A tree of Nudt16 and Syndesmos identifies them as paralogs. Amino acid sequences were aligned, graphed and analyzed as in Methods section. The tree was visualized with TreeView. Bootstrap values are shown. Vertebrates and invertebrates are separated and phylogenetic relationships are comparable to the generic tree of evolution. Syndesmos sequences branch from the tetrapod lineage of the Nudt16 sequences.

Amino acid alignment of putative Nudt16 orthologs from a sampling of the organisms found to have this protein. The alignment reveals three areas of highly conserved sequence, indicated by the green, red and blue boxes. A functional role based on structure for each conserved region is described in more detail in the text. To be designated as a Nudt16 homolog, sequences had to have the NUDIX domain (in red box) and the other two domains. Gray residues indicate identity; yellow highlighted residues show similarity. The accession number for each sequence used is listed in Supplementary Table 1.

A ‘standard’ phylogenetic tree. This tree illustrates the relative position of the organisms examined here and is provided as a quick reference for taxonomic relationships, based on the Tree of Life Web Project. (A) The Kingdom Animalia/Metazoa was searched. Only those branches for organisms containing a Nudt16 ortholog are shown. (B) An expansion of the vertebrate branch reveals the conservation of Nudt16 and appearance of Syndesmos. The ‘N’ indicates branches with organisms containing Nudt16 orthologs, ‘S’ indicates branches containing Syndesmos and ‘asterisk’ indicates the likely gene duplication event; acknowledging the possibility that it may have occurred earlier with high rates of subsequent gene loss. Sequences ‘below’ the ‘asterisk’ have only the Nudt16 protein.

Insect Nudt16 protein is active for decapping RNA. Cap-labeled U8 RNA was incubated in the presence of buffer, Mn²⁺ and protein (as indicated) for 30 min at 37°C (reaction indicated below). Samples of the reactions were spotted on a TLC, which was developed and then visualized on a phosphorimager. The Xenopus (X29/X-Nudt16) protein, present as a positive control, released m⁷GDP from the cap-labeled RNA. The two human proteins, which varied by an unrelated amino terminal extension, were both efficient at decapping the U8 RNA. Insect (sharpshooter) Nudt16 protein hydrolyzed the RNA to release the m⁷GPD cap comigrating with that cleaved by the other orthologs. The human H-Syndesmos protein displays no decapping activity.

Nudt16 and Syndesmos are closely related members of a gene family. (A) Alignment of a portion of the Syndesmos protein from 17 organisms (denoted by yellow box on right edge) and the corresponding region of Nudt16 from 10 organisms (marked with the blue box on right edge). All Syndesmos orthologs have a repeated glycine/leucine sequence (orange box) in place of the glutamic acid residues (heavy red box) required for catalysis in the NUDIX domain (red box). The green box is the conserved region in Nudt16 proteins, equivalent to the green box in Figure 1. Syndesmos paralogs have all three conserved regions but lack a functional NUDIX domain. The central parts of the proteins are aligned here. The accession numbers are in Supplementary Table 1. (B) Genomic organization of the orthologs in human. Dashed boxes indicate the transcription unit, solid boxes are exons, horizontal lines are introns and the conserved domains are color coded as per Figure 1. Note Nudt16 has a longer 5′-UTR and 3′-UTR than Syndesmos.

Structural comparison of paralogous proteins. (A) Molecular modeling of the orthologs and paralogs of Xenopus Nudt16p. The Xray crystal structure of Xenopus X29/Nudt16p (PDB: 2A8P) was used to model the human and sharpshooter Nudt16p proteins and human Syndesmos. Selected conserved residues were highlighted in the ribbon models to correctly align the models relative to each other. The blue strand (CVTHFY) is indicated with blue asterisks in Figure 4. The black asterisk is a conserved Phe residue near the catalytic site, while the red asterisk is a second conserved Phe residue indicated in Figure 4. The yellow line in the Xenopus protein is oriented across the dimer interface; the monomer above the line is oriented and positioned relative to the other three proteins. (B) Electrostatic charge potential of structures. Once the proteins were correctly oriented, electrostatic charge potential was calculated. Blue is positively charged surface and red is negatively charged. The yellow line in the Xenopus protein is oriented across the dimer interface as in (A).

Functional comparison of paralogous proteins. (A) Chemical cross-linking to examine homodimer formation. Over-expressed, purified proteins were untreated or incubated with the reversible chemical cross-linker DTSSP (as indicated). One half of the treated samples were heated in the presence of reducing agent, BME to reverse the cross-link. All samples resolved on a NuPAGE gel. The 30 kDa Xenopus protein cross-linked to form a ∼60 kDa dimer and larger multimers, as indicated. These resolved to monomers under reducing conditions. Human Nudt16 formed a dimer which also resolved to a monomer under reducing conditions. Human Syndesmos did not appreciably alter mobility in the presence of the cross-linker, although some of the higher molecular weight contaminating proteins did alter mobility. Molecular weight markers are present on the far right and left with values indicated on the right. (B) RNA–protein cross-linking examined RNA binding. RNA and protein were incubated, then exposed (+) or not (−) to UV. After RNaseA digestion samples were resolved on NuPAGE gels and exposed to a phosphorplate. U8 snoRNA was cross-linked to Xenopus Nudt16 protein in a UV-dependent manner, resulting in labeled protein at 30 kDa. Human Nudt16 forms UV-dependent cross-links, migrating slightly faster, consistent with the smaller size of the human protein (see panel A). The human Syndesmos protein forms a faint UV-dependent cross-link migrating at 30 KDa, indicated by the asterisk and a higher order cross-link (indicated by the bracket) over 70 kDa. Molecular weight markers (M) are as indicated.