User:Alexis Neyman/Sandbox 1 - Revision history

Alexis Neyman at 20:48, 24 April 2018

Alexis Neyman — Tue, 24 Apr 2018 20:48:56 GMT

←Older revision		Revision as of 20:48, 24 April 2018
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	=== RRM Stability===		=== RRM Stability===
-	SRp20 has a <scene name='78/781963/Hydrophobic_core_residues/1'>hydrophobic core</scene>, which may contribute to the stability of the protein. A previous study, looking at the RRM in [https://en.wikipedia.org/wiki/TARDBP TDP-43] has suggested that the hydrophobic core may be a strong contributing factor to the protein’s stability <ref>PMID:24497641</ref>. In a different study, it was determined that, in the U11/U12-65K protein, the β-sheet packs against the two α-helices ~~by way of~~ hydrophobic interactions and that the resulting stabilization could be critical for the proper folding and orientation of elements for RNA binding <ref>PMID:19447915</ref>. Due to the conservative nature of RRMs, it could be speculated that the hydrophobic core found in SRp20, between the β-sheet and two α-helices, could contribute to the stability of its RRM in a similar fashion. However, additional studies need to completed, focusing specifically on SRp20, to confirm this supposition.	+	SRp20 has a <scene name='78/781963/Hydrophobic_core_residues/1'>hydrophobic core</scene>, which may contribute to the stability of the protein. A previous study, looking at the RRM in [https://en.wikipedia.org/wiki/TARDBP TDP-43] has suggested that the hydrophobic core may be a strong contributing factor to the protein’s stability <ref>PMID:24497641</ref>. In a different study, it was determined that, in the U11/U12-65K protein, the β-sheet packs against the two α-helices via hydrophobic interactions and that the resulting stabilization could be critical for the proper folding and orientation of elements for RNA binding <ref>PMID:19447915</ref>. Due to the conservative nature of RRMs, it could be speculated that the hydrophobic core found in SRp20, between the β-sheet and two α-helices, could contribute to the stability of its RRM in a similar fashion. However, additional studies need to completed, focusing specifically on SRp20, to confirm this supposition.

	===RRM Specificity===		===RRM Specificity===

Alexis Neyman at 17:53, 23 April 2018

Alexis Neyman — Mon, 23 Apr 2018 17:53:54 GMT

←Older revision		Revision as of 17:53, 23 April 2018
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	=== Poor Solubility Problem ===		=== Poor Solubility Problem ===
-	The SRp20 protein has poor [https://en.wikipedia.org/wiki/Solubility solubility] in its free state. This made it impossible to determine the structure of SRp20 using HSQC Spectroscopy without a modification to the free state protein. This problem was resolved by purifying the proteins after fusing the RRM (RNA-recognition motif) with the immunoglobulin G-binding domain 1 of Streptococcal [https://en.wikipedia.org/wiki/Protein_G Protein G] GB1 solubility tag (Figure 2) <ref name="Hargous">PMID:17036044</ref>. [[Image:Figure_Three_Solubility_Tag_Edited.jpg\|~~250~~ px\|~~left~~\|thumb\|Figure 2: SRp20 with the solubility IgG tag and the RNA ligand are shown. The solubility tag is at the N' terminus, in front of the RRM. While in the actual protein, the RRM is at the N'terminus, in front of the RS domain. Image created using ''Pymol'']]	+	The SRp20 protein has poor [https://en.wikipedia.org/wiki/Solubility solubility] in its free state. This made it impossible to determine the structure of SRp20 using HSQC Spectroscopy without a modification to the free state protein. This problem was resolved by purifying the proteins after fusing the RRM (RNA-recognition motif) with the immunoglobulin G-binding domain 1 of Streptococcal [https://en.wikipedia.org/wiki/Protein_G Protein G] GB1 solubility tag (Figure 2) <ref name="Hargous">PMID:17036044</ref>. [[Image:Figure_Three_Solubility_Tag_Edited.jpg\|300 px\|right\|thumb\|Figure 2: SRp20 with the solubility IgG tag and the RNA ligand are shown. The solubility tag is at the N' terminus, in front of the RRM. While in the actual protein, the RRM is at the N'terminus, in front of the RS domain. Image created using ''Pymol'']]

