User:Wally Novak/Sandbox Whitaker

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U5 snRNP

1 Function
2 Structural Highlights
- 2.1 Brr2 Structural Highlights
- 2.2 Prp8 Structural Highlights
3 Protein Interactions

Function

General Overview

The U5 snRNP consists of several proteins which are highly conserved from yeast to humans. These proteins consist of , , , Prp28, Snu40/52K, and ^[1], however, only Prp8, Snu114, and Brr2 remain in complex with the U5 snRNA while the spliceosome is activated ^[2]. These proteins are necessary for the remodeling of the spliceosome and may play a role in maintaining catalytic activity at the active center of the spliceosome ^[2]. Assembly of the U5 snRNP is followed by the formation of the U4/U6.U5 which is the last remaining complex necessary to form the complete spliceosome and is therefore vital for its function.

U5 snRNA

The U5 snRNA interacts directly with the 5’ exon before the first step of splicing and with the 5’ and 3’ exons following the first step of splicing ^[3]. These U5–exon interactions are essential for tethering and aligning the exons for ligation during the second step of splicing ^[3].

Brr2

Brr2 is an ATPase required for unwinding U4/U6 base-pairing before the first step of splicing and for unwinding U2/U6 base-pairing after the second step of splicing ^[4]. These conformational changes catalyzed by Brr2 are essential for the progression of the spliceosome cycle.

Prp8

Prp8 is believed to be the master regulator of the splicing cycle by regulating the spliceosomal DExD/H-box RNA dependent ATPases, specifically Brr2 ^[5]. Consistent with this regulatory role it has been shown that the C-terminus of Prp8 activates Brr2 helicase function and inhibits Brr2's U4/U6-dependent ATPase activity in vitro ^[6]. Structural studies of the Prp8 C-terminus have identified an RNase H-like domain within Prp8 and it has been proposed that this RNase H-domain may form the active site of the spliceosome ^[2].

Snu114

is the only GTPase associated with the spliceosome and it has multiple contacts with Prp8 as well as U5 snRNA and Brr2 but its role in spliceosome function is not yet well-defined. ^[2].

Structural Highlights

Four sequences are highly conserved within the U5 snRNA and it is believed that these sequences play a primary role in the proteins binding capabilities. The four regions are the , Internal loop 1, Stem-closing internal loop 1, and Sm protein binding site ^[7]. spans nucleotides 84 to 110 (S. cerevisiae) and consists of a highly conserved 11 nucleotide block with complementary sequences on either side ^[7]. These complementary sequences enable local base-pairing to form the stem of the loop ^[7]. The U5 snRNA stem-loop 1 is necessary for tethering and aligning exons for ligation during the second step of splicing. The U5 snRNA and internal loop 1 (IL1) are also highly conserved between yeast and humans, both in size and in structure ^[7]. The U5 snRNA IL1 was found to be the most important region for the association of Prp8, Snu114 and Brr2 with the U5 snRNA ^[8]. Genetic analysis also identified the U5 snRNA IL1 as being important for Brr2 function ^[7]. Overall, the U5 snRNA IL1 appears to be critical for the association of the U5 snRNP proteins Prp8, Snu114 and Brr2 with the U5 snRNA to form the ^[7].

Brr2 Structural Highlights

Recent structural analysis of Brr2 has revealed that parts of the Sec63 like cassettes of the Brr2 helicase domains resembles the DNA helicase Hel308, hinting at an RNA unwinding action of Brr2 similar to that of the DNA unwinding by helicase Hel308 ^[2]. The activity of Brr2 is regulated by the guanine nucleotide state of Snu114 ^[2].

Prp8 Structural Highlights

Prp8 is a 280 kDa protein component of the U5 snRNP and is also part of the U4/U6.U5 tri-snRNP ^[2]. Prp8 forms a complex with the assembly factor Aar2 and Snu114 without the U5 snRNA in the cytoplasm, however, Aar2 is replaced with Brr2 in the nucleus ^[2].

