Ma XY et al. (2020), Structures of mammalian GLD-2 proteins reveal m...

XB-ART-57438

Nucleic Acids Res 2020 Sep 04;4815:8782-8795. doi: 10.1093/nar/gkaa578.

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

Ma XY , Zhang H , Feng JX , Hu JL , Yu B , Luo L , Cao YL , Liao S , Wang J , Gao S .

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The stability and processing of cellular RNA transcripts are efficiently controlled via non-templated addition of single or multiple nucleotides, which is catalyzed by various nucleotidyltransferases including poly(A) polymerases (PAPs). Germline development defective 2 (GLD-2) is among the first reported cytoplasmic non-canonical PAPs that promotes the translation of germline-specific mRNAs by extending their short poly(A) tails in metazoan, such as Caenorhabditis elegans and Xenopus. On the other hand, the function of mammalian GLD-2 seems more diverse, which includes monoadenylation of certain microRNAs. To understand the structural basis that underlies the difference between mammalian and non-mammalian GLD-2 proteins, we determine crystal structures of two rodent GLD-2s. Different from C. elegans GLD-2, mammalian GLD-2 is an intrinsically robust PAP with an extensively positively charged surface. Rodent and C. elegans GLD-2s have a topological difference in the β-sheet region of the central domain. Whereas C. elegans GLD-2 prefers adenosine-rich RNA substrates, mammalian GLD-2 can work on RNA oligos with various sequences. Coincident with its activity on microRNAs, mammalian GLD-2 structurally resembles the mRNA and miRNA processor terminal uridylyltransferase 7 (TUT7). Our study reveals how GLD-2 structurally evolves to a more versatile nucleotidyltransferase, and provides important clues in understanding its biological function in mammals.

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Genes referenced: tspan7 tut4