	=== RNA Interactions ===		=== RNA Interactions ===

Alexis Neyman at 17:51, 23 April 2018

Alexis Neyman — Mon, 23 Apr 2018 17:51:58 GMT

←Older revision		Revision as of 17:51, 23 April 2018
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	== Relationship to 9G8 ==		== Relationship to 9G8 ==
-	<scene name='78/781963/Rrm_motif/1'>SRp20</scene> and splicing factor <scene name='78/781963/9g8_rrm/1'>9G8</scene> are both sequence specific RNA binding proteins (Figure 4) and are the smallest members of the Serine-and-Arginine Rich (SR) protein family. [[Image:Combined_SRp20_and_9G8_Image.jpg\|~~300~~ px\|left\|thumb\|Figure 4: Comparing SRp20 and 9G8 RRMs and sequence alignments. Structural images created using ''Pymol'']] Both RNA Recognition Motifs (RRMs) have a similar βαββαβ topology. SRp20 and 9G8 are 80% identical. The sequence alignment shows the alignment of the RRMs of SRp20 and 9G8 <ref name="Hargous">PMID:17036044</ref>(Figure 4). SRp20 binds pyrimidine rich areas while 9G8 binds purine rich areas.This difference in binding comes from the fact that 9G8 has a [https://en.wikipedia.org/wiki/Zinc_finger zinc knuckle] that recognizes GAC triplets <ref name="Cava">PMID:10094314 </ref>. 9G8s RRM is followed by a zinc knuckle and then the SR domain whereas SRp20s RRM is followed directly by the SR domain. When 9G8 lacks a zinc knuckle, it binds pyrimidine-rich sequences like SRp20 <ref name="Hargous">PMID:17036044</ref>. The zinc knuckle of 9G8 contains glycine residues at positions 5 and 8 and charged residues at positions 6 and 13 that are highly conserved <ref name="Cava">PMID:10094314 </ref>. Due to the poor solubility problem, a structure for the zinc knuckle of 9G8 is not available to show in an image.	+	<scene name='78/781963/Rrm_motif/1'>SRp20</scene> and splicing factor <scene name='78/781963/9g8_rrm/1'>9G8</scene> are both sequence specific RNA binding proteins (Figure 4) and are the smallest members of the Serine-and-Arginine Rich (SR) protein family. [[Image:Combined_SRp20_and_9G8_Image.jpg\|350 px\|left\|thumb\|Figure 4: Comparing SRp20 and 9G8 RRMs and sequence alignments. Structural images created using ''Pymol'']] Both RNA Recognition Motifs (RRMs) have a similar βαββαβ topology. SRp20 and 9G8 are 80% identical. The sequence alignment shows the alignment of the RRMs of SRp20 and 9G8 <ref name="Hargous">PMID:17036044</ref>(Figure 4). SRp20 binds pyrimidine rich areas while 9G8 binds purine rich areas.This difference in binding comes from the fact that 9G8 has a [https://en.wikipedia.org/wiki/Zinc_finger zinc knuckle] that recognizes GAC triplets <ref name="Cava">PMID:10094314 </ref>. 9G8s RRM is followed by a zinc knuckle and then the SR domain whereas SRp20s RRM is followed directly by the SR domain. When 9G8 lacks a zinc knuckle, it binds pyrimidine-rich sequences like SRp20 <ref name="Hargous">PMID:17036044</ref>. The zinc knuckle of 9G8 contains glycine residues at positions 5 and 8 and charged residues at positions 6 and 13 that are highly conserved <ref name="Cava">PMID:10094314 </ref>. Due to the poor solubility problem, a structure for the zinc knuckle of 9G8 is not available to show in an image.
	</StructureSection>		</StructureSection>
-	~~== Disease ==~~	+	== Cancer ==
-	===Cancer===	+
	There have been findings that support the role of SRp20 in cellular proliferation/maturation. It was discovered that there was an over expression of SRp20 in breast cancer tissues. When SRp20 was reduced in cancer cells via [https://en.wikipedia.org/wiki/Small_interfering_RNA siRNA], targets SRp20 mRNA, there was reduction in cell proliferation and increase in [https://en.wikipedia.org/wiki/Apoptosis cellular apoptosis]. For example, it was speculated that SRp20 might be involved in alternative splicing of [https://en.wikipedia.org/wiki/FOXM1 ''FoxM1''], a transcription factor involved in cellular proliferation, by either the inclusion or exclusion of exon 9 in ''FoxM1'' transcript. If exon 9 was excluded from the ''FoxM1'' mRNA via SRp20, then there was an increase in ''FoxM1'' expression, cellular proliferation, and reduction in cell apoptosis<ref name="Jia">PMID:21179588</ref>. Apoptosis is a necessary function to maintain homeostasis, and an imbalance in the regulation in apoptosis can lead to uncontrolled cell proliferation and tumor development. Due to the alternative splicing functionality of SRp20, it effects many other genes involved in cancer such as [https://en.wikipedia.org/wiki/CD44 ''CD44''] gene, [https://en.wikipedia.org/wiki/PKM2 ''PK-M''] gene, [https://en.wikipedia.org/wiki/Tau_protein ''TAU''] gene, [https://en.wikipedia.org/wiki/P53 ''TP53''] gene, and involved in [https://en.wikipedia.org/wiki/Wnt_signaling_pathway WnT signaling pathway]<ref name="corbo">PMID:23685143</ref>. Although it has been understood that SRp20 plays a crucial role in cancer cells, the mechanism by which SRp20 affects these genes, and how its structure contributes to the development of oncogenic genes, is still unclear<ref name="Jia">PMID:21179588</ref><ref name="Jang">PMID:24321384</ref>.		There have been findings that support the role of SRp20 in cellular proliferation/maturation. It was discovered that there was an over expression of SRp20 in breast cancer tissues. When SRp20 was reduced in cancer cells via [https://en.wikipedia.org/wiki/Small_interfering_RNA siRNA], targets SRp20 mRNA, there was reduction in cell proliferation and increase in [https://en.wikipedia.org/wiki/Apoptosis cellular apoptosis]. For example, it was speculated that SRp20 might be involved in alternative splicing of [https://en.wikipedia.org/wiki/FOXM1 ''FoxM1''], a transcription factor involved in cellular proliferation, by either the inclusion or exclusion of exon 9 in ''FoxM1'' transcript. If exon 9 was excluded from the ''FoxM1'' mRNA via SRp20, then there was an increase in ''FoxM1'' expression, cellular proliferation, and reduction in cell apoptosis<ref name="Jia">PMID:21179588</ref>. Apoptosis is a necessary function to maintain homeostasis, and an imbalance in the regulation in apoptosis can lead to uncontrolled cell proliferation and tumor development. Due to the alternative splicing functionality of SRp20, it effects many other genes involved in cancer such as [https://en.wikipedia.org/wiki/CD44 ''CD44''] gene, [https://en.wikipedia.org/wiki/PKM2 ''PK-M''] gene, [https://en.wikipedia.org/wiki/Tau_protein ''TAU''] gene, [https://en.wikipedia.org/wiki/P53 ''TP53''] gene, and involved in [https://en.wikipedia.org/wiki/Wnt_signaling_pathway WnT signaling pathway]<ref name="corbo">PMID:23685143</ref>. Although it has been understood that SRp20 plays a crucial role in cancer cells, the mechanism by which SRp20 affects these genes, and how its structure contributes to the development of oncogenic genes, is still unclear<ref name="Jia">PMID:21179588</ref><ref name="Jang">PMID:24321384</ref>.

