User:Mark Macbeth/Sandbox2: Difference between revisions

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~~==Sex-Lethal Protein==~~

== Background ==

'''Sex Lethal Protein''' (Sxl) is a splicing repressor in the male developmental pathway of [~~https~~://en.wikipedia.org/wiki/Sex-determination_system sex determination] of the common fruit fly, ''[~~https~~://en.wikipedia.org/wiki/Drosophila_melanogaster Drosophila melanogaster]''<ref name="Handa">PMID: 10217141</ref>. <scene name='78/783145/Sxl/1'>Sxl</scene> regulates [~~https~~://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 [~~https~~://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 [~~https~~://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"/>.

'''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 [http://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"/>.

== Significance ==

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[[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].]]

The Sxl RNA splicing targets encode for the transformer (''tra'') and the male-sex lethal (''msl-2'') proteins. ''Tra'' is a splicing activator for the female developmental pathway, and ''msl-2'' modulates [~~https~~://en.wikipedia.org/wiki/X_chromosome X chromosome] application in male fruit flies. The mechanism for how Sxl targets these pathways differs slightly. In both mechanisms, Sxl occupies the 3' splice site and prevents [~~https~~://en.wikipedia.org/wiki/U2AF2 U2AF] from binding. This causes the U2AF splicing factor to bind at a downstream splice site encoding proteins in the female developmental pathway. In ''msl-2'' targeting, Sxl also blocks the binding of another regulatory splicing factor, Rox8, and the [~~https~~://en.wikipedia.org/wiki/SnRNP U1 snRNP] at the 5’ splice site<ref name="Penalva">. Sxl can also control its own splicing pattern to conserve female expression. It does so by binding to [~~https~~://en.wikipedia.org/wiki/Exon Exon] 3 of its own RNA and creating an RNP complex to eliminate this exon. After removal of Exon 3, Sxl becomes active and female expression is maintained.

The Sxl RNA splicing targets encode for the transformer (''tra'') and the male-sex lethal (''msl-2'') proteins. ''Tra'' is a splicing activator for the female developmental pathway, and ''msl-2'' modulates [http://en.wikipedia.org/wiki/X_chromosome X chromosome] application in male fruit flies. The mechanism for how Sxl targets these pathways differs slightly. In both mechanisms, Sxl occupies the 3' splice site and prevents [http://en.wikipedia.org/wiki/U2AF2 U2AF] from binding. This causes the U2AF splicing factor to bind at a downstream splice site encoding proteins in the female developmental pathway. In ''msl-2'' targeting, Sxl also blocks the binding of another regulatory splicing factor, Rox8, and the [http://en.wikipedia.org/wiki/SnRNP U1 snRNP] at the 5’ splice site<ref name="Penalva"/>. Sxl can also control its own splicing pattern to conserve female expression. It does so by binding to [http://en.wikipedia.org/wiki/Exon Exon] 3 of its own RNA and creating an RNP complex to eliminate this exon. After removal of Exon 3, Sxl becomes active and female expression is maintained.

== Structure ==

[[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.]]

[[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 [http://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.

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=== Alternative Splicing Pathways ===

[[Image:Sxl mechanism alternativesplicing figure version2.jpg|300px|left|thumb| '''Figure 3.''' 2-dimensional representation of alternative splicing repression by Sxl on the ''tra'' and ''msl-2'' genes.]]

The alternative splicing pathways of Sxl differ, but both involve repression at the 3' splice site<ref name="Handa"/><ref name="Black">. The ''tra'' expression pathway only involves the 3' splice site, while the ''msl-2'' pathway involves both the 3' splice site and the 5' splice site. Both mechanisms cause U2AF binding downstream with lower affinity (Fig. 3)<ref name="Black"/>. U2AF is a more general splicing factor than Sxl, and prefers cytidine-containing poly-uridine pre-mRNA sequences, so Sxl binds to the guanosine-containing pre-mRNA with a 104-fold greater affinity<ref name="Handa"/>.

The alternative splicing pathways of Sxl differ, but both involve repression at the 3' splice site<ref name="Handa"/><ref name="Black"/>. The ''tra'' expression pathway only involves the 3' splice site, while the ''msl-2'' pathway involves both the 3' splice site and the 5' splice site. Both mechanisms cause U2AF binding downstream with lower affinity (Fig. 3)<ref name="Black"/>. U2AF is a more general splicing factor than Sxl, and prefers cytidine-containing poly-uridine pre-mRNA sequences, so Sxl binds to the guanosine-containing pre-mRNA with a 104-fold greater affinity<ref name="Handa"/>.

=== Autoregulation ===

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=== ''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"/>.

