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Sex lethal: RNA-binding proteinSex lethal: RNA-binding protein

Introduction Figure 1. Sxl sex determination and dosage compensation pathways. Functionality of Sxl is determined by the number of X chromosomes. The functional protein (XX) causes a cascade that leads to female structures and behaviors, while the nonfunctional protein results in the default male structures and behaviors. 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 . Sxl binds specifically to the continuous single-stranded RNA sequence 5’-UGUUUUUUU . 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 pathway that results in the development of female structures and behaviors (Figure 1). 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 . The pre-mRNA transcript of Male-specific lethal 2 (Msl-2) is the downstream target through which Sxl regulates dosage compensation. Active Sxl protein (in females) binds to two recognition elements on Msl-2 causing the retention of the first intron in the 5’UTR of Msl-2. Sxl protein bound at this intron then blocks translation, preventing expression of Msl-2 in females . With no Msl-2 protein present, the Male-specific lethal complex cannot form and carry out its function of upregulating expression of genes on the X chromosome (Figure 1). Structure RRMs Figure 2. Electropositive binding cleft. Electropositive region shown in blue and electronegative region shown in red. The electropositive binding cleft accommodates the phosphodiester backbone of the RNA molecule to which Sxl binds. The sex-lethal protein (Sxl) is 354 amino acid residues long. It is composed of two highly conserved regions called (RRMs) that function as a monomeric unit. Each RRM is approximately 90 amino acids long with a four stranded sheet and two . The β-pleated sheets from each RRM interact with the RNA ligand, while the α-helices interact with each other to shape the protein . The RRMs interact at : between the side chain of Lys 197 and the main chain carbonyl of Val 238 and between the side chains of Tyr 131 and Gln 239. The two RRMs are connected via an interdomain linker. The linker often forms a short 310 helix from Gly205 to Thr211.The interaction between the RRMs and the ligand is facilitated by the v-shaped formed by the β-pleated sheet of each RRM. The v-shaped cleft is strongly electropositive which also assists in ligand binding (Figure 2). The presence of two RRMs increases RNA binding specificity by allowing for an elongated and continuous binding site . The Ligand Figure 3. Intramolecular ring stacking of U7 (top) and U8 (bottom) The RNA bound by Sxl is 9 nucleotides long—UGUUUUUUU. 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. interact with the strongly electropositive v-shaped cleft, while 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's of U5 and U6 and the phosphate group of U8, as well as between the 2’OH of U7 and the phosphate group of U5. This is kink is also facilitated by the only intramolecular stacking pair in the ligand, U7 and U8 (Figure 3). 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 intramolecular stacking regions and the large number of C2’-endo conformations deem this ligand unique. RNA binding 1. Hydrogen bonding with RNA bases: There are numerous hydrogen bonds between the residues and the nucleotide bases. RRM2 creates three hydrogen bonds with U3 and G4 with residues and residue . It also forms two hydrogen bonds with U5 with residues . RRM1 creates five hydrogen bonds with U6, U7, and U8 with residues . It also creates seven hydrogens bonds to U9, U10, and U11 with residues and residues . 2. Hydrogen bonding with the RNA backbone: There are an unusual abundance of interactions between the protein and the RNA backbone of the ligand. RRM2 residues hydrogen bond to the backbone of U3, U6 and U9. RRM1 residues hydrogen bond to the backbone of U9 and U11. 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 respectively. In RRM1, residues are involved in favorable intermolecular stacking of aromatic rings with nucleotides U6-U11. Autoregulation Figure 4. Alternative splicing mechanism for autoregulation of Sxl. The presence of Sxl causes the alternative splicing of exon 3 in the female mRNA transcript. The retention of exon 3, along with the premature stop codon it contains, leads to a truncated and inactive Sxl protein in males. Sxl controls its own levels of expression via positive and negative autoregulation. Sxl binds its own pre-mRNA transcript in a similar manner as its downstream targets, Tra and Msl2. Through binding to its recognition element, Sxl causes a 3’ splice site to be skipped. Alternative splicing occurs utilizing a 3’ splice site further downstream, cleaving out a premature stop codon within Exon 3 and preventing truncation and inactivation (Figure 4). This is a pathway of positive autoregulation, as functional Sxl protein must be present to cause proper processing of the pre-mRNA. The negative autoregulation pathway of Sxl proceeds via repression of its own translation. The Sxl transcript contains the target polyuridine sequence within its 3’UTR. Sxl binds this target, and blocks translation. Negative autoregulation allows maintenance of a stable and standard Sxl protein concentration. An excess of Sxl increases the degree of translation repression because more Sxl protein are present to potentially bind at the 3’UTR, while a shortage allows for more unrepressed translation. Mutation In fruit flies, dosage compensation is achieved by hyperexpression of the single X chromosome found in the male genome. The single X chromosome is hyperexpressed and regulated by a group of genes known as the Male-specific lethal genes. The genes involved work together to produce a complex known as the Male-specific lethal complex (Msl complex). One gene that is important in forming this complex is the gene that codes for the Msl-2 protein. Without this protein, the Msl complex cannot form . Males exclusively make the Msl-2 protein and therefore exclusively create the Msl complex. Females cannot make the complex because the Sxl protein prevents the production of the Msl-2 protein. It does this by alternatively splicing an intron near the 5’ UTR. In males this intron is included and in females it is not . Mutations of the Sxl protein can affect the formation of this complex and lead to death for females—hence the name sex lethal. If the Sxl gene is mutated in females, the formation of the Msl-2 complex will not be inhibited. If there is a functional Msl-2 complex in females then both of the X chromosomes will be hyperexpressed . The overexpression of genes on the X chromosome is a fatal phenomenon ultimately caused by a mutation of the Sxl protein. Although a mutation to the Sxl gene is harmful to females, it has no effect on males because the functional Sxl gene is not expressed—a mutation in males has no phenotypic effect.

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