Protein Interactions

The U5 snRNP is one of the five subunits which come together to form the spliceosome and is, therefore, a key protein for eukaryotic mRNA processing. U5 snRNP is a complex which consists of U5 snRNA, Brr2, Snu114, and Prp8. Prp8 also interacts with other proteins of the spliceosome, the snRNAs and extensively with the pre-mRNA ^[2]. It has been shown that Prp8 crosslinks directly to the pre-mRNA 5’ splice site, the branch site, and the 3’ splice site as well as U5 and U6 snRNAs localizing it to the heart of the spliceosome ^[2]. In yeast, Prp8 makes extensive direct contacts with the U5 snRNA including the highly conserved U5 loop 1 and IL1 ^[2].

Following successful assembly of the U5 snRNP, a consisting of U4, U5 and U6 is formed which is then incorporated into the active spliceosome.

References

↑ Stevens SW, Barta I, Ge HY, Moore RE, Young MK, Lee TD, Abelson J. Biochemical and genetic analyses of the U5, U6, and U4/U6 x U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae. RNA. 2001 Nov;7(11):1543-53. PMID:11720284
↑ ^2.00 ^2.01 ^2.02 ^2.03 ^2.04 ^2.05 ^2.06 ^2.07 ^2.08 ^2.09 ^2.10 Galej WP, Oubridge C, Newman AJ, Nagai K. Crystal structure of Prp8 reveals active site cavity of the spliceosome. Nature. 2013 Jan 31;493(7434):638-43. doi: 10.1038/nature11843. Epub 2013 Jan 23. PMID:23354046 doi:http://dx.doi.org/10.1038/nature11843
↑ ^3.0 ^3.1 O'Keefe RT, Newman AJ. Functional analysis of the U5 snRNA loop 1 in the second catalytic step of yeast pre-mRNA splicing. EMBO J. 1998 Jan 15;17(2):565-74. doi: 10.1093/emboj/17.2.565. PMID:9430647 doi:http://dx.doi.org/10.1093/emboj/17.2.565
↑ Hahn D, Beggs JD. Brr2p RNA helicase with a split personality: insights into structure and function. Biochem Soc Trans. 2010 Aug;38(4):1105-9. doi: 10.1042/BST0381105. PMID:20659012 doi:http://dx.doi.org/10.1042/BST0381105
↑ Collins CA, Guthrie C. The question remains: is the spliceosome a ribozyme? Nat Struct Biol. 2000 Oct;7(10):850-4. doi: 10.1038/79598. PMID:11017191 doi:http://dx.doi.org/10.1038/79598
↑ Maeder C, Kutach AK, Guthrie C. ATP-dependent unwinding of U4/U6 snRNAs by the Brr2 helicase requires the C terminus of Prp8. Nat Struct Mol Biol. 2009 Jan;16(1):42-8. doi: 10.1038/nsmb.1535. Epub 2008 Dec, 21. PMID:19098916 doi:http://dx.doi.org/10.1038/nsmb.1535
↑ ^7.0 ^7.1 ^7.2 ^7.3 ^7.4 ^7.5 Frank DN, Roiha H, Guthrie C. Architecture of the U5 small nuclear RNA. Mol Cell Biol. 1994 Mar;14(3):2180-90. PMID:8114748
↑ Nancollis V, Ruckshanthi JP, Frazer LN, O'Keefe RT. The U5 snRNA internal loop 1 is a platform for Brr2, Snu114 and Prp8 protein binding during U5 snRNP assembly. J Cell Biochem. 2013 Dec;114(12):2770-84. doi: 10.1002/jcb.24625. PMID:23857713 doi:http://dx.doi.org/10.1002/jcb.24625

