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DrosophilaSmaug and its orthologs comprise a family of mRNA repressor proteins that exhibit various functions during animal development. Smaug proteins contain a characteristic RNA-binding sterile-α motif (SAM) domain and a conserved but uncharacterized N-terminal domain (NTD). Here, we resolved the crystal structure of the NTD of the human SAM domain-containing protein 4A (SAMD4A, a.k.a. Smaug1) to 2.0 Å resolution, which revealed its composition of a homodimerization D-subdomain and a subdomain with similarity to a PHAT domain. Furthermore, we show thatDrosophilaSmaug directly interacts with theDrosophilagermline inducer Oskar and with the Hedgehog signaling transducer Smoothened through its D-PHAT domain. We determined the crystal structure of the D-PHAT domain of Smaug in complex with a Smoothened α-helical peