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From Proteopedia

Introduction

The spliceosome is a complex piece of machinery responsible for catalyzing the removal of pre-mRNA introns before the material is shipped out of the nucleus to be translated into protein. This large catalytic complex is composed of 5 snRNAs (U1, U2, U4, U5 and U6) and protein factors including Prp8. Prp8 is the only known protein of the spliceosome that makes contact with all its critical catalytic components (U5 and U6 snRNAs, 5’-ss, 3’-ss, and BP)^[1]. Through these contacts Prp8 regulates two trans-esterification reactions that allow for the release of an intron and the ligation of two neighboring exons of pre-mRNA. Eukaryotes are able to construct multiple proteins from a single gene through these SN2 reactions.

The human Prp8 protein is 2,335 residues in length and yeast Prp8 is 2,413 residues^[2]. These two proteins share 61 percent of their amino acid sequence, emphasizing the evolutionary conservation of the Prp8 protein throughout many species^[1]. Mutations within the structure of Prp8 have shown the importance of the protein which can cause diseases such as Retinitis Pigmentosa when function is lost. ^[1].

Splicing

Assembly of splicing machinery takes place during the process of intron removal. First the U1 snRNP is recruited to the 5’ splice site (5'ss) where it base pairs, while U2 attaches to the branch point. The branch point is a nucleotide, usually adenosine, that will participate in the first nucleophilic reaction that forms a lariat structure. Next, the U4/U5.U6 tri-snRNP assembles and attaches itself to the complex, making the pre-catalytic complex B. The U6 snRNA then base pairs with the 5’ss, displacing the U1 snRNA and releasing U4 with the aid of accessory proteins. U1 and U4 are then free to dissociate from the complex.

After release of U1 and U4 the spliceosome is catalytically active and referred to as complex B*. The Prp8 protein then takes on the role of converting the now active spliceosome to a catalytically-active spliceosome, which can carry out the first trans-esterification reaction: nucleophilic attack of the branch point on the 5’ss. In this reaction the 5’ss is attacked by the 2’-hyroxyl group of the branch point adenosine, resulting in a free exon and an intron-exon2 lariat ^[3]. Next, a second nucleophilic attack takes place, in which the 3’ splice site is attacked by the 3’-hydroxyl group of the free exon. This leads to the release of the lariat intron and ligation of the two exons. The spliceosome complex can now be released.

Structure

The Prp8 protein has multiple domains: the RNaseH-like, Jab1/MPN, Aar2, and a large domain further divided into a type II endonuclease and large polymerase-like domains^[1]. Most of these domains received their names because of significant sequence similarities with domains of other proteins.

The type II endonuclease domain is made up of 5 β-sheets surrounded by 3 α-helices that interact with the polymerase domain through a small linker domain^[4]. The large polymerase domain is subdivided into palm, finger, and thumb domains^[4]. Its palm is similar in sequence to that of bacterial reverse transcriptase, so it is often referred to as the reverse transcriptase domain^[4]. The thumb domain , is characterized by an antiparallel β-sheet and three helix bundles^[4].

The RNaseH-like and Jab1/MPN domains are connected by disordered linkers, and stabilized by Aar2, a U5 snRNP assembly factor^[1]. The C-terminal tail of Aar domain reaches out from its main body in order to interact with the junction between RNaseH and Jab1/MPN. Through this contact Aar2 binds together a β-barrel of Jab1/MPN and β-hairpin from RNaseH domain using a parallel β-sheet. Also, through the Aar2 domain, RNaseH and Jab1/MPN domains are able to interact with the large polymerase domain^[4]. Once the Prp8 protein is imported into the nucleus Aar2 is replaced by a Brr2 domain, an integral U5 snRNP component that is responsible for unwinding the U4/U6 snRNP duplex. This exchange may alter the position of the domains with respect to each other ^[4]. The active site of Prp8 has been mapped to the RNaseH-like and Large domains and mutations to this region have revealed ^[4].

Nuclear localization signals (NLS) can be found on the Prp8 protein at its N-terminus within the first . These NLS are made of two clusters of positively charged residues separated by a region of 10-15 variable amino acids. A bromodomain also exist at the N-terminal region from residue 200 to 315. This region consists of 4 α-helices and two loops^[5].

Function

The assembly of spliceosome proteins is assisted by the bromodomain, which are able to recognize acetylated lysine residues. Because many histone and spliceosomal proteins are acetylated at these residues, the bromodomain is able to facilitate their assembly^[5].

The RNase-H domain acts as the RNA binding site of Prp8. It interacts with U4/U6 snRNA through a binding site at the base of a hairpin loop which resembles many features of ribosomal stabilizing structures and RNase H folds in DNA repair enzymes^[6]. Research has shown that this sticks out of its globular domain and plays a critical role in stabilizing intermediates of the first catalytic step. It does so by inserting its extension into the U2/U5/U6 pre-mRNA complex and stabilizing it^[2]. When it is time to move onto the second catalytic step, the first step complex is disrupted and the reaction continues. The interactions between the β-finger and the complex are weak to ensure that this disruption can take place with little resistance^[2]

Before transition can take place between the two catalytic steps, the activated complex B* must be prepared. This is done through the activity of the Brr2 complex. The RNase-H domain, which blocked Brr2’s single stranded loading sight on U4/U6 complex, blocks Brr2’s helicase activity^[6]. The disruption of the RNas-H interaction allows for the binding of , which joins the spliceosome complex after it exits the nucleus. Brr2 then catalyzes the ATP dependent unwinding of U4 snRNA from the U4/U6 complex. After carrying out this reaction, Brr2 remains attached to the spliceosome and will later assist in the dissociation of the U2/U6 complex during spliceosome breakdown ^[6]. The Prp8 protein interacts with Brr2 through its c-terminal.

The activity of the Brr2 complex is regulated by Jab1/MPN. The Jab1/MPN belongs to a class of deubiquitinating enzymes that allows it to bind ubiquitin. It has been suggested that Jab1 may interact with ubiquitinated splicing factors that function to regulate splicing activity^[7] The tail of Jab1/MPN has charged residues that interact with Brr2’s RNA pocket. When induced by a signal, Jab1 releases its tail from the binding motif, triggering the helicase activities of Brr2 and releasing it from its locked conformation ^[6]. Jab1 has also been found to stabilize the U4/U5/U6 triple snRNP complex.

Many of the folds within the reverse transcriptase domain resemble the structure of polymerase enzymes. The domain is missing two of three catalytic aspartate residues though, which diminishes its nucleic acid synthesis abilities ^[5]. The remainder of the last catalytic residue allows for the domain to bind with a metal ion and carry out transfer reactions involving nucleotides and phophoryl groups plus hydrolysis of phosphoester bonds^[5].

After the work of Brr2 and the release of U1 and U4, the spliceosome is now activated and can proceed to catalytic step one. With the additional help from Prp2p and Prp16p, both transesterification reactions can take place, and the intron is removed^[1].

Disease

Mutation to the Prp8 gene has been linked to autosomal dominant Retinitis Pigmentosa-13 ^[8]. This is the result of nine different missense mutations acting on the C-terminus within 7 highly conserved amino acid residues. Mutation to the C-terminus of Prp8 inhibits the interaction of Brr2, resulting in lost ability to unwind the U4/U6 complex. The disease is characterized by degeneration of the photoreceptors in the retina, causing severe vision impairment^[8].

References

1. ↑ ^{1.0 1.1 1.2 1.3 1.4 1.5} Galej, Wojciech P, Andrew J. Newman, Thi Hoang Duong Nguyen, Nagai Kiyoshi. "Structural Studies of the Spliceosome: Zooming into the Heart of the Machine." Current Opinion In Structural Biology 25.Theory and simulation / Macromolecular
2. ↑ ^{2.0 2.1 2.2} Yang, Kui, Annie Xeroux, Lingdi Zhang, Rui Zhao, and Tao Xu. "Crystal Structure of the β-Finger Domain of Prp8 Reveals Analogy to Ribosomal Proteins." Proceedings of the National Academy of Sciences of the United States of America 2008: 13817. JSTOR Journals. Web. 13 Apr. 2015.
3. ↑ Wahl, Markus C., Cindy L. Will, and Reinhard Lührmann. "Review: The Spliceosome: Design Principles of a Dynamic RNP Machine." Cell 136.(2009): 701-718. ScienceDirect. Web. 13 Apr. 2015.machines (2014): 57-66. ScienceDirect. Web. 13 Apr. 2015.
4. ↑ ^{4.0 4.1 4.2 4.3 4.4 4.5 4.6} Galej, Wojciech P., Andrew J. Newman, Chris Oubridge, and Kiyoshi Nagai. "Crystal Structure of Prp8 Reveals Active Site Cavity of the Spliceosome." Nature 493.7434 (2013): 638-643. Academic Search Complete. Web. 13 Apr. 2015.
5. ↑ ^{5.0 5.1 5.2 5.3} Mozaffari-Jovin, Sina, Hsiao HH, Luhrmann R, Santos KF, Urlaub H, Wahl MC, and Will CL. "The Prp8 Rnase H-Like Domain Inhibits Brr2-Mediated U4/U6 Snrna Unwinding by Blocking Brr2 Loading onto the U4 Snrna." Genes & Development 26.21 (2012): 2422-2434. MEDLINE. Web. 13 Apr. 2015.12.
6. ↑ ^{6.0 6.1 6.2 6.3} Mozaffari-Jovin, Sin. "Mechanism of Regulation of Spliceosome Activation by Brr2 and Prp8 and Links to Retinal Disease." (2012): n. pag.
7. ↑ Bellare, Priya, Guthrie C, Kutach AK, Rines AK, Sontheimer EJ. "Ubiquitin Binding by a Variant Jab1/MPN Domain in the Essential Pre-Mrna Splicing Factor Prp8p." RNA (New York, N.Y.) 12.2 (2006): 292-302. MEDLINE. Web. 13 Apr. 2015.
8. ↑ ^{8.0 8.1} Boon, Kum-Loong, Chris F. Inglehearn, David J. Barrass, Jean D. Beggs, Parastoo Ehsani, Richard J. Grainger, and Tatsiana Auchynnikara. "Prp8 Mutations that Cause Human Retinitis Pigmentosa Lead to a U5 SnRNP Maturation Defect in Yeast." Nature Structural & Molecular Biology 14.11 (2007): 1077-1083. Academic Search Complete. Web. 13 Apr. 2015.