Cid1 Poly(U) Polymerase
Created by Tomihiro Ono
Cid1 Poly(U) Polymerase (PDB ID: 4ep7) is a polymerase found in Schizosaccharomyces pombe 972h- that catalyzes the formation of poly-uridyl 3’-end tails in mRNA (Munoz-Tello et al, p.4). In E. Coli, poly(A) and poly(U) addition plays a role in directing RNase E mRNA-degradation activity (Huang et al, p.1). In Saccharomyces cerevisiae, polyadenylation of non-coding, misfolded or hypermodified RNAs leads to recruitment of the nuclear exosome and subsequent degradation (Etheridge, p.6). In mice, Arabidopsis, and the Epstein-Barr virus, mRNA degradation is directed by miRNA recognizing a post-translational 3’-poly(U) addition (Shen & Goodman, p.2). Likewise, Cid1 Poly(U) Polymerase plays a crucial step in mRNA degradation in Schizosaccharomyces pombe by adding the poly(U) tail, and this polyuridylation-dependent degradation has been reported in degradation of other poly(A)-minus RNAs as well. Thus, study of this polymerase has critical significance for understanding the degradation of histone mRNAs, miRNAs, and more generally cellular RNA molecules (Munoz-Tello et al., p.3). In addition, there are possible applications of research in this protein to the development of inhibitors of structurally similar proteins. A number of structural similarities between Cid1 and Trypanosoma terminal Uridyl transferases (TUTases) have been highlighted, which may be useful in the development of TUTase inhibitor trypanocides (Aphasizhev et al., p.2).
The molecular weight of Cid1 is 4656.89 Da, and the isoelectric point (pI) is 7.77 (Artimo et al., p.5). These values were calculated from ExPASy, a bioinformatics service that has multiple tools and resources hosted by the Swiss Institute of Bioinformatics. In particular, the Compute pI/MW tool takes a polypeptide sequence and analyzes its residue constituents to output an estimate of the molecular weight and isoelectric point.
PSI-BLAST, a program used to search for sequenced proteins that have primary structure homologies with the protein of interest, was applied to this protein. The first three proteins in the query result—distinct from the protein of interest—were “Cid (Caffeine-induced Death protein)” from Schizosaccharomyces japonicas yFS275 with an E value of 1E-131; PAP/OAS1 Substrate-Binding Domain-Containing Protein from Fomitiporia mediterranea MF3/22 with an E value of 4E-128; and Cid1 from Cryptococcus neoformans var. neoformans with an E value of 4E-126 (Altschul et al., p.4). The E value is calculated from the possibility that two sequence homologies arose by pure chance, which is in turn an indicator of how similar the structures are. Homologies in primary structure can be useful for speculating about evolutionary relationships and criticalities of residues.
In addition, the tertiary structure of the protein was compared with other proteins via the Dali Server. The Dali Server allows for a computational search of proteins that are structurally homologous on the tertiary structure level, but not necessarily at the primary structure level. The comparison is again useful for speculating on evolutionary origins and structural criticalities. The Dali query for Cid1 Poly(U) Polymerase yielded “Minor Editisome-TUTase” (PDB ID: 3HJ1) from Trypanosoma brucei with a Z-score of 30.1; “tRNA nucleotidyltransferase” (PDB ID: 1F5A) from Archaeoglobus fulgidus with a Z-score of 19.0; and “Interleukin Enhancer-Binding Factor 2” (PDB ID: 2Q0E) from Mus musculus with a Z-score of 20.3 (Holm & Rosenstrom, p.7). The Z-score is an indicator of structural similarity as calculated by the Dali server, and a comparison with a Z-score above 2 is generally said to be a “significant similarity” comparison. In this study, Cid1 Poly(U) Polymerase from Schizosaccharomyces pombe 972h- will be compared against Minor Editisome-Associated TUTase from Trypanosoma brucei since the two proteins show significant tertiary structure homology (Z-score: 30.1). Also, more information is available for TUTase, whereas all of the results from the PSI-BLAST query did not have PDB entries and/or their structural criticalities were not as well-characterized.