Alexis Neyman at 16:01, 23 April 2018

Alexis Neyman — Mon, 23 Apr 2018 16:01:06 GMT

←Older revision		Revision as of 16:01, 23 April 2018
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	=Biological Structure of SRp20=		=Biological Structure of SRp20=
-	~~<StructureSection load='2i2yRRM.pdb' size='350' side='right' caption='SRp20 Structure' scene='78/781963/Opening_scene/1'>~~	+

	== Introduction ==		== Introduction ==
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	== Structure ==		== Structure ==
		+	<StructureSection load='2i2yRRM.pdb' size='350' side='right' caption='SRp20 Structure' scene='78/781963/Opening_scene/1'>__NoTOC__
	[[Image:Figure_1_edited_2D_SRp20.jpg\|250 px\|right\|thumb\|Figure 1: SRp20 RRM and SR domains are shown]] The structure of SRp20 was determined by heteronuclear single quantum coherence ([https://en.wikipedia.org/wiki/Heteronuclear_single_quantum_coherence_spectroscopy HSQC]) NMR. The structure is composed of one RNA recognition motif (RRM) at the N-terminus and one Ser/Arg (SR) domain at the C-terminus where the Ser residues are phosphorylated<ref name="corbo">PMID:23685143</ref>. The RRM of SRp20 demonstrates the β1α1β2β3α2β3 topology seen in other [https://en.wikipedia.org/wiki/RNA_recognition_motif RRMs]. The role of the RRM region is to provide substrate specificity where SRp20 interacts with splicing enhancing sequences in mRNA. There have been no determined 3D structures of the SR domain thus it is unclear what its exact role is. However, there has been some speculation that it might be involved in aiding protein-protein interactions in the spliceosome. It contains 164 amino acids, half belonging to the RRM and other half to the SR domain (Figure 1). SRp20 has a molecular weight of 19 kDA<ref name="corbo">PMID:23685143</ref>.		[[Image:Figure_1_edited_2D_SRp20.jpg\|250 px\|right\|thumb\|Figure 1: SRp20 RRM and SR domains are shown]] The structure of SRp20 was determined by heteronuclear single quantum coherence ([https://en.wikipedia.org/wiki/Heteronuclear_single_quantum_coherence_spectroscopy HSQC]) NMR. The structure is composed of one RNA recognition motif (RRM) at the N-terminus and one Ser/Arg (SR) domain at the C-terminus where the Ser residues are phosphorylated<ref name="corbo">PMID:23685143</ref>. The RRM of SRp20 demonstrates the β1α1β2β3α2β3 topology seen in other [https://en.wikipedia.org/wiki/RNA_recognition_motif RRMs]. The role of the RRM region is to provide substrate specificity where SRp20 interacts with splicing enhancing sequences in mRNA. There have been no determined 3D structures of the SR domain thus it is unclear what its exact role is. However, there has been some speculation that it might be involved in aiding protein-protein interactions in the spliceosome. It contains 164 amino acids, half belonging to the RRM and other half to the SR domain (Figure 1). SRp20 has a molecular weight of 19 kDA<ref name="corbo">PMID:23685143</ref>.

