User:Mark Macbeth/Sandbox2

<StructureSection load='1b7f' size='350' side='right' caption='Sex-Lethal protein' scene=''>

'''Sex Lethal Protein''' (Sxl) is a splicing repressor in the male developmental pathway of [http://en.wikipedia.org/wiki/Sex-determination_system sex determination] of the common fruit fly, ''[http://en.wikipedia.org/wiki/Drosophila_melanogaster Drosophila melanogaster]''<ref name="Handa">PMID: 10217141</ref>. <scene name='78/783145/Sxl/1'>Sxl</scene> regulates [http://en.wikipedia.org/wiki/Alternative_splicing alternative splicing] pathways to promote the expression of female sex-linked proteins. In eukaryotes, splicing is carried out via the [https://en.wikipedia.org/wiki/Spliceosome spliceosome], a [http://en.wikipedia.org/wiki/Ribozyme ribozyme]-protein complex which binds to the 5’ and 3’ splice sites. As Sxl is a splicing repressor, it prevents the binding of the [http://en.wikipedia.org/wiki/U2AF2 U2AF] and [https://en.wikipedia.org/wiki/U1_spliceosomal_RNA U1 subunits] of the spliceosome at their respective splice sites, which represses the alternative splicing mechanism<ref name="Penalva">PMID: 12966139</ref>. As a result, the fruit fly expressing Sxl will produce mRNA transcripts encoding proteins for the female developmental pathway<ref name="Handa"/>.

[[Image:Sex Lethal Protein Structural Overview with Labels.png|300px|right|thumb| '''Figure 1.''' Structural overview of Sxl. RNA ligand colored in green is recognized and bound, while RNA ligand colored in grey is not bound. Image created in PyMol. Structure shown is [https://www.rcsb.org/structure/1b7f PDB:1b7f].]]

[[Image:Sex lethal protein electrostatic surface representation.png|300px|right|thumb| '''Figure 2.''' Three-dimensional representation of Sex-lethal protein showing the electropositive binding pocket and the bound RNA ligand. Pre-mRNA residues binding to Sxl shown in green, non-binding residues shown in grey. Structure shown is [https://www.rcsb.org/structure/1b7f PDB:1b7f]. Image created in PyMol.]]

Sxl is composed of two asymmetric RNA binding domains (RBD1 and RBD2) which recognize a poly-uridine site in the pre-mRNA transcript<ref name="Handa"/>. Each RBD is comprised of two alpha helices and one antiparallel four-stranded β sheet<ref name="Handa"/> (Fig. 1). The β sheets face each other, lining the electropositive V-shaped cleft<ref name="Handa"/>. The inter-domain linker forms a distorted 3₁₀ helix which helps form the V-shaped cleft into which the pre-mRNA sequence binds<ref name="Handa"/><ref name="Black">doi: 10.1146/annurev.biochem.72.121801.161720</ref>. Sxl binds to UGUUUUUUU sequence of GUUGUUUUUUUU in the ''tra'' pre-mRNA<ref name="Handa"/><ref name="Black"/>. RBD1 binds U6-U11 and RBD2 binds U3, G4, and U5. Although the two RBDs do not interact with each other, this nine-ribonucleotide sequence must be recognized continuously to allow Sxl to bind, preventing U2AF from binding at the 3’ splice site<ref name="Handa"/>. The binding of Sxl to the pre-mRNA occurs in an electropositive pocket due to extensive interactions with the RNA phosphate backbone and negatively charged residues<ref name="Handa"/>. Since Sxl binds primarily with the phosphate backbone, the protein residues are not highly conserved.

Sxl is capable of autoregulation of its expression<ref name="Black"/>. The Sxl gene is transcribed in male flies, but the inclusion of exon 3 results in a premature stop codon, producing an inactive, truncated protein. The same Sxl promoter is active in female flies, but an additional (briefly active) Sxl promoter produces a transcript with exon 3 removed, resulting in an active Sxl protein which will initiate other female-specific splicing cascades<ref name="Black"/>.

=== ''Tra'' ===

In alternative splicing of the ''tra'' gene, Sxl binds at the 3' poly-uridine site (Fig. 3). This causes U2AF to bind downstream and the spliceosome transcribes the following exon<ref name="Penalva"/>. In the absence of Sxl, the normal gene for male development is transcribed. The exon contains a stop codon which results in a truncated, non-functional protein<ref name="Black"/>. In the presence of Sxl, this exon is spliced, so the stop codon is skipped<ref name="Black"/> (Fig. 3). This enables translation of an active ''tra'' protein<ref name="Black"/>.