Crystallization experiments have revealed the structure of Cid1 Poly(U) Polymerase to have two subunits, making it a homodimer of identical chains A and B, which is presumably close to the structure it takes under physiological conditions. (Munoz-Tello et al., p.1). Its secondary structure consists of an assortment of 11 alpha helices and several beta helices that form a sheet. The CAT (catalytic) domain contains a five-stranded beta sheet with two alpha helices on one side and the catalytic site on the other side. The CD (central) domain has six alpha helices. The catalytic site is surrounded by alpha helices and a section of the beta helices. Some known critical residues include Arg 139, which plays a role in substrate selection and stabilization, and Asn-171 / His-336, which aid recognition of the UTP substrate by creating hydrogen bonds with the UTP molecule and inducing conformational changes. Phe-332 properly positions the nucleotide recognition motif loop once the substrate is bound by the protein, and Tyr-205 provides a surface upon which the nucleotide substrate can rest. Asp-101, Asp-103, and Asp-160 form a catalytic aspartic acid triad in the active site of the polymerase. Lys-193 and Lys-197 are also known to be critical, presumably because they keep the substrate locked in the active site with hydrogen bond interactions while the catalytic reaction is occurring. There are no known inter-residue bonds and interactions (Munoz-Tello et al., p.3-6).
The associated ligands of the protein include magnesium ions and uridine 5’-triphosphate. The Mg2+ ions, present in each chain, assists in coordinating the phosphate oxygen molecules in UTP to adopt a particular conformation in the active site. The uridine 5’-triphosphate is the monomer for poly(U) polymerization of the target RNA (Munoz-Tello et al., p.5). The product of the polymerase taking in a 3’-end tail of an mRNA molecule and several UTPs is an mRNA molecule with an additional poly(U) 3’-tail.
The proposed conformational changes involved in this protein are as follows: upon UTP association, the CAT domain closes on the CD domain, twisting a helix which locks the Phe-332 residue in a hydrophobic pocket. This conformational change assures, among other things, that the catalytic triad is held in close proximity to the UTP, which allows enough time for hydrolysis and addition to the target RNA (Munoz-Tello et al., p.5). At this time, there are no drugs specifically targeted to this protein, and thus no conformational changes from inhibitor association. The popular commercial option for TUTase inhibition is at the genetic level through siRNAs, and an inhibitor at the enzymatic level has yet to be widely introduced (Santa Cruz Biotechnology, p.1).
The comparison protein, Minor Editisome-Associated TUTase from Trypanosoma brucei, is a crucial enzyme for RNA editing via template-independent 3’ polyuridylation of mRNAs and insertion of a poly(U) sequence into guide RNAs (Stagno et al., 2010, p.8). It has three conserved motifs in the catalytic core region with an aspartic acid triad (Asp-342, Asp-344, and Asp-548), is localized in the mitochondrion, and has both distributive and processive functions in polymerization of mRNA. Mutational lesions in this protein lead to severe growth defects, implicating that TUTase is an instrumental component of RNA editing in Trypanosoma (Aphasizhev et al., p.3). It is part of the mitochondrial transcriptome and shows no appreciable conformational change upon UTP binding (Stagno et al., 2007, p.3).
We can see that even from the similar poly(U) functions of the two proteins, Cid1 and TUTase are similar. In fact, Cid1 was originally classified as a cytoplasmic PAP with a robust TUTase activity (Stagno et al., 2010, p.7). Structurally, we also see that the two proteins are similar. Cid1 has seven Mg2+ binding sites, whereas TUTase only has two. The protein length is different as well, with TUTase having a ~60 residues worth of extra alpha helices on the exterior, a difference that presumably does not have any significant effect on function judging from the similarity in the function of these two proteins. In both proteins, crucial catalytic regions such as the aspartic acid triad are conserved, with both proteins possessing a core set of 12 secondary structures, roughly in the same orientation. TUTase has the aforementioned small helices on the exterior (Holm & Rosenstrom, p.3).
The superimposition of the two proteins makes the slight structural differences more apparent. The orange chain is the Cid1 protein, and the teal chain is the TUTase protein. Clearly, the two proteins share many tertiary characteristics and the major differences are all on the exterior, signifying that the exterior part is not crucial to the common functionalities. Conversely, the central structure of the protein is markedly conserved in these proteins from distinctly different organisms (Schizosaccharomyces pombe 972h- and Trypanosoma brucei), implicating that the structure seen is essential for poly(U) function.