Prp8: Difference between revisions

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The reverse transcriptase-like domain is located approximately halfway through Prp8 and extends through residues 950 - 1220 <ref name='Prp8, the pivotal protein of the spliceosomal catalytic center evolved from a retroelement-encoded reverse transcriptase'>DOI:10.1261/rna.2396011</ref>. This domain is only reverse transcriptase-like because it does contain the conventional core set of alpha-helices and beta-strands that are a general characteristic of RT domains, but lacks key motifs that would normally confer its catalytic activity <ref name='Prp8, the pivotal protein of the spliceosomal catalytic center evolved from a retroelement-encoded reverse transcriptase'>DOI:10.1261/rna.2396011</ref>. However, the Prp8 RT-like domain still possesses the potential to bind RNA <ref name='Prp8, the pivotal protein of the spliceosomal catalytic center evolved from a retroelement-encoded reverse transcriptase'>DOI:10.1261/rna.2396011</ref>. Prp8 also contains an RNase H-like domain towards its C-terminal end (residues 1836 - 2092) <ref name='Mechanism for aar2p function as a U5 snRNP assembly factor'>PMID:21764848</ref>. The central region of the RNase H-like domain consists of a mixed alpha/beta fold, which resembles the overall shape of a mitten <ref name='Mechanism for aar2p function as a U5 snRNP assembly factor'>PMID:21764848</ref>. Following this analogy, the central 6 stranded beta sheets and alpha helices are the palm with a beta hairpin as the thumb and alpha helices as fingers. This is represented by the portion of the PDB figure that has yellow apha helices with blue beta sheets and the blue unstructured protein chain. The groove created by the mitten like structure is lined with positive surface potential but also lacks key residues for RNase activity which is perfect for binding RNA, and has a very similar structure to the reverse transcriptase-like domain mentioned above <ref name='Structure and function of an RNase H domain at the heart of the spliceosome'>DOI:10.1038/emboj.2008.209</ref>. The RNase domain possesses conserved charged residues at D1853/1781, D1854/1782, and Q1907/1835 (yeast/human numbering) which are thought to mediate interactions with the negatively charged backbone of an RNA molecule <ref name='Structure and function of an RNase H domain at the heart of the spliceosome'>DOI:10.1038/emboj.2008.209</ref>. Finally, the final domain one the C-terminal end of Prp8 is a metalloprotease/Jab1-like domain which extends through residues 2112 - 2413 and is represented by the dark and bright blue portions of the PDB figure. Its overall structure consists of an oval shaped fold containing 12 beta strands, four alpha helices, and four 310 helices <ref name='Structure of a Multipartite Protein-Protein'>DOI:10.1016/j.molcel.2007.01.023</ref>. They form a mixed beta-barrel made up of seven strands surrounded by the remaining alpha and beta elements <ref name='Structure of a Multipartite Protein-Protein'>DOI:10.1016/j.molcel.2007.01.023</ref>. This domain also contains unique Prp8 residue substitutions; the commonplace metalloenzyme motif is replaced by Q-X-H-X-Q-X-S-E which allows the domain to coordinate protein-protein contacts instead of a metal ion <ref name='Structure of a Multipartite Protein-Protein'>DOI:10.1016/j.molcel.2007.01.023</ref>.

*Note - Thus far only C-terminal regions of Prp8 have been crystallized which is why only one PDB is represented by this page.

*Note - Thus far only C-terminal regions of Prp8 have been crystallized which is why only one PDB out of the half a dozen available is represented on this page.