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="Penalva"/>. 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 ===

@@ Line 1: / Line 1: @@
-==Sex-Lethal Protein==
 <StructureSection load='1b7f' size='350' side='right' caption='Sex-Lethal protein' scene=''>
 == Background ==
-'''Sex Lethal Protein''' (Sxl) is a splicing repressor in the male developmental pathway of [https://en.wikipedia.org/wiki/Sex-determination_system sex determination] of the common fruit fly, ''[https://en.wikipedia.org/wiki/Drosophila_melanogaster Drosophila melanogaster]''<ref name="Handa">PMID: 10217141</ref>. <scene name='78/783145/Sxl/1'>Sxl</scene> regulates [https://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 [https://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 [https://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"/>.
+'''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 [http://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"/>.
 == Significance ==
@@ Line 9: / Line 8: @@
 [[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].]]
-The Sxl RNA splicing targets encode for the transformer (''tra'') and the male-sex lethal (''msl-2'') proteins. ''Tra'' is a splicing activator for the female developmental pathway, and ''msl-2'' modulates [https://en.wikipedia.org/wiki/X_chromosome X chromosome] application in male fruit flies. The mechanism for how Sxl targets these pathways differs slightly. In both mechanisms, Sxl occupies the 3' splice site and prevents [https://en.wikipedia.org/wiki/U2AF2 U2AF] from binding. This causes the U2AF splicing factor to bind at a downstream splice site encoding proteins in the female developmental pathway. In ''msl-2'' targeting, Sxl also blocks the binding of another regulatory splicing factor, Rox8, and the [https://en.wikipedia.org/wiki/SnRNP U1 snRNP] at the 5’ splice site<ref name="Penalva">. Sxl can also control its own splicing pattern to conserve female expression. It does so by binding to [https://en.wikipedia.org/wiki/Exon Exon] 3 of its own RNA and creating an RNP complex to eliminate this exon. After removal of Exon 3, Sxl becomes active and female expression is maintained.
+The Sxl RNA splicing targets encode for the transformer (''tra'') and the male-sex lethal (''msl-2'') proteins. ''Tra'' is a splicing activator for the female developmental pathway, and ''msl-2'' modulates [http://en.wikipedia.org/wiki/X_chromosome X chromosome] application in male fruit flies. The mechanism for how Sxl targets these pathways differs slightly. In both mechanisms, Sxl occupies the 3' splice site and prevents [http://en.wikipedia.org/wiki/U2AF2 U2AF] from binding. This causes the U2AF splicing factor to bind at a downstream splice site encoding proteins in the female developmental pathway. In ''msl-2'' targeting, Sxl also blocks the binding of another regulatory splicing factor, Rox8, and the [http://en.wikipedia.org/wiki/SnRNP U1 snRNP] at the 5’ splice site<ref name="Penalva"/>. Sxl can also control its own splicing pattern to conserve female expression. It does so by binding to [http://en.wikipedia.org/wiki/Exon Exon] 3 of its own RNA and creating an RNP complex to eliminate this exon. After removal of Exon 3, Sxl becomes active and female expression is maintained.
 == Structure ==
-[[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.]]
+[[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 [http://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 3<sub>10</sub> 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.
@@ Line 18: / Line 18: @@
 === Alternative Splicing Pathways ===
 [[Image:Sxl mechanism alternativesplicing figure version2.jpg|300px|left|thumb| '''Figure 3.''' 2-dimensional representation of alternative splicing repression by Sxl on the ''tra'' and ''msl-2'' genes.]]
-The alternative splicing pathways of Sxl differ, but both involve repression at the 3' splice site<ref name="Handa"/><ref name="Black">. The ''tra'' expression pathway only involves the 3' splice site, while the ''msl-2'' pathway involves both the 3' splice site and the 5' splice site. Both mechanisms cause U2AF binding downstream with lower affinity (Fig. 3)<ref name="Black"/>. U2AF is a more general splicing factor than Sxl, and prefers cytidine-containing poly-uridine pre-mRNA sequences, so Sxl binds to the guanosine-containing pre-mRNA with a 10<sup>4</sup>-fold greater affinity<ref name="Handa"/>.
+The alternative splicing pathways of Sxl differ, but both involve repression at the 3' splice site<ref name="Handa"/><ref name="Black"/>. The ''tra'' expression pathway only involves the 3' splice site, while the ''msl-2'' pathway involves both the 3' splice site and the 5' splice site. Both mechanisms cause U2AF binding downstream with lower affinity (Fig. 3)<ref name="Black"/>. U2AF is a more general splicing factor than Sxl, and prefers cytidine-containing poly-uridine pre-mRNA sequences, so Sxl binds to the guanosine-containing pre-mRNA with a 10<sup>4</sup>-fold greater affinity<ref name="Handa"/>.
 === Autoregulation ===
@@ Line 28: / Line 28: @@
 === ''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"/>.
+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="Penalva"/>. 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 ===