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At this point the spliceosome is now a catalytically active 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’ splice site. 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 <ref name="Wahl2009">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.</ref>. Next a second nucleophilic attach 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.  

The Prp8 protein has multiple domains: the RNaseH-like, Jab1/MPN, Aar2, and a large domain consisting of type II endonuclease and the large polymerase-like domains <ref name="Galej5" />. Most of these domains received their names because of significant sequence similarities they have with domains of other proteins.  

The two domains of the large domain are connected through a linker. The type II endonuclease domain (residues 1650 – 1810) is made up of 5 β-sheets surrounded by 3 α-helices <ref name="Galej2013">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.</ref>. The large polymerase domain (residues 885-1375) is subdivided into the palm, finger, thumb and endonuclease domains<ref name="Galej2013">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.</ref>. The palm domain (residues 1048-1182) is similar in sequence to that of bacterial reverse transcriptase, so it is often referred to as the reverse transcriptase domain<ref name="Galej2013" />. The thumb domain (residues 1257-1375), is characterized by an antiparallel β-sheet and three helix bundles<ref name="Galej2013" />.

The RNaseH-like (1840-2090) and Jab1/MPN (2150-2396) domains are connected by disordered linkers, and stabilized by the Aar domain, a U5 snRNP assembly factor<ref name="Galej5" />. 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. In a way, it zips together a β-barrel of Jab1/MPN and β-hairpin from RNaseH domain using a parallel β-sheet. Also, through the Aar domain, RNaseH and Jab1/MPN domains are able to interact with the large polymerase domain<ref name="Galej2013" />. Once the Prp8 protein is imported into the nucleus the Aar domain 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 <ref name="Galej2013" />.  

Nuclear localization signals (NLS) can be found on the Prp8 protein at its N-terminus within the first 500 amino acids. 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<ref name="Moz2012">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.</ref>.   

The assembly of spliceosome proteins is assisted by the bromodomain. These bromodomains 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<ref name="Moz2012" />.  

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<ref name="Sin2012" />. Research has shown that his β-hairpin finger 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[3]. 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<ref name="Yang2008" />

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 RNas-H domain, which blocked Brr2’s single stranded loading sight on U4/U6 complex, blocks Brr2’s helicase activity<ref name="Sin2012">Mozaffari-Jovin, Sin. "Mechanism of Regulation of Spliceosome Activation by Brr2 and Prp8 and Links to Retinal Disease." (2012): n. pag.</ref>. The disruption of the RNas-H interaction allows for the binding of Brr2, 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 <ref name=Sin2012 />. 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<ref>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.</ref> 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 <ref name="Sin2012" />. 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 <ref name="Moz2012" />. 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<ref name="Moz2012" />.   

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<ref name="Galej5" />.