Line 35:		Line 36:
	== Relationship to 9G8 ==		== Relationship to 9G8 ==
	<scene name='78/781963/Rrm_motif/1'>SRp20</scene> and splicing factor <scene name='78/781963/9g8_rrm/1'>9G8</scene> are both sequence specific RNA binding proteins (Figure 4) and are the smallest members of the Serine-and-Arginine Rich (SR) protein family. [[Image:Combined_SRp20_and_9G8_Image.jpg\|300 px\|left\|thumb\|Figure 4: Comparing SRp20 and 9G8 RRMs and sequence alignments. Structural images created using ''Pymol'']] Both RNA Recognition Motifs (RRMs) have a similar βαββαβ topology. SRp20 and 9G8 are 80% identical. The sequence alignment shows the alignment of the RRMs of SRp20 and 9G8 <ref name="Hargous">PMID:17036044</ref>(Figure 4). SRp20 binds pyrimidine rich areas while 9G8 binds purine rich areas.This difference in binding comes from the fact that 9G8 has a [https://en.wikipedia.org/wiki/Zinc_finger zinc knuckle] that recognizes GAC triplets <ref name="Cava">PMID:10094314 </ref>. 9G8s RRM is followed by a zinc knuckle and then the SR domain whereas SRp20s RRM is followed directly by the SR domain. When 9G8 lacks a zinc knuckle, it binds pyrimidine-rich sequences like SRp20 <ref name="Hargous">PMID:17036044</ref>. The zinc knuckle of 9G8 contains glycine residues at positions 5 and 8 and charged residues at positions 6 and 13 that are highly conserved <ref name="Cava">PMID:10094314 </ref>. Due to the poor solubility problem, a structure for the zinc knuckle of 9G8 is not available to show in an image.		<scene name='78/781963/Rrm_motif/1'>SRp20</scene> and splicing factor <scene name='78/781963/9g8_rrm/1'>9G8</scene> are both sequence specific RNA binding proteins (Figure 4) and are the smallest members of the Serine-and-Arginine Rich (SR) protein family. [[Image:Combined_SRp20_and_9G8_Image.jpg\|300 px\|left\|thumb\|Figure 4: Comparing SRp20 and 9G8 RRMs and sequence alignments. Structural images created using ''Pymol'']] Both RNA Recognition Motifs (RRMs) have a similar βαββαβ topology. SRp20 and 9G8 are 80% identical. The sequence alignment shows the alignment of the RRMs of SRp20 and 9G8 <ref name="Hargous">PMID:17036044</ref>(Figure 4). SRp20 binds pyrimidine rich areas while 9G8 binds purine rich areas.This difference in binding comes from the fact that 9G8 has a [https://en.wikipedia.org/wiki/Zinc_finger zinc knuckle] that recognizes GAC triplets <ref name="Cava">PMID:10094314 </ref>. 9G8s RRM is followed by a zinc knuckle and then the SR domain whereas SRp20s RRM is followed directly by the SR domain. When 9G8 lacks a zinc knuckle, it binds pyrimidine-rich sequences like SRp20 <ref name="Hargous">PMID:17036044</ref>. The zinc knuckle of 9G8 contains glycine residues at positions 5 and 8 and charged residues at positions 6 and 13 that are highly conserved <ref name="Cava">PMID:10094314 </ref>. Due to the poor solubility problem, a structure for the zinc knuckle of 9G8 is not available to show in an image.
-		+	</StructureSection>
	== Disease ==		== Disease ==
	===Cancer===		===Cancer===

Alexis Neyman at 15:58, 23 April 2018

Alexis Neyman — Mon, 23 Apr 2018 15:58:07 GMT

←Older revision		Revision as of 15:58, 23 April 2018
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	=== RRM Stability===		=== RRM Stability===
-	SRp20 has a ~~<scene name='78/781963/Hydrophobic_core/1'>hydrophobic core</scene>~~ <scene name='78/781963/Hydrophobic_core_residues/1'>hydrophobic core</scene>, which may contribute to the stability of the protein. A previous study, looking at the RRM in [https://en.wikipedia.org/wiki/TARDBP TDP-43] has suggested that the hydrophobic core may be a strong contributing factor to the protein’s stability <ref>PMID:24497641</ref>. In a different study, it was determined that, in the U11/U12-65K protein, the β-sheet packs against the two α-helices by way of hydrophobic interactions and that the resulting stabilization could be critical for the proper folding and orientation of elements for RNA binding <ref>PMID:19447915</ref>. Due to the conservative nature of RRMs, it could be speculated that the hydrophobic core found in SRp20, between the β-sheet and two α-helices, could contribute to the stability of its RRM in a similar fashion. However, additional studies need to completed, focusing specifically on SRp20, to confirm this supposition.	+	SRp20 has a <scene name='78/781963/Hydrophobic_core_residues/1'>hydrophobic core</scene>, which may contribute to the stability of the protein. A previous study, looking at the RRM in [https://en.wikipedia.org/wiki/TARDBP TDP-43] has suggested that the hydrophobic core may be a strong contributing factor to the protein’s stability <ref>PMID:24497641</ref>. In a different study, it was determined that, in the U11/U12-65K protein, the β-sheet packs against the two α-helices by way of hydrophobic interactions and that the resulting stabilization could be critical for the proper folding and orientation of elements for RNA binding <ref>PMID:19447915</ref>. Due to the conservative nature of RRMs, it could be speculated that the hydrophobic core found in SRp20, between the β-sheet and two α-helices, could contribute to the stability of its RRM in a similar fashion. However, additional studies need to completed, focusing specifically on SRp20, to confirm this supposition.