=== ''Msl-2'' ===

The alternative splicing of ''msl-2'' is reliant on Sxl binding to both the 5' and 3' splice sites (Fig. 3). Sxl binds at the 3' splice site, replacing U2AF as in ''tra'' splicing. Sxl also competes with [http://www.uniprot.org/uniprot/Q26281 Rox8], which binds to the first intron. As a result, Sxl prevents splicing of the first intron of the ''msl-2'' primary transcript. Sxl also binds to the poly- U sequences of the 3' UTR to repress translation (Fig.3)<ref name="Panalva">doi:10.1128/mmbr.67.3.343-359.2003</ref>. When Sxl targets ''msl-2'', the first intron is retained<ref name="Black"/>. However, the retained intron is in the [https://en.wikipedia.org/wiki/Untranslated_region 5' UTR] and does not affect the reading frame<ref name="Black"/>.

=== Structural Basis for Recognition of Poly-U Sequences ===

The structural interactions with regards to the targeting of the 5' splice site and of its own mRNA transcript are much less understood than the competition of Sxl with U2AF at the 3' splice site. All the RNA-protein interactions described here refer to ''tra'' pre-mRNA-Sxl interactions.

The <scene name='78/783145/Arg_252_interaction_with_u3_g4/6'>Arg252 interaction with U3 and G4</scene> is crucial to pre-mRNA binding; a mutation of Arg252 to alanine eliminated the ability of Sxl to bind RNA<ref name="Handa"/>.

The ligand pre-mRNA sequence forms a characteristic <scene name='78/783145/U5_u6_u7_loop/2'>loop</scene> at U5, U6, and U7. This interaction is stabilized by π stacking between the G4 and U5 nucleotides and residues <scene name='78/783145/Aromatic_stacking/3'>Tyr214 and Phe256</scene>, respectively<ref name="Handa"/>. The nucleobases are exposed to residues on Sxl due to the 2’ endo conformation of all the nucleotides except for U8, which maintains a 3’ endo conformation<ref name="Handa"/>.

The U6 residue is recognized as part of the RNA <scene name='78/783145/U5_u6_u7_loop/2'>loop</scene> by <scene name='78/783145/Molecule_base_origin/4'>Arg195</scene><ref name="Handa"/>. The Arg195 amide hydrogen-bonds to the O2' of U6 and the U6 N3H hydrogen bonds to the Arg195 carbonyl oxygen<ref name="Handa"/>.

In the RNA <scene name='78/783145/U5_u6_u7_loop/2'>loop</scene>, the U7 and U8 bases are involved in π <scene name='78/783145/U7_u8_stack/1'>stacking</scene>, stabilizing the unusual 3' endo conformation of the U8 sugar<ref name="Handa"/>. U8 is further stabilized via hydrogen bonding <scene name='78/783145/U8_with_s165_and_y166/1'>interactions with Ser165 and Tyr166</scene><ref name="Handa"/>. The amine group of U8 hydrogen bonds to the the carbonyl oxygens of both Ser165 and Tyr166 <ref name="Handa"/>.

<scene name='78/783145/N130_interaction_with_u9/3'>U9</scene> is recognized by the interdomain linker <ref name="Handa"/>. This interaction is a salt bridge between the Asn130 side chain and a phosphate oxygen of U9<ref name="Handa"/>. U9 is further stabilized by a second <scene name='78/783145/U9_with_interdomain_linker/1'>salt bridge</scene> between the U9 O2' and the side chain of Arg202 and the U9 O4' and the Lys197 side chain<ref name="Handa"/>.

U9 facilitates the stabilization of U10, which is also recognized by the interdomain linker. <scene name='78/783145/Arg_258_interaction_w_u9_u10/3'>Arg-258 interacts with U9 and U10</scene> to form a salt bridge.

U11 is recognized by Arg155. The O2' of U11 <scene name='78/783145/R155_intxn_with_u11/3'>interacts with Arg155</scene> to form a hydrogen bond<ref name="Handa"/>.

The above interactions are relevant in that Sxl recognizes the specific pre-mRNA based mostly on interactions with the sugar-phosphate backbones<ref name="Handa"/>. Many proteins with [https://en.wikipedia.org/wiki/RNA_recognition_motif RNA recognition motifs] are specific in the interactions they form with the bases of the RNA recognized. In contrast, Sxl has a high specificity despite primarily interacting with the phosphate backbone.