Drosophila Sister-of-Sex-lethal is a repressor of translation

Abstract

The RNA-binding protein Sex-lethal (Sxl) is an important post-transcriptional regulator of sex determination and dosage compensation in female Drosophila. To prevent the assembly of the MSL dosage compensation complex in female flies, Sxl acts as a repressor of male-specific lethal-2 (msl-2) mRNA translation. It uses two distinct and mutually reinforcing blocks to translation that operate on the 5 ′ ′ ′ ′ ′ and 3 ′ ′ ′ ′ ′ untranslated regions (UTRs) of msl-2 mRNA, respectively. While 5 ′ ′ ′ ′ ′ UTR-mediated translational control involves an upstream open reading frame, 3 ′ ′ ′ ′ ′ UTR-mediated regulation strictly requires the co-repressor protein Upstream of N-ras (Unr), which is recruited to the transcript by Sxl. We have identified the protein Sister-of-Sex-lethal (Ssx) as a novel repressor of translation with Sxl-like activity. Both proteins have a comparable RNA-binding specificity and can associate with uracil-rich RNA regulatory elements present in msl-2 mRNA. Moreover, both repress translation when bound to the 5 ′ ′ ′ ′ ′ UTR of msl-2. However, Ssx is inactive in 3 ′ ′ ′ ′ ′ UTR-mediated regulation, as it cannot engage the co-repressor protein Unr. The difference in activity maps to the first RNA-recognition motif (RRM) of Ssx. Conversion of three amino acids within this domain into their Sxl counterpart results in a gain of function and repression via the 3 ′ ′ ′ ′ ′ UTR, allowing detailed insights into the evolutionary origin of the two proteins and into the molecular requirements of an important translation regulatory pathway.

Purification of cross-linked RNA-protein complexes by phenol-toluol extraction

“…Furthermore, abundant cellular proteins unrelated to RNA-binding such as beta-actin are not detectable in the PTex fraction. To further demonstrate the efficient removal of non-cross-linked proteins, we spiked into the cell lysates a recombinant RNA-binding protein (the central domain of Drosophila melanogaster Sex-lethal, denoted Sxl-RBD4 17 ), after UV cross-linking. PTex efficiently removes ∼99% of the free spike-in RBP (as determined by densitometry compared to the input; Fig.…”

“…Recombinant Sxl-RBD4 protein (Sxl amino acids 122–301) was purified essentially as described before 17 . In brief, after IPTG induction for 4 h at 23 °C in E. coli (BL21Star [Invitrogen] transformed with the Rosetta 2 plasmid [Merck]), cells were lysed in a buffer containing 20 mM Tris-HCl pH 7.5, 1 M NaCl, 0.2 mM EDTA, 1 mM DTT, cOmplete Protease Inhibitor Cocktail [Roche] followed by centrifugation for 20 min at 12,000× g .…”

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

“…Protein purification Recombinant Sxl-RBD4 protein (Sxl amino acids 122-301) was purified essentially as described before (22). In brief, after IPTG induction for 4h at 23°C in E. coli (BL21Star [Invitrogen] transformed with the Rosetta 2 plasmid [Merck]), cells were lysed in a buffer containing 20 mM Tris/Cl pH 7.5, 1M NaCl, 0.2 mM EDTA, 1 mM DTT, cOmplete Protease Inhibitor Cocktail [Roche] followed by centrifugation for 20min at 12,000 x g. The cleared lysate was then subjected to GSH-affinity chromatography using an ÄKTA FPLC system.…”

“…Furthermore, abundant cellular proteins unrelated to RNA-binding such as beta-actin are not detectable in the PTex fraction. To further demonstrate the efficient removal of non-cross-linked proteins, we spiked into the cell lysates a recombinant RNA-binding protein (the central domain of Drosophila melanogaster Sex-lethal, denoted Sxl-RBD4 (22)), after UV-cross-linking. PTex efficiently removes ∼99% of the free spike-in RBP (as determined by densitometry compared to the input; Fig.…”

DrosophilaSister-of-Sex-lethal reinforces a male-specific gene expression pattern by controllingSex-lethalalternative splicing

“…Transposon insertion into the Ssx locus however is immunocompromising and the mutant flies quickly succumb to gram-positive bacterial infection, but not to infection with gram-negative pathogens, suggesting a function in immunity ( 11 ). We have previously reported, that Ssx can associate with msl-2 mRNA to repress its translation like its paralog Sxl ( 12 ).…”