=Function of Prp8=

Following suite with its unique structural makeup, Prp8 also possesses the potential to perform a variety of functions at the heart of the spliceosome. Above all else, Prp8 has been strongly implicated as a large scaffold which coordinates the active catalytic components of the spliceosome <ref name='Prp8: At the heart of the spliceosome'>DOI:10.1261/rna.2220705</ref>. Prp8 acts as a cofactor for RNA splicing because it is required for the formation of the active (complex B*) spliceosome and for subsequent first and second transesterification reactions <ref name='Prp8: At the heart of the spliceosome'>DOI:10.1261/rna.2220705</ref>. Prp8's bromodomain has been evolutionarily modified to recognize acetylated lysine residues, and given that many spliceosomal proteins are acetylated at lysine residues, it is likely that Prp8 mediates the assembly of spliceosome proteins and protein-RNA contacts through recognition of the lysine residues <ref name='Prp8, the pivotal protein of the spliceosomal catalytic center, evolved from a retroelement-encoded reverse transcriptase'>PMID:21441348 </ref>. One example of such an interaction is Prp8's bromodomain mediated contact with Snu114p, which is a GTPase involved in RNA remodeling during the catalytic cycle of splicing <ref name='Prp8, the pivotal protein of the spliceosomal catalytic center, evolved from a retroelement-encoded reverse transcriptase'>PMID:21441348 </ref>.

Prp8's reverse transcritase and RNase H-like domains are postulated to function as recognition, interaction, and handover domains for snRNAs catalytically active in the spliceosome and portions of the pre-mRNA substrate <ref name='Mechanism for aar2p function as a U5 snRNP assembly factor'>PMID:21764848</ref>. Crosslinking experiments have shown that U5 and U6 snRNAs, as well as the GU dinucleotide of the 5' splice site, polypyrimidine tract (close to the intron branch point), and the 3' splice site all interact with or close to the reverse transcriptase and RNase H-like domains of Prp8 which supports the scaffolding and coordination role of Prp8 in pre-mRNA splicing <ref name='Structure and function of an RNase H domain at the heart of the spliceosome'>DOI:10.1038/emboj.2008.209</ref><ref name='splicing factor prp8 governs u4/u6 rna unwinding'>PMID:10024880</ref><ref name='roles of prp8 protein in the assembly of splicing complexes'>PMID:1396567<ref>.

