From Proteopedia

proteopedia linkproteopedia link

Contents 1 Sex Lethal 1.1 Introduction 1.2 Structure 1.3 Autoregulation 1.4 Mutation 1.5 References

Sex Lethal

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

spinqualitylabelsall models Sex lethal (PDB: 1B7F) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image RRMs 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 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 [4]. 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[2]. The two RRMs are connected via an . 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 [2]. The presence of two RRMs increases RNA binding specificity by allowing for an elongated and continuous binding site [4]. The Ligand Figure 2: Intramolecular Ring Stacking of U7 and U8 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 within the strongly electropositive v-shaped cleft, while are bound to a positively charged surface on RRM2 [2]. 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. 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[2]. RNA binding 1. Hydrogen bonding with RNA bases: There are numerous hydrogen bonds between the residues and the nucleotide bases. RRM2 creates five hydrogen bonds with U3-G4-U5 at residues . RRM1 creates nine hydrogen bonds between U6-U7-U8-U9-U10-U11 at residues [2]. 2. Hydrogen bonding with 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 . RRM1 creates two hydrogen bonds with the U9 backbone at [2]. 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[2].

Autoregulation

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^[1]. This is a pathway of positive autoregulation, as functional Sxl protein must be present to cause proper processing of the pre-mRNA. Sxl's negative autoregulation pathway 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^[1]. 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

References

1. ↑ ^{1.0 1.1 1.2 1.3 1.4} Black DL. Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem. 2003;72:291-336. doi: 10.1146/annurev.biochem.72.121801.161720., Epub 2003 Feb 27. PMID:12626338 doi:http://dx.doi.org/10.1146/annurev.biochem.72.121801.161720
2. ↑ ^{2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7} Handa N, Nureki O, Kurimoto K, Kim I, Sakamoto H, Shimura Y, Muto Y, Yokoyama S. Structural basis for recognition of the tra mRNA precursor by the Sex-lethal protein. Nature. 1999 Apr 15;398(6728):579-85. PMID:10217141 doi:10.1038/19242
3. ↑ Georgiev P, Chlamydas S, Akhtar A. Drosophila dosage compensation. Fly 2011;5(2):147-154. https://doi.org/10.4161/fly.5.2.14934
4. ↑ ^{4.0 4.1} Clery A, Blatter M, Allain FH. RNA recognition motifs: boring? Not quite. Curr Opin Struct Biol. 2008 Jun;18(3):290-8. doi: 10.1016/j.sbi.2008.04.002. PMID:18515081 doi:http://dx.doi.org/10.1016/j.sbi.2008.04.002