Structural and functional characterization of multiple myeloma associated cytoplasmic poly(A) polymerase FAM46C
Hong Zhang1, Shi-Hui Zhang1, Jia-Li Hu1,2
1State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong, 510060, P. R. China.
Background:
Multiple 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.
Methods:
We 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.
Results:
We 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.
Conclusions:
FAM46C 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.
Insights
Family with sequence similarity 46, member C (FAM46C) acts as a tumor suppressor in multiple myeloma (MM). MM-related mutations in FAM46C disrupt its poly(A) polymerase activity, impacting cell apoptosis and disease progression.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Multiple myeloma (MM) is a bone marrow malignancy linked to frequent mutations in the FAM46C gene.
- FAM46C is identified as a non-canonical poly(A) polymerase (PAP) with tumor-suppressive functions in MM.
- Understanding FAM46C's structure and mutation effects is crucial for MM research.
Purpose of the Study:
- To elucidate the structural features of the non-canonical PAP, FAM46C.
- To investigate how MM-related mutations affect FAM46C's structural and biochemical properties.
- To advance understanding of FAM46C mutation-driven MM occurrence.
Main Methods:
- Purification and crystallization of a mammalian FAM46C construct.
- Structural analysis, site-directed mutagenesis, and biochemical assays to characterize FAM46C's PAP activity.
- Comparison with its homolog FAM46B and structural analysis of MM-related FAM46C mutations.
Main Results:
- Determined the crystal structure of mammalian FAM46C, revealing ATP consumption and A-rich RNA substrate extension.
- FAM46C exhibits weaker PAP activity than FAM46B, influenced by specific residue variations.
- MM-associated mutations at catalytic or RNA-binding sites abolish or compromise FAM46C's PAP activity and its anti-apoptotic effect.
Conclusions:
- FAM46C functions as a prokaryotic-like PAP with a preference for A-rich RNA substrates.
- Distinct enzymatic efficiency exists between FAM46C and FAM46B due to residue differences.
- Missense mutations in FAM46C associated with MM result in diverse structural and biochemical alterations.
