Introduction

Sex lethal (Sxl) is an RNA-binding protein that plays a vital role in sex determination and dosage compensation in Drosophila melanogaster, the common fruit fly ^[1]. Sxl binds specifically to the continuous single-stranded RNA sequence 5’-UGUUUUUUU ^[2]. Functional copies of Sxl are expressed only in female fruit flies, where they induce sex-specific splicing patterns in the transcript of the Transformer (Tra) gene that lead to its function. Tra initiates a cascade that results in the development of female structures and behaviors. Sxl binds to its recognition element in the Tra pre-mRNA transcript, thereby blocking association of the splicing factor U2AF at the nearby splice site. Without the association of this essential splicing factor, the 3’ splice site shifts downstream, causing the removal of a premature stop codon and preventing truncation and inactivation of the Tra protein ^[1]. The pre-mRNA transcript of Male-specific lethal 2 (Msl2) is the downstream target through which Sxl regulates dosage compensation. Active Sxl protein (in females) binds to two recognition elements on Msl2 causing the retention of the first intron in the 5’UTR of Msl2. Sxl bound at this intron then blocks translation, preventing expression of Msl2 in females ^[1]. Without Msl2 protein, the Male specific lethal complex cannot form and carry out its function of upregulating expression of genes on the X chromosome ^[3].

Structure

RRMs

The sex-lethal protein (Sxl) is 354 amino acid residues long. It is composed of two highly conserved regions called RNA recognition motifs (RRMs) that function as a monomeric unit. Each RRM is approximately 90 amino acids long with a four stranded β-pleated sheet and two α-helices. The β-pleated sheets from each RRM interact with the RNA ligand, while the α-helices interact with each other to shape the protein.CLERY Only two interactions between the two RRMs have been observed: between the side chain of Lys 197 and the main chain carbonyl of Val 238 and the side chains of Tyr 131 and Gln 239. The presence of two RRMs increases RNA binding specificity by allowing for an elongated and continuous binding site.CLERY The two RRMs are connected via an interdomain linker. The linker often forms a short 310 α-helix from Gly205 to Thr211.The interaction between the RRMs and the ligand is facilitated by the v-shaped cleft formed by the β-pleated sheet of each RRM. The v-shaped cleft is strongly electropositive which also assists in ligand binding.HANDA

The Ligand

The RNA ligand bound to Sxl is 12 nucleotides long—GUUGUUUUUUUU. This ligand lacks intramolecular base pairs—a characteristic that would typically assist in RNA recognition—and therefore presents with many unique features. Sxl fixes U3-U11 in a specific elongated conformation. U6-U11 interact within the strongly electropositive v-shaped cleft, while U3-G4-U5 are bound to a positively charged surface on RRM2. The kink in the middle of the ligand is created by hydrogen bonds between the 2’OH of U5 and U6 with the phosphate group of U8 and the 2’OH of U7 and the phosphate group of U5. This is kink is also facilitated by the only intermolecular stacking pair in the ligand between U7 and U8. All of the nucleotides besides U8 are in the C2’-endo conformation. This orientation allows the bases to be highly exposed to the protein and therefore increases specificity. The low number of intermolecular stacking regions and the large number of C2’-endo conformations deem this ligand unique.

RNA binding

1. Hydrogen binding to the RNA backbone: There is an unusual abundance of interactions between the protein and the RNA backbone of the ligand. RRM2 creates four hydrogen bonds with U3 and G4 backbones at Y214, N241, R252, and R258. RRM1 creates two hydrogen bonds with the U9 backbone at N130, R155. 2. Hydrogen binding to the bases: There are numerous hydrogen bonds between the residues and the nucleotide bases. RRM2 creates five hydrogen bonds with U3-G4-U5 at the main chains of N217, R252, and A289 and the side chains of N212 and Q239. RRM1 creates nine hydrogen bonds between U6-U7-U8-U9-U10-U11 at the main chains ofS165, R195, R202, G204 and the side chains of N126, Q134, R155, K197 and R202. 3. Intermolecular stacking: Intermolecular stacking between the aromatic side chains and the nucleotide bases also contributes to RNA binding. In RRM2, U3-G4-U5 stack with V254, Y214, and F256 respectively. In RRM1, U6 stacks with Y131 and R195, U9 stacks with N130, and U11 stacks with F170.

Mutation