	===RRM Specificity===		===RRM Specificity===

Alexis Neyman at 15:56, 23 April 2018

Alexis Neyman — Mon, 23 Apr 2018 15:56:31 GMT

←Older revision		Revision as of 15:56, 23 April 2018
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	=== RRM Stability===		=== RRM Stability===
-	SRp20 has a <scene name='78/781963/Hydrophobic_core/1'>hydrophobic core</scene>, which may contribute to the stability of the protein. A previous study, looking at the RRM in [https://en.wikipedia.org/wiki/TARDBP TDP-43] has suggested that the hydrophobic core may be a strong contributing factor to the protein’s stability <ref>PMID:24497641</ref>. In a different study, it was determined that, in the U11/U12-65K protein, the β-sheet packs against the two α-helices by way of hydrophobic interactions and that the resulting stabilization could be critical for the proper folding and orientation of elements for RNA binding <ref>PMID:19447915</ref>. Due to the conservative nature of RRMs, it could be speculated that the hydrophobic core found in SRp20, between the β-sheet and two α-helices, could contribute to the stability of its RRM in a similar fashion. However, additional studies need to completed, focusing specifically on SRp20, to confirm this supposition.	+	SRp20 has a <scene name='78/781963/Hydrophobic_core/1'>hydrophobic core</scene> <scene name='78/781963/Hydrophobic_core_residues/1'>hydrophobic core</scene>, which may contribute to the stability of the protein. A previous study, looking at the RRM in [https://en.wikipedia.org/wiki/TARDBP TDP-43] has suggested that the hydrophobic core may be a strong contributing factor to the protein’s stability <ref>PMID:24497641</ref>. In a different study, it was determined that, in the U11/U12-65K protein, the β-sheet packs against the two α-helices by way of hydrophobic interactions and that the resulting stabilization could be critical for the proper folding and orientation of elements for RNA binding <ref>PMID:19447915</ref>. Due to the conservative nature of RRMs, it could be speculated that the hydrophobic core found in SRp20, between the β-sheet and two α-helices, could contribute to the stability of its RRM in a similar fashion. However, additional studies need to completed, focusing specifically on SRp20, to confirm this supposition.

	===RRM Specificity===		===RRM Specificity===

Alexis Neyman at 16:48, 17 April 2018

Alexis Neyman — Tue, 17 Apr 2018 16:48:43 GMT

←Older revision		Revision as of 16:48, 17 April 2018
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	== Structure ==		== Structure ==
-	[[Image:Figure_1_edited_2D_SRp20.jpg\|250 px\|right\|thumb\|Figure 1: SRp20 RRM and RS domains are shown]] The structure of SRp20 was determined by heteronuclear single quantum coherence ([https://en.wikipedia.org/wiki/Heteronuclear_single_quantum_coherence_spectroscopy HSQC]) NMR. The structure is composed of one RNA recognition motif (RRM) at the N-terminus and one Ser/Arg (SR) domain at the C-terminus where the Ser residues are phosphorylated<ref name="corbo">PMID:23685143</ref>. The RRM of SRp20 demonstrates the β1α1β2β3α2β3 topology seen in other [https://en.wikipedia.org/wiki/RNA_recognition_motif RRMs]. The role of the RRM region is to provide substrate specificity where SRp20 interacts with splicing enhancing sequences in mRNA. There have been no determined 3D structures of the SR domain thus it is unclear what its exact role is. However, there has been some speculation that it might be involved in aiding protein-protein interactions in the spliceosome. It contains 164 amino acids, half belonging to the RRM and other half to the SR domain (Figure 1). SRp20 has a molecular weight of 19 kDA<ref name="corbo">PMID:23685143</ref>.	+	[[Image:Figure_1_edited_2D_SRp20.jpg\|250 px\|right\|thumb\|Figure 1: SRp20 RRM and SR domains are shown]] The structure of SRp20 was determined by heteronuclear single quantum coherence ([https://en.wikipedia.org/wiki/Heteronuclear_single_quantum_coherence_spectroscopy HSQC]) NMR. The structure is composed of one RNA recognition motif (RRM) at the N-terminus and one Ser/Arg (SR) domain at the C-terminus where the Ser residues are phosphorylated<ref name="corbo">PMID:23685143</ref>. The RRM of SRp20 demonstrates the β1α1β2β3α2β3 topology seen in other [https://en.wikipedia.org/wiki/RNA_recognition_motif RRMs]. The role of the RRM region is to provide substrate specificity where SRp20 interacts with splicing enhancing sequences in mRNA. There have been no determined 3D structures of the SR domain thus it is unclear what its exact role is. However, there has been some speculation that it might be involved in aiding protein-protein interactions in the spliceosome. It contains 164 amino acids, half belonging to the RRM and other half to the SR domain (Figure 1). SRp20 has a molecular weight of 19 kDA<ref name="corbo">PMID:23685143</ref>.

