A structural biology view on the enzymes involved in eukaryotic mRNA turnover.

The cellular environment contains numerous ribonucleases that are dedicated to process mRNA transcripts that have been targeted for degradation. Here, we review the three dimensional structures of the ribonuclease complexes (Pan2-Pan3, Ccr4-Not, Xrn1, exosome) and the mRNA decapping enzymes (Dcp2, DcpS) that are involved in mRNA turnover. Structures of major parts of these proteins have been experimentally determined. These enzymes and factors do not act in isolation, but are embedded in interaction networks which regulate enzyme activity and ensure that the appropriate substrates are recruited. The structural details of the higher order complexes that form can, in part, be accurately deduced from known structural data of sub-complexes. Interestingly, many of the ribonuclease and decapping enzymes have been observed in structurally different conformations. Together with experimental data, this highlights that structural changes are often important for enzyme function. We conclude that the known structural data of mRNA decay factors provide important functional insights, but that static structural data needs to be complemented with information regarding protein motions to complete the picture of how transcripts are turned over. In addition, we highlight multiple aspects that influence mRNA turnover rates, but that have not been structurally characterized so far.

The crystal structure of Staufen1 in complex with a physiological RNA sheds light on substrate selectivity.

During mRNA localization, RNA-binding proteins interact with specific structured mRNA localization motifs. Although several such motifs have been identified, we have limited structural information on how these interact with RNA-binding proteins. Staufen proteins bind structured mRNA motifs through dsRNA-binding domains (dsRBD) and are involved in mRNA localization in Drosophila and mammals. We solved the structure of two dsRBDs of human Staufen1 in complex with a physiological dsRNA sequence. We identified interactions between the dsRBDs and the RNA sugar-phosphate backbone and direct contacts of conserved Staufen residues to RNA bases. Mutating residues mediating nonspecific backbone interactions only affected Staufen function in Drosophila when in vitro binding was severely reduced. Conversely, residues involved in base-directed interactions were required in vivo even when they minimally affected in vitro binding. Our work revealed that Staufen can read sequence features in the minor groove of dsRNA and suggests that these influence target selection in vivo.

mRNA localization in metazoans: A structural perspective.

Asymmetric localization of mRNAs is a widespread gene regulatory mechanism that is crucial for many cellular processes. The localization of a transcript involves multiple steps and requires several protein factors to mediate transport, anchoring and translational repression of the mRNA. Specific recognition of the localizing transcript is a key step that depends on linear or structured localization signals, which are bound by RNA-binding proteins. Genetic studies have identified many components involved in mRNA localization. However, mechanistic aspects of the pathway are still poorly understood. Here we provide an overview of structural studies that contributed to our understanding of the mechanisms underlying mRNA localization, highlighting open questions and future challenges.

The bicoid mRNA localization factor Exuperantia is an RNA-binding pseudonuclease.

Anterior patterning in Drosophila is mediated by the localization of bicoid (bcd) mRNA at the anterior pole of the oocyte. Exuperantia (Exu) is a putative exonuclease (EXO) associated with bcd and required for its localization. We present the crystal structure of Exu, which reveals a dimeric assembly with each monomer consisting of a 3'-5' EXO-like domain and a sterile alpha motif (SAM)-like domain. The catalytic site is degenerate and inactive. Instead, the EXO-like domain mediates dimerization and RNA binding. We show that Exu binds RNA directly in vitro, that the SAM-like domain is required for RNA binding activity and that Exu binds a structured element present in the bcd 3' untranslated region with high affinity. Through structure-guided mutagenesis, we show that Exu dimerization is essential for bcd localization. Our data demonstrate that Exu is a noncanonical RNA-binding protein with EXO-SAM-like domain architecture that interacts with its target RNA as a homodimer.

Structural basis for the nuclear export activity of Importin13.

Importin13 (Imp13) is a bidirectional karyopherin that can mediate both import and export of cargoes. Imp13 recognizes several import cargoes, which include the exon junction complex components Mago-Y14 and the E2 SUMO-conjugating enzyme Ubc9, and one known export cargo, the translation initiation factor 1A (eIF1A). To understand how Imp13 can perform double duty, we determined the 3.6-Å crystal structure of Imp13 in complex with RanGTP and with eIF1A. eIF1A binds at the inner surface of the Imp13 C-terminal arch adjacent and concomitantly to RanGTP illustrating how eIF1A can be exported by Imp13. Moreover, the 3.0-Å structure of Imp13 in its unbound state reveals the existence of an open conformation in the cytoplasm that explains export cargo release and completes the export branch of the Imp13 pathway. Finally, we demonstrate that Imp13 is able to bind and export eIF1A in vivo and that its function is essential.

The C-terminal domains of human TNRC6A, TNRC6B, and TNRC6C silence bound transcripts independently of Argonaute proteins.

Proteins of the GW182 family are essential components of the miRNA pathway in animal cells. Vertebrate genomes encode three GW182 paralogs (TNRC6A, TNRC6B, and TNRC6C), which may be functionally redundant. Here, we show that the N-terminal GW-repeat-containing regions of all three TNRC6s interact with the four human Argonaute proteins (AGO1-AGO4). We also show that TNRC6A, TNRC6B, and TNRC6C silence the expression of bound mRNAs. This activity is mediated by their C-terminal silencing domains, and thus, is independent of the interaction with AGO1-AGO4. Silencing by TNRC6A, TNRC6B, and TNRC6C is effected by changes in protein expression and mRNA stability that can, in part, be attributed to deadenylation. Our findings indicate that TNRC6A, TNRC6B, and TNRC6C are recruited to miRNA targets through an interaction between their N-terminal domain and an Argonaute protein; the TNRC6s then promote translational repression and/or degradation of miRNA targets through a C-terminal silencing domain.