FAM46 proteins are novel eukaryotic non-canonical poly(A) polymerases

Abstract

FAM46 proteins, encoded in all known animal genomes, belong to the nucleotidyltransferase (NTase) fold superfamily. All four human FAM46 paralogs (FAM46A, FAM46B, FAM46C, FAM46D) are thought to be involved in several diseases, with FAM46C reported as a causal driver of multiple myeloma; however, their exact functions remain unknown. By using a combination of various bioinformatics analyses (e.g. domain architecture, cellular localization) and exhaustive literature and database searches (e.g. expression profiles, protein interactors), we classified FAM46 proteins as active non-canonical poly(A) polymerases, which modify cytosolic and/or nuclear RNA 3' ends. These proteins may thus regulate gene expression and probably play a critical role during cell differentiation. A detailed analysis of sequence and structure diversity of known NTases possessing PAP/OAS1 SBD domain, combined with state-of-the-art comparative modelling, allowed us to identify potential active site residues responsible for catalysis and substrate binding. We also explored the role of single point mutations found in human cancers and propose that FAM46 genes may be involved in the development of other major malignancies including lung, colorectal, hepatocellular, head and neck, urothelial, endometrial and renal papillary carcinomas and melanoma. Identification of these novel enzymes taking part in RNA metabolism in eukaryotes may guide their further functional studies.

Figure 1.

Phylogenetic tree of representative FAM46 protein sequences. Maximum likelihood (ML) analysis for selected 29 family members was carried out using the LG+G model. The approximate likelihood ratio test Shimodaira–Hasegawa-like (SH-like) branch supports above 0.5 are shown. Branches with support lower than 0.5 were collapsed.

Figure 2.

Multiple sequence alignment of human FAM46 proteins, human non-canonical poly(A) polymerases (TUT1-7) and all representative structures possessing both the NTase and PAP/OAS1 SBD domains. Only conserved regions of the domains are shown. Sequences are labelled with PDB code or UniProt ID. The numbers of excluded residues are specified in parentheses. Residue conservation is denoted with the following scheme: uncharged, highlighted in yellow; polar, highlighted in grey; invariant active site residues involved in catalysis, highlighted in black; critical substrate binding residues, highlighted in red. Locations of observed and predicted secondary structure elements are marked above the corresponding alignment blocks. Abbreviations: PAP, poly(A) polymerase; TUTase, terminal uridylyltransferase; CCA, CCA-adding enzyme; OAS, oligoadenylate synthetase; cGAS, cyclic GMP-AMP synthase; NF45 and NF90, nuclear factors NF45 and NF90; Utp22, U3 small nucleolar RNA-associated protein 22; MiD51 and MiD49, mitochondrial dynamics proteins MiD51 and MID49; Ss, S. scrofa; Tb, T. brucei; Af, A. fulgidus; Hs, H. sapiens; Mm, M. musculus; Sp, S. pombe; Sc, S. cerevisiae; Vc, V. cholerae; Bt, B. taurus. Sequence-to-structure alignment for FAM46 proteins can be assigned higher confidence in the NTase domain.

Figure 3.

Comparison of 3D model of human FAM46C and available structures of non-canonical poly(A) polymerase Trf4p (pdb|3nyb) and mitochondrial dynamics protein MiD51 (pdb|4oaf). Regions in MiD51 responsible for protein–protein interactions and their potential counterpart in FAM46C are coloured pink. The region between the fourth and fifth core α-helices of PAP/OAS1 SBD in FAM46C and Trf4p (critical for nucleobase binding) is shown in green. The region not modelled in FAM46C (70 amino acids) is denoted by red dots. The conserved active site carboxylates are shown in blue.

Figure 4.

Comparison of the active sites of FAM46C, poly(A) polymerase (Pap1, pdb|1fa0), poly(U) polymerase (Cid1, pdb|4fhp), CCA-adding enzyme (pdb|4x4r), OAS (OAS1, pdb|4rwo) and cyclic GMP-AMP synthase (Mb21d1, pdb|4k97). Only NTase and PAP/OAS1 SBD domains are shown. The region not modelled in FAM46C (70 amino acids) is denoted by red dots. Conserved amino acids critical for catalysis and substrate binding are shown in blue.

Figure 5.

Missense mutations in FAM46 family members found in cancer patients and hem6 mouse. The positions of the corresponding single point mutations, mapped onto a 3D model of human FAM46C, are shown as spheres. The spheres are coloured according to JSD score, which refers to the amino acid conservation in FAM46 family.