=Evolution of Prp8=

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 The reverse transcriptase-like domain is located approximately halfway through Prp8 and extends through residues 950 - 1220 <ref name='Prp8, the pivotal protein of the spliceosomal catalytic center evolved from a retroelement-encoded reverse transcriptase'>DOI:10.1261/rna.2396011</ref>. This domain is only reverse transcriptase-like because it does contain the conventional core set of alpha-helices and beta-strands that are a general characteristic of RT domains, but lacks key motifs that would normally confer its catalytic activity <ref name='Prp8, the pivotal protein of the spliceosomal catalytic center evolved from a retroelement-encoded reverse transcriptase'>DOI:10.1261/rna.2396011</ref>. However, the Prp8 RT-like domain still possesses the potential to bind RNA <ref name='Prp8, the pivotal protein of the spliceosomal catalytic center evolved from a retroelement-encoded reverse transcriptase'>DOI:10.1261/rna.2396011</ref>. Prp8 also contains an RNase H-like domain towards its C-terminal end (residues 1836 - 2092) <ref name='Mechanism for aar2p function as a U5 snRNP assembly factor'>PMID:21764848</ref>. The central region of the RNase H-like domain consists of a mixed alpha/beta fold, which resembles the overall shape of a mitten <ref name='Mechanism for aar2p function as a U5 snRNP assembly factor'>PMID:21764848</ref>. Following this analogy, the central 6 stranded beta sheets and alpha helices are the palm with a beta hairpin as the thumb and alpha helices as fingers. This is represented by the portion of the PDB figure that has yellow apha helices with blue beta sheets and the blue unstructured protein chain. The groove created by the mitten like structure is lined with positive surface potential but also lacks key residues for RNase activity which is perfect for binding RNA, and has a very similar structure to the reverse transcriptase-like domain mentioned above <ref name='Structure and function of an RNase H domain at the heart of the spliceosome'>DOI:10.1038/emboj.2008.209</ref>. The RNase domain possesses conserved charged residues at D1853/1781, D1854/1782, and Q1907/1835 (yeast/human numbering) which are thought to mediate interactions with the negatively charged backbone of an RNA molecule <ref name='Structure and function of an RNase H domain at the heart of the spliceosome'>DOI:10.1038/emboj.2008.209</ref>. Finally, the final domain one the C-terminal end of Prp8 is a metalloprotease/Jab1-like domain which extends through residues 2112 - 2413 and is represented by the dark and bright blue portions of the PDB figure. Its overall structure consists of an oval shaped fold containing 12 beta strands, four alpha helices, and four 310 helices <ref name='Structure of a Multipartite Protein-Protein'>DOI:10.1016/j.molcel.2007.01.023</ref>. They form a mixed beta-barrel made up of seven strands surrounded by the remaining alpha and beta elements <ref name='Structure of a Multipartite Protein-Protein'>DOI:10.1016/j.molcel.2007.01.023</ref>. This domain also contains unique Prp8 residue substitutions; the commonplace metalloenzyme motif is replaced by Q-X-H-X-Q-X-S-E which allows the domain to coordinate protein-protein contacts instead of a metal ion <ref name='Structure of a Multipartite Protein-Protein'>DOI:10.1016/j.molcel.2007.01.023</ref>.
-*Note - Thus far only C-terminal regions of Prp8 have been crystallized which is why only one PDB is represented by this page.
+*Note - Thus far only C-terminal regions of Prp8 have been crystallized which is why only one PDB out of the half a dozen available is represented on this page.
 =Function of Prp8=
 Following suite with its unique structural makeup, Prp8 also possesses the potential to perform a variety of functions at the heart of the spliceosome. Above all else, Prp8 has been strongly implicated as a large scaffold which coordinates the active catalytic components of the spliceosome <ref name='Prp8: At the heart of the spliceosome'>DOI:10.1261/rna.2220705</ref>. Prp8 acts as a cofactor for RNA splicing because it is required for the formation of the active (complex B*) spliceosome and for subsequent first and second transesterification reactions <ref name='Prp8: At the heart of the spliceosome'>DOI:10.1261/rna.2220705</ref>. Prp8's bromodomain has been evolutionarily modified to recognize acetylated lysine residues, and given that many spliceosomal proteins are acetylated at lysine residues, it is likely that Prp8 mediates the assembly of spliceosome proteins and protein-RNA contacts through recognition of the lysine residues <ref name='Prp8, the pivotal protein of the spliceosomal catalytic center, evolved from a retroelement-encoded reverse transcriptase'>PMID:21441348 </ref>. One example of such an interaction is Prp8's bromodomain mediated contact with Snu114p, which is a GTPase involved in RNA remodeling during the catalytic cycle of splicing <ref name='Prp8, the pivotal protein of the spliceosomal catalytic center, evolved from a retroelement-encoded reverse transcriptase'>PMID:21441348 </ref>.
+Prp8's reverse transcritase and RNase H-like domains are postulated to function as recognition, interaction, and handover domains for snRNAs catalytically active in the spliceosome and portions of the pre-mRNA substrate <ref name='Mechanism for aar2p function as a U5 snRNP assembly factor'>PMID:21764848</ref>. Crosslinking experiments have shown that U5 and U6 snRNAs, as well as the GU dinucleotide of the 5' splice site, polypyrimidine tract (close to the intron branch point), and the 3' splice site all interact with or close to the reverse transcriptase and RNase H-like domains of Prp8 which supports the scaffolding and coordination role of Prp8 in pre-mRNA splicing <ref name='Structure and function of an RNase H domain at the heart of the spliceosome'>DOI:10.1038/emboj.2008.209</ref><ref name='splicing factor prp8 governs u4/u6 rna unwinding'>PMID:10024880</ref><ref name='roles of prp8 protein in the assembly of splicing complexes'>PMID:1396567<ref>.
 =Evolution of Prp8=