	=== Poor Solubility Problem ===		=== Poor Solubility Problem ===

Alexis Neyman at 16:48, 17 April 2018

Alexis Neyman — Tue, 17 Apr 2018 16:48:17 GMT

←Older revision		Revision as of 16:48, 17 April 2018
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	== Structure ==		== Structure ==
-	[[Image:~~2D_RRM_and_RS_SRp20_with_fun_shapes3~~.~~png~~\|250 px\|right\|thumb\|Figure 1: SRp20 RRM and RS domains are shown]] The structure of SRp20 was determined by heteronuclear single quantum coherence ([https://en.wikipedia.org/wiki/Heteronuclear_single_quantum_coherence_spectroscopy HSQC]) NMR. The structure is composed of one RNA recognition motif (RRM) at the N-terminus and one Ser/Arg (SR) domain at the C-terminus where the Ser residues are phosphorylated<ref name="corbo">PMID:23685143</ref>. The RRM of SRp20 demonstrates the β1α1β2β3α2β3 topology seen in other [https://en.wikipedia.org/wiki/RNA_recognition_motif RRMs]. The role of the RRM region is to provide substrate specificity where SRp20 interacts with splicing enhancing sequences in mRNA. There have been no determined 3D structures of the SR domain thus it is unclear what its exact role is. However, there has been some speculation that it might be involved in aiding protein-protein interactions in the spliceosome. It contains 164 amino acids, half belonging to the RRM and other half to the SR domain (Figure 1). SRp20 has a molecular weight of 19 kDA<ref name="corbo">PMID:23685143</ref>.	+	[[Image:Figure_1_edited_2D_SRp20.jpg\|250 px\|right\|thumb\|Figure 1: SRp20 RRM and RS domains are shown]] The structure of SRp20 was determined by heteronuclear single quantum coherence ([https://en.wikipedia.org/wiki/Heteronuclear_single_quantum_coherence_spectroscopy HSQC]) NMR. The structure is composed of one RNA recognition motif (RRM) at the N-terminus and one Ser/Arg (SR) domain at the C-terminus where the Ser residues are phosphorylated<ref name="corbo">PMID:23685143</ref>. The RRM of SRp20 demonstrates the β1α1β2β3α2β3 topology seen in other [https://en.wikipedia.org/wiki/RNA_recognition_motif RRMs]. The role of the RRM region is to provide substrate specificity where SRp20 interacts with splicing enhancing sequences in mRNA. There have been no determined 3D structures of the SR domain thus it is unclear what its exact role is. However, there has been some speculation that it might be involved in aiding protein-protein interactions in the spliceosome. It contains 164 amino acids, half belonging to the RRM and other half to the SR domain (Figure 1). SRp20 has a molecular weight of 19 kDA<ref name="corbo">PMID:23685143</ref>.

	=== Poor Solubility Problem ===		=== Poor Solubility Problem ===

Alexis Neyman at 16:45, 17 April 2018

Alexis Neyman — Tue, 17 Apr 2018 16:45:47 GMT

(Difference between revisions)

Alexis Neyman at 16:12, 17 April 2018

Alexis Neyman — Tue, 17 Apr 2018 16:12:56 GMT

←Older revision		Revision as of 16:12, 17 April 2018
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	== Structure ==		== Structure ==
-	[[Image:2D_RRM_and_RS_SRp20_with_fun_shapes3.png\|250 px\|right\|thumb\|Figure 1: SRp20 RRM and RS domains are shown]] The structure of SRp20 was determined by heteronuclear single quantum coherence ([https://en.wikipedia.org/wiki/Heteronuclear_single_quantum_coherence_spectroscopy HSQC]) NMR. The structure is composed of one RNA recognition motif (RRM) at the N-terminus and one Arg/Ser (AR) domain at the C-terminus where the Ser residues are phosphorylated<ref name="corbo">PMID:23685143</ref>. The RRM of SRp20 demonstrates the β1α1β2β3α2β3 topology seen in other [https://en.wikipedia.org/wiki/RNA_recognition_motif RRMs]. The role of the RRM region is to provide substrate specificity where SRp20 interacts with splicing enhancing sequences in mRNA. There have been no determined 3D structures of the RS domain thus it is unclear what its exact role is. However there have been some speculation that it might be involved in aiding protein-protein interactions in the spliceosome. It contains 164 amino acids, half belonging to the RRM and other half to the RS domain (Figure 1). SRp20 has a molecular weight of 19 kDA<ref name="corbo">PMID:23685143</ref>.	+	[[Image:2D_RRM_and_RS_SRp20_with_fun_shapes3.png\|250 px\|right\|thumb\|Figure 1: SRp20 RRM and RS domains are shown]] The structure of SRp20 was determined by heteronuclear single quantum coherence ([https://en.wikipedia.org/wiki/Heteronuclear_single_quantum_coherence_spectroscopy HSQC]) NMR. The structure is composed of one RNA recognition motif (RRM) at the N-terminus and one Arg/Ser (AR) domain at the C-terminus where the Ser residues are phosphorylated<ref name="corbo">PMID:23685143</ref>. The RRM of SRp20 demonstrates the β1α1β2β3α2β3 topology seen in other [https://en.wikipedia.org/wiki/RNA_recognition_motif RRMs]. The role of the RRM region is to provide substrate specificity where SRp20 interacts with splicing enhancing sequences in mRNA. There have been no determined 3D structures of the RS domain thus it is unclear what its exact role is. However, there have been some speculation that it might be involved in aiding protein-protein interactions in the spliceosome. It contains 164 amino acids, half belonging to the RRM and other half to the RS domain (Figure 1). SRp20 has a molecular weight of 19 kDA<ref name="corbo">PMID:23685143</ref>.

