FAM46B is a prokaryotic-like cytoplasmic poly(A) polymerase essential in human embryonic stem cells

Abstract

Family with sequence similarity (FAM46) proteins are newly identified metazoan-specific poly(A) polymerases (PAPs). Although predicted as Gld-2-like eukaryotic non-canonical PAPs, the detailed architecture of FAM46 proteins is still unclear. Exact biological functions for most of FAM46 proteins also remain largely unknown. Here, we report the first crystal structure of a FAM46 protein, FAM46B. FAM46B is composed of a prominently larger N-terminal catalytic domain as compared to known eukaryotic PAPs, and a C-terminal helical domain. FAM46B resembles prokaryotic PAP/CCA-adding enzymes in overall folding as well as certain inter-domain connections, which distinguishes FAM46B from other eukaryotic non-canonical PAPs. Biochemical analysis reveals that FAM46B is an active PAP, and prefers adenosine-rich substrate RNAs. FAM46B is uniquely and highly expressed in human pre-implantation embryos and pluripotent stem cells, but sharply down-regulated following differentiation. FAM46B is localized to both cell nucleus and cytosol, and is indispensable for the viability of human embryonic stem cells. Knock-out of FAM46B is lethal. Knock-down of FAM46B induces apoptosis and restricts protein synthesis. The identification of the bacterial-like FAM46B, as a pluripotent stem cell-specific PAP involved in the maintenance of translational efficiency, provides important clues for further functional studies of this PAP in the early embryonic development of high eukaryotes.

Structures of mammalian GLD-2 proteins reveal molecular basis of their functional diversity in mRNA and microRNA processing

“…We next investigated the preference of rnGLD-2 for nucleotides and substrate RNAs. The consumption of ATP/GTP/UTP/CTP in the presence of A 15 oligo substrate was individually monitored in time course based on an HPLC-based quantification assay ( 47 ). After 12 min, around 75% ATP and 40% GTP was consumed by rnGLD-2, whereas over 90% UTP and CTP remained unhydrolyzed, suggesting that rnGLD-2 prefers ATP over other nucleotides during polyadenylation (Figure 5A ).…”

“…While cytoplasmic polyadenylation in mammalian development is a pivotal event, it is possible that other PAPs take over or share this responsibility in mammals. For example, members of FAM46 proteins, recently reported as active non-canonical PAPs ( 47 , 56 ), showed specifically increased expression during gametogenesis and early embryonic development. The fact that mammalian GLD-2s are intrinsically potent PAPs and stay in a constitutive active state suggests that they may not need GLD-3- or RNP-8-like partners as in C. elegans to stimulate PAP activity.…”

Functions and mechanisms of RNA tailing by metazoan terminal nucleotidyltransferases

“…Importantly, human TENT5B has almost exclusive preference for ATP incorporation, suggesting that TENT5 proteins are highly unlikely to perform mixed RNA tailing. Biochemical analysis revealed that human TENT5B prefers substrate RNAs that are adenosine‐rich close to the very 3′ end, suggesting that TENT5 proteins preferentially act on already polyadenylated mRNAs (Hu et al, 2020). The functional relevance of cytoplasmic polyadenylation by TENT5 ncPAP is only beginning to emerge because it is a relatively newly discovered family of TENTs (Figure 4) (Kuchta et al, 2016).…”

“…As mentioned above, the structure of xtTENT5B protein has been solved, and the biochemical properties of TENT5B have recently been reported (Hu et al, 2020). However, few studies have described the functional relevance of this ncPAP.…”

“…Large‐scale transcriptomic analyses revealed that TENT5B is specifically expressed in human embryonic stem cells (hESCs) and human induced pluripotent stem cells, and its level is sharply downregulated during differentiation (Hu et al, 2020). In hESCs, TENT5B knockout is lethal, whereas knockdown leads to a lower abundance of newly synthesized proteins and the severely compromised viability of hESCs.…”