Dataset Information

Structural and Functional Analyses of the FAM46C/Plk4 Complex.

ABSTRACT: FAM46C, a non-canonical poly(A) polymerase, is frequently mutated in multiple myeloma. Loss of function of FAM46C promotes cell survival of multiple myeloma, suggesting a tumor-suppressive role. FAM46C is also essential for fastening sperm head and flagellum, indispensable for male fertility. The molecular mechanisms of these functions of FAM46C remain elusive. We report the crystal structure of FAM46C to provide the basis for its poly(A) polymerase activity and rationalize mutations associated with multiple myeloma. In addition, we found that FAM46C interacts directly with the serine/threonine kinase Plk4, the master regulator of centrosome duplication. We present the structure of FAM46C in complex with the Cryptic Polo-Box 1-2 domains of Plk4. Our structure-based mutational analyses show that the interaction with Plk4 recruits FAM46C to centrosomes. Our data suggest that Plk4-mediated localization of FAM46C enables its regulation of centrosome structure and functions, which may underlie the roles for FAM46C in cell proliferation and sperm development.

SUBMITTER: Chen H

PROVIDER: S-EPMC7415566 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Structural and Functional Analyses of the FAM46C/Plk4 Complex.

Chen Hua H Lu Defen D Shang Guijun G Gao Guoming G Zhang Xuewu X

Structure (London, England : 1993) 20200519 8

FAM46C, a non-canonical poly(A) polymerase, is frequently mutated in multiple myeloma. Loss of function of FAM46C promotes cell survival of multiple myeloma, suggesting a tumor-suppressive role. FAM46C is also essential for fastening sperm head and flagellum, indispensable for male fertility. The molecular mechanisms of these functions of FAM46C remain elusive. We report the crystal structure of FAM46C to provide the basis for its poly(A) polymerase activity and rationalize mutations associated ...[more]

PMID: 32433990

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Project description:BackgroundMultiple myeloma (MM) is a hematologic malignancy characterized by the accumulation of aberrant plasma cells within the bone marrow. The high frequent mutation of family with sequence similarity 46, member C (FAM46C) is closely related with the occurrence and progression of MM. Recently, FAM46C has been identified as a non-canonical poly(A) polymerase (PAP) that functions as a tumor suppressor in MM. This study aimed to elucidate the structural features of this novel non-canonical PAP and how MM-related mutations affect the structural and biochemical properties of FAM46C, eventually advancing our understandings towards FAM46C mutation-related MM occurrence.MethodsWe purified and crystallized a mammalian FAM46C construct, and solved its structure. Next, we characterized the property of FAM46C as a PAP through a combination of structural analysis, site-directed mutagenesis and biochemical assays, and by comparison with its homolog FAM46B. Finally, we structurally analyzed MM-related FAM46C mutations and tested the enzymatic activity of corresponding mutants.ResultsWe determined the crystal structure of a mammalian FAM46C protein at 2.35 Å, and confirmed that FAM46C preferentially consumed adenosine triphosphate (ATP) and extended A-rich RNA substrates. FAM46C showed a weaker PAP activity than its homolog FAM46B, and this difference was largely dependent on the residue variance at particular sites. Of them, residues at positions 77, 290, and 298 of mouse FAM46C were most important for the divergence in enzymatic activity. Among the MM-associated FAM46C mutants, those residing at the catalytic site (D90G and D90H) or putative RNA-binding site (I155L, S156F, D182Y, F184L, Y247V, and M270V) showed abolished or compromised PAP activity of FAM46C, while N72A and S248A did not severely affect the PAP activity. FAM46C mutants D90G, D90H, I155L, S156F, F184L, Y247V, and M270V had significantly lower inhibitory effect on apoptosis of RPMI-8226 cells as compared to wild-type FAM46C.ConclusionsFAM46C is a prokaryotic-like PAP with preference for A-rich RNA substrates, and showed distinct enzymatic efficiency with its homolog FAM46B. The MM-related missense mutations of FAM46C lead to various structural and biochemical outcomes to the protein.

Project description:BackgroundThe post-genomic era is characterised by a torrent of biological information flooding the public databases. As a direct consequence, similarity searches starting with a single query sequence frequently lead to the identification of hundreds, or even thousands of potential homologues. The huge volume of data renders the subsequent structural, functional and evolutionary analyses very difficult. It is therefore essential to develop new strategies for efficient sampling of this large sequence space, in order to reduce the number of sequences to be processed. At the same time, it is important to retain the most pertinent sequences for structural and functional studies.ResultsAn exhaustive analysis on a large scale test set (284 protein families) was performed to compare the efficiency of four different sampling methods aimed at selecting the most pertinent sequences. These four methods sample the proteins detected by BlastP searches and can be divided into two categories: two customisable methods where the user defines either the maximal number or the percentage of sequences to be selected; two automatic methods in which the number of sequences selected is determined by the program. We focused our analysis on the potential information content of the sampled sets of sequences using multiple alignment of complete sequences as the main validation tool. The study considered two criteria: the total number of sequences in BlastP and their associated E-values. The subsequent analyses investigated the influence of the sampling methods on the E-value distributions, the sequence coverage, the final multiple alignment quality and the active site characterisation at various residue conservation thresholds as a function of these criteria.ConclusionThe comparative analysis of the four sampling methods allows us to propose a suitable sampling strategy that significantly reduces the number of homologous sequences required for alignment, while at the same time maintaining the relevant information concerning the active site residues.

Dataset Information

Structural and Functional Analyses of the FAM46C/Plk4 Complex.

Publications

Structural and Functional Analyses of the FAM46C/Plk4 Complex.

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