	=== Poor Solubility Problem ===		=== Poor Solubility Problem ===
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	1H-15N HSQC results showed a large hydrophobic β-sheet on the RRM binding to the RNA with all four bases interacting with one of the four aromatic residues via hydrophobic interactions <ref name="Hargous">PMID:17036044</ref>. [https://en.wikipedia.org/wiki/Beta_hairpin β-hairpin] amino acids are hydrogen bonded to bases on nucleic acid targets <ref name="Clery">PMID:18515081</ref>. This suggests that the β-hairpin plays a role in SRp20 selectivity for specific ligands. The researchers used a smaller peptide chain to reduce the NMR broadening seen with longer peptides (allowing for structure determination), with the consequence of reduced binding affinity.		1H-15N HSQC results showed a large hydrophobic β-sheet on the RRM binding to the RNA with all four bases interacting with one of the four aromatic residues via hydrophobic interactions <ref name="Hargous">PMID:17036044</ref>. [https://en.wikipedia.org/wiki/Beta_hairpin β-hairpin] amino acids are hydrogen bonded to bases on nucleic acid targets <ref name="Clery">PMID:18515081</ref>. This suggests that the β-hairpin plays a role in SRp20 selectivity for specific ligands. The researchers used a smaller peptide chain to reduce the NMR broadening seen with longer peptides (allowing for structure determination), with the consequence of reduced binding affinity.

-	The ligand used was <scene name='78/781963/Looking_at_the_ligand/1'>CAUC</scene>. The conformation of U3 and C4 shows that U3 bulges out while C4 partially stacks over A2. Interactions with the RRM that ~~the researchers saw~~ were that <scene name='78/781963/C1_and_tyr_13/3'>C1 stacks with Tyr 13</scene> in β1 and <scene name='78/781963/A2_phe_50/2'>A2 stacks with Phe 50</scene> in β3. These aromatic side chains form hydrophobic interactions with the ligand when stacked (Figure 3). Also, the residue <scene name='78/781963/C1_a2_phe48/2'>F48 inserts between the sugar rings of C1 and A2</scene>. <scene name='78/781963/C1_binding_pocket3/1'>C1 is recognized definitively by the RRM</scene>. The amino proton of C1 hydrogen bonds with the carbonyl oxygen of Leu 80 and the side-chain carbonyl oxygen of Glu 79. The N3 of C1 hydrogen bonds with the amide of Asn 82, and the O2 of C1 hydrogen bonds with the hydroxyl group of Ser 81<ref name="Hargous">PMID:17036044</ref>.	+	The ligand used was <scene name='78/781963/Looking_at_the_ligand/1'>CAUC</scene>. The conformation of U3 and C4 shows that U3 bulges out while C4 partially stacks over A2. Interactions with the RRM that were noticed were that <scene name='78/781963/C1_and_tyr_13/3'>C1 stacks with Tyr 13</scene> in β1 and <scene name='78/781963/A2_phe_50/2'>A2 stacks with Phe 50</scene> in β3. These aromatic side chains form hydrophobic interactions with the ligand when stacked (Figure 3). Also, the residue <scene name='78/781963/C1_a2_phe48/2'>F48 inserts between the sugar rings of C1 and A2</scene>. <scene name='78/781963/C1_binding_pocket3/1'>C1 is recognized definitively by the RRM</scene>. The amino proton of C1 hydrogen bonds with the carbonyl oxygen of Leu 80 and the side-chain carbonyl oxygen of Glu 79. The N3 of C1 hydrogen bonds with the amide of Asn 82, and the O2 of C1 hydrogen bonds with the hydroxyl group of Ser 81<ref name="Hargous">PMID:17036044</ref>.