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	Figure 1. Overall structure of GLD-2. (A) Schematic representation showing the domain organization of mammalian and Caenorhabditiselegans GLD-2 homologs. Borders of the domains are indicated by residue numbers. hs, Homo sapiens; mm, Mus musculus; rn, Rattus norvegicus; ce, Caenorhabditis elegans. (B) Cartoon representation of rnGLD-2, colored as in A. The identity of each helix and Î²-strand are indicated. (C) The topology diagram of rnGLD-2. Secondary structural elements are not drawn to scale. Elements of rnGLD-2 are named and colored as in B.
	Figure 2. Structure comparison between rnGLD-2 and ceGLD-2. (A) Structural superposition of rnGLD-2 with ceGLD-2 complexed with GLD-3 (left, PDB code: 4zrl) or with RNP-8 (right, PDB code: 5jnb). The Î²6âÎ²9 regions are indicated. (B andC) Structural details of ceGLD-2 at the tip of the central domain when complexed with RNP-8 (B) or with GLD-3 (C). (D andE) The Î²-sheet at the central domain of rnGLD-2 (D) and Cid1 (E). (F) Sequence alignment for Î²8âÎ²9 of GLD-2s from various species and corresponding Î²10âÎ²11 of Cid1. Residues after Î²9 of ceGLD-2 that are missing in the structural model are indicated by a dashed line. (G) Comparison of the Î²-sheet at the central domain for rnGLD-2, ceGLD-2 and Cid1 shown as topology diagrams. The ceGLD-2 specific insert is indicated as a blue dashed line. Note the difference in the arrangement of Î²8 and Î²9 between rnGLD-2 and ceGLD-2.
	Figure 3. rnGLD-2 is a potent PAP. (A) The PAP activity of rnGLD-2 in the presence of various divalent cations. A total of 400 nM rnGLD-2 was incubated with 500 nM 5â€² biotinylated A15 RNA oligo and 500 Î¼M ATP. For each sample, 2 mM indicated divalent ion or EDTA was supplied to the reaction. w/o, without protein. (B) Protein concentration-dependent PAP activity of hsGLD-2, mmGLD-2, rnGLD-2 and ceGLD-2. (C) Comparison between truncated and full-length hsGLD-2 in PAP activity. (D) Overall structural comparison between rnGLD-2 and ceGLD-2/RNP-8 complex from the backside of the catalytic domain, where the interaction site of ceGLD-2 and RNP-8 can be seen. Frames indicate the areas shown in panels (Eâ€“H). (Eâˆ’H) Detailed structure differences between rnGLD-2 and ceGLD-2 explaining the discrepancy of their PAP activity. Color as in D. (E) The unique 310 helix (Î·1) of rnGLD-2 and its relative position to RNP-8. (F) Interaction between Î·1 and Î±4 buries the local hydrophobic cluster of rnGLD-2. (G) The N-terminal tip of rnGLD-2 shelters the hydrophobic residues on Î±1 and Î±5. (H) The histidine cluster on Î·1 and non-conserved Arg295 of rnGLD-2. (I) Electrostatic surface potential comparison at the backside of catalytic center for rnGLD-2 (left) and ceGLD-2 complexed with GLD-3 (middle) or RNP-8 (right). Locations of the N-terminal tip (N) and 310 helix (Î·1) of rnGLD-2 are outlined by yellow dashes, and the histidine cluster and Arg295 are indicated. GLD-3 and RNP-8 are shown in cartoon representation with 50% transparency. (J) Non-conserved surface residues between rnGLD-2 and ceGLD-2 that may affect substrate binding. (K) PAP activity of rnGLD-2 with mutations regarding the residue difference from ceGLD-2 as shown in (J).
	Figure 4. Important residues for ATP coordination of rnGLD-2. (AâC) Comparison between rnGLD-2 and other PAPs at adenosine nucleotide binding site. Residues of Bos taurus (bt)PAPÎ± (PDB code: 1f5a, A), Saccharomyces cerevisiae (sc)PAP (PDB code: 2q66, B) and Gallus gallus mitochondria (ggMT)PAP (PDB code: 5a30, C) involved in binding 3â²-dATP, ATP and ATPÎ³S are individually superimposed with corresponding residues of rnGLD-2. (D) PAP activity of rnGLD-2 with mutations regarding the residues potentially involved in ATP coordination as shown in AâC.
	Figure 5. Substrate preference of rnGLD-2. (A) Time-course NTP consumption of rnGLD-2 with A15. Error bars indicate s.d. (n = 3). (B) Time-course ATP consumption of rnGLD-2 with various 15-mer RNA oligos. (C) NTP consumption of rnGLD-2 with various 15-mer RNA oligos. (D) Nucleotidyltransferase assays showing the preference of rnGLD-2 on various substrates and different nucleotides. 400 nM rnGLD-2, 500 nM 5â€² biotinylated 15-mer RNA substrates with various sequences, and 500 Î¼M ATP/UTP/GTP/CTP were used.
	Figure 6. Similarity between rnGLD-2 and TUT7CM. (A) NTase activity of rnGLD-2 on different miRNA substrates. A total of 400 nM rnGLD-2, 500 nM various 5â²-biotinylated miRNA and 500 Î¼M ATP/UTP/GTP/CTP were used. (B) Overall structure comparison between rnGLD-2 and TUT7CM in complex with UMPNPP/U2 (upper, PDB code: 5w0n) and with UTP/dsRNA (lower, PDB code: 5w0o). (C) Comparison between rnGLD-2 and TUT7CM at UTP binding site. Residues of TUT7CM (PDB code: 5w0o) involved in binding UTP is superimposed with corresponding residues of rnGLD-2. (D) Uridylation activity of rnGLD-2 with mutations regarding the residues potentially involved in UTP coordination. (E) Comparison between rnGLD-2 and TUT7CM at substrate binding site. Residues of TUT7CM (PDB code: 5w0n) involved in binding the U2 substrate is superimposed with corresponding residues of rnGLD-2. (F) Adenylation and uridylation activity of rnGLD-2 with mutations regarding the residues potentially involved in substrate binding.
	Figure 7. Analysis of positively charged surface residues of rnGLD-2. (A) The electrostatic surface potential of rnGLD-2. Some positively charged residues are specified. Two positively charged patches and the catalytic cleft are indicated. (B) The surface conservation plots of rnGLD-2 within the GLD-2 family (left) and among other NTases including MTPAP, Cid1, TUT4 and TUT7 (right). (C) PAP activity of rnGLD-2 with mutations of positively charged surface residues as in A. (D) EMSA results showing the substrate affinity of rnGLD-2 with mutations of positively charged surface residues as in A.

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Adams, PHENIX: a comprehensive Python-based system for macromolecular structure solution. 2010, Pubmed