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

Abstract

<jats:title>Abstract</jats:title> <jats:p>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.</jats:p>

Structural and functional characterization of multiple myeloma associated cytoplasmic poly(A) polymerase FAM46C

“…Many non‐canonical PAPs can extend the 3’ end of various substrate RNAs with different nucleotides, which underlies their biological functions. For example, mammalian PAP germline development defective‐2 (GLD‐2) was recently shown to be able to adenylate and uridylate RNA oligos with diverse sequences, and this feature is consistent with its cellular role, a RNA‐processing enzyme for both mRNAs and microRNAs (miRNAs) [ 51 ]. Another two groups of non‐canonical PAPs, yeast Cid1 and mammalian terminal uridylyltransferase 4/7 (TUT4/7), specifically add uridine to various RNA substrates including mRNAs, miRNAs, or U6 small nuclear RNA [ 52 , 53 , 54 , 55 ].…”

“…For example, the well‐studied GLD‐2 can polyadenylate mRNAs and adenylate/uridylate miRNAs in Huh7 cells [ 59 , 60 , 61 , 62 , 63 ]. Although the partners are known to be important in regulating the activity and substrate specificity of PAPs [ 64 ], the feature of substrate promiscuity for GLD‐2 seems to be intrinsic according to the in vitro elongation experiment [ 51 ]. TUT7, whose catalytic core is structurally similar to GLD‐2, can also work on miRNA with various sequences [ 54 , 55 , 65 , 66 ].…”

“…TUT7, whose catalytic core is structurally similar to GLD‐2, can also work on miRNA with various sequences [ 54 , 55 , 65 , 66 ]. With substantial structural difference from GLD‐2, FAM46C and FAM46B seem to preferentially polyadenylate on RNA substrates with poly(A) tails (Figure 4A ) [ 51 ]. This relatively strict in vitro substrate preference indicates that FAM46 proteins mainly work on mRNA processing.…”