	It was also noted that <scene name='78/781963/A2_syn_conformation/1'>A2</scene> adopts an unusual syn conformation. U3 interacts with <scene name='78/781963/U3_hydrophobic_interactions/2'>Phe 48, Trp 40, Ala 42,</scene> and with the β2-3 loop of the RRM. These residues are all hydrophobic, offering a large hydrophobic surface that helps bind the ligand, as well as prevents the solvent from binding. Additionally, C4 is maintained in its position by a <scene name='78/781963/C4_a2_h_bond/1'>hydrogen bond between C4 amino group and the A2 2’ oxygen</scene> <ref name="Hargous">PMID:17036044</ref>. [[Image:Figure_4_C1_and_A2_interactions_Edited2.png\|300 px\|left\|thumb\|Figure 3: C1 and A2 on the RNA ligand interacting with hydrophobic residues (Tyr 13, Phe 50, Phe 48) in the RRM domain of the SRp20 protein. Image created using ''Pymol'']]		It was also noted that <scene name='78/781963/A2_syn_conformation/1'>A2</scene> adopts an unusual syn conformation. U3 interacts with <scene name='78/781963/U3_hydrophobic_interactions/2'>Phe 48, Trp 40, Ala 42,</scene> and with the β2-3 loop of the RRM. These residues are all hydrophobic, offering a large hydrophobic surface that helps bind the ligand, as well as prevents the solvent from binding. Additionally, C4 is maintained in its position by a <scene name='78/781963/C4_a2_h_bond/1'>hydrogen bond between C4 amino group and the A2 2’ oxygen</scene> <ref name="Hargous">PMID:17036044</ref>. [[Image:Figure_4_C1_and_A2_interactions_Edited2.png\|300 px\|left\|thumb\|Figure 3: C1 and A2 on the RNA ligand interacting with hydrophobic residues (Tyr 13, Phe 50, Phe 48) in the RRM domain of the SRp20 protein. Image created using ''Pymol'']]
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	== Relationship to 9G8 ==		== Relationship to 9G8 ==
-	[[Image:Combined_SRp20_and_9G8_Image.jpg\|300 px\|left\|thumb\|Figure 4: Comparing SRp20 and 9G8 RRMs and sequence alignments. Structural images created using ''Pymol'']] <scene name='78/781963/Rrm_motif/1'>SRp20</scene> and splicing factor <scene name='78/781963/9g8_rrm/1'>9G8</scene> are both sequence specific RNA binding proteins (Figure 4) and are the smallest members of the Serine-and-Arginine Rich (SR) protein family. Both RNA Recognition Motifs (RRMs) have a similar βαββαβ topology. SRp20 and 9G8 are 80% identical. The sequence alignment shows the alignment of the RRMs of SRp20 and 9G8 <ref name="Hargous">PMID:17036044</ref>. SRp20 binds pyrimidine rich areas while 9G8 binds purine rich areas.This difference in binding comes from the fact that 9G8 has a [https://en.wikipedia.org/wiki/Zinc_finger zinc knuckle] that recognizes GAC triplets <ref name="Cava">PMID:10094314 </ref>. 9G8s RRM is followed by a zinc knuckle and then the SR domain whereas SRp20s RRM is followed directly by the SR domain. When 9G8 lacks a zinc knuckle, it binds pyrimidine-rich sequences like SRp20 <ref name="Hargous">PMID:17036044</ref>. The zinc knuckle of 9G8 contains glycine residues at positions 5 and 8 and charged residues at positions 6 and 13 that are highly conserved <ref name="Cava">PMID:10094314 </ref>. Due to the poor solubility problem, a structure for the zinc knuckle of 9G8 is not available to show in an image.	+	[[Image:Combined_SRp20_and_9G8_Image.jpg\|300 px\|left\|thumb\|Figure 4: Comparing SRp20 and 9G8 RRMs and sequence alignments. Structural images created using ''Pymol'']] <scene name='78/781963/Rrm_motif/1'>SRp20</scene> and splicing factor <scene name='78/781963/9g8_rrm/1'>9G8</scene> are both sequence specific RNA binding proteins (Figure 4) and are the smallest members of the Serine-and-Arginine Rich (SR) protein family. Both RNA Recognition Motifs (RRMs) have a similar βαββαβ topology. SRp20 and 9G8 are 80% identical. The sequence alignment shows the alignment of the RRMs of SRp20 and 9G8 <ref name="Hargous">PMID:17036044</ref>(Figure 4). SRp20 binds pyrimidine rich areas while 9G8 binds purine rich areas.This difference in binding comes from the fact that 9G8 has a [https://en.wikipedia.org/wiki/Zinc_finger zinc knuckle] that recognizes GAC triplets <ref name="Cava">PMID:10094314 </ref>. 9G8s RRM is followed by a zinc knuckle and then the SR domain whereas SRp20s RRM is followed directly by the SR domain. When 9G8 lacks a zinc knuckle, it binds pyrimidine-rich sequences like SRp20 <ref name="Hargous">PMID:17036044</ref>. The zinc knuckle of 9G8 contains glycine residues at positions 5 and 8 and charged residues at positions 6 and 13 that are highly conserved <ref name="Cava">PMID:10094314 </ref>. Due to the poor solubility problem, a structure for the zinc knuckle of 9G8 is not available to show in an image.

	== Disease ==		== Disease ==