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Wally Novak

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@@ Line 11: / Line 11: @@
 ===Prp8===
 <Structure load='3e66' size='400' frame='true' align='right' caption='Prp8'/>
-Prp8 is believed to be the master regulator of the splicing cycle by regulating the spliceosomal DExD/H-box RNA dependent ATPases, specifically Brr2 <ref>PMID:11017191</ref>. Consistent with this regulatory role it has been shown that the C-terminus of Prp8 activates Brr2 helicase function and inhibits Brr20 s U4/U6-dependent ATPase activity in vitro <ref>PMID:19098916</ref>. Structural studies of the Prp8 C-terminus have identified an RNase H-like domain within Prp8 and it has been proposed that this RNase H-domain may form the active site of the spliceosome <ref name="Irish" />.
+Prp8 is believed to be the master regulator of the splicing cycle by regulating the spliceosomal DExD/H-box RNA dependent ATPases, specifically Brr2 <ref>PMID:11017191</ref>. Consistent with this regulatory role it has been shown that the C-terminus of Prp8 activates Brr2 helicase function and inhibits Brr2's U4/U6-dependent ATPase activity in vitro <ref>PMID:19098916</ref>. Structural studies of the Prp8 C-terminus have identified an RNase H-like domain within Prp8 and it has been proposed that this RNase H-domain may form the active site of the spliceosome <ref name="Irish" />.
 ===Snu114===
 <scene name='79/798410/Snu114/1'>Snu114</scene> is the only GTPase associated with the spliceosome and it has multiple contacts with Prp8 as well as U5 snRNA and Brr2 but its role in spliceosome function is not yet well-defined.  <ref name="Irish" />.
 ==Structural Highlights==
-Four sequences are highly conserved within the U5 snRNA and it is believed that these sequences play a primary role in the proteins binding capabilities. The four regions are the Stem-loop 1, Internal loop 1, Stem-closing internal loop 1, and Sm protein binding site <ref name="Frank">PMID:8114748</ref>. Stem-loop 1 spans nucleotides 84 to 110 (S. cerevisiae) and consists of a highly conserved 11 nucleotide block with complementary sequences on either side <ref name="Frank" />. These complementary sequences enable local base-pairing to form the stem of the loop <ref name="Frank" />. The U5 snRNA stem-loop 1 is necessary for tethering and aligning exons for ligation during the second step of splicing. The U5 snRNA stem 1 and internal loop 1 (IL1) are also highly conserved between yeast and humans, both in size and in structure <ref name="Frank" />. The U5 snRNA IL1 was found to be the most important region for the association of Prp8, Snu114 and Brr2 with the U5 snRNA <ref>PMID:23857713</ref>. Genetic analysis also identified the U5 snRNA IL1 as being important for Brr2 function <ref name="Frank" />. Overall, the U5 snRNA IL1 appears to be critical for the association of the U5 snRNP proteins Prp8, Snu114 and Brr2 with the U5 snRNA to form the <scene name='79/798410/Prp8_snu114_brr2_5gan/2'>U5 snRNP</scene> <ref name="Frank" />.
+Four sequences are highly conserved within the U5 snRNA and it is believed that these sequences play a primary role in the proteins binding capabilities. The four regions are the <scene name='79/798410/Stem_loop/1'>Stem-loop 1</scene>, Internal loop 1, Stem-closing internal loop 1, and Sm protein binding site <ref name="Frank">PMID:8114748</ref>. <scene name='79/798410/Stem_loop/1'>Stem-loop 1</scene> spans nucleotides 84 to 110 (S. cerevisiae) and consists of a highly conserved 11 nucleotide block with complementary sequences on either side <ref name="Frank" />. These complementary sequences enable local base-pairing to form the stem of the loop <ref name="Frank" />. The U5 snRNA stem-loop 1 is necessary for tethering and aligning exons for ligation during the second step of splicing. The U5 snRNA <scene name='79/798410/Stem_loop/1'>stem 1</scene> and internal loop 1 (IL1) are also highly conserved between yeast and humans, both in size and in structure <ref name="Frank" />. The U5 snRNA IL1 was found to be the most important region for the association of Prp8, Snu114 and Brr2 with the U5 snRNA <ref>PMID:23857713</ref>. Genetic analysis also identified the U5 snRNA IL1 as being important for Brr2 function <ref name="Frank" />. Overall, the U5 snRNA IL1 appears to be critical for the association of the U5 snRNP proteins Prp8, Snu114 and Brr2 with the U5 snRNA to form the <scene name='79/798410/Prp8_snu114_brr2_5gan/2'>U5 snRNP</scene> <ref name="Frank" />.
 ===Brr2 Structural Highlights===
 <Structure load='3hib' size='400' frame='true' align='right' caption='Brr2'/>