have normal “Smooth” eyes (representative image shown in panel B-b1). Flies that lack endogenous belle specifically in eyes and do not express any transgenic Belle (wIR; ey-Gal4, UAS-FLP/+; FRT82B, belle⁴⁷¹¹⁰/FRT82B, GMR-hid) have “Rough” eyes (representative image shown in panel C-c1). (B–D) Representative images of (B) smooth, (C) rough, and (D) rough and degenerated eyes. Each panel shows the eye image of flies with a corresponding annotation in the table shown in (A).

FIGURE 3.

Ectopic expression of Belle ATPase mutants has dominant toxic effects on female fertility. Female fertility assays using flies expressing Belle transgene in germlines (MTD-Gal4 > Belle). (A) The numbers of eggs laid by test females crossed with OregonR wild-type males and (B) the hatching rates from the eggs. Mean ± SD (n = 3 biological replicates). P-value <0.05 (two-sided Student's t-test) are indicated by (*). (C) Representative images of dissected ovaries.

FIGURE 4.

Ectopic expression of Belle ATPase mutants decreases Cyclin B protein level in ovaries. (A) Representative images of western blot using ovary lysates of MTD-Gal4 > w¹¹¹⁸ and MTD-Gal4 > Belle flies. (B) Quantification of band intensities of western blot. Mean ± SD (n = 3 or 4 biological replicates). P-value <0.05 (two-sided Student's t-test) are indicated by (*). (C) Relative levels of cyclin A/B/B3, cdk1 and vasa mRNAs in MTD-Gal4 > Belle ovaries compared with controls (MTD-Gal4 > w¹¹¹⁸) determined by high-throughput sequencing. Mean ± SD (n = 3 biological replicates).

FIGURE 5.

Belle posttranscriptionally promotes protein translation in an ATPase activity-dependent manner. (A) GFP-5x BoxB reporter structure, harboring a ubiquitous tubulin promoter, the GFP-coding sequence, and a 3′-UTR containing five BoxB hairpins. λN-HA-fused control peptide and Belle under a UASP promoter were expressed in germline cells using MAT67-Gal4 driver. (B) Confocal images of GFP signal in stage 14 oocytes. (C) Representative images of western blots using ovary lysates. (D) Quantification of western blots band intensities. GFP protein levels normalized with α-tubulin are shown. (E) Relative abundance of GFP-5xBoxB mRNA normalized by gapdh mRNA determined by qRT-PCR.

FIGURE 6.

The Belle amino-terminal IDR is required for posttranscriptional promotion of protein translation. (A) Confocal images of GFP signal in stage 14 oocytes. (B) Representative images of western blots using ovary lysates. (C) Quantification of western blots band intensities. GFP protein levels normalized with α-tubulin are shown. (D) Relative abundance of GFP-5xBoxB mRNA normalized by gapdh mRNA determined by qRT-PCR.

FIGURE 7.

Belle binds endogenous cyclin B mRNA in ovaries. (A–D) Fold enrichment of mRNAs [(A): cyclin A, (B): cyclin B, (C): cyclin B3, (D): cdk1] relative to a control rp49 mRNA that were coimmunoprecipitated with transgenic Belle expressed specifically in germlines in ovaries (MTD-Gal4 > Belle) normalized by w¹¹¹⁸ negative control (MTD-Gal4 > w¹¹¹⁸), determined by RNA-immunoprecipitation followed by qRT-PCR. Mean ± SD (n = 3 biological replicates). P-value <0.05 (two-sided Student's t-test) is indicated by (*).

FIGURE 8.

Model of Belle mechanism. (A). Wild-type Belle binds a specific subset of mRNAs. Using its ATPase activity, Belle promotes translation of bound mRNAs. (B) Belle ATPase mutants remain bound to a specific subset of mRNAs and therefore blocking access of endogenous wild-type Belle. Without its ATPase activity, Belle cannot promote translation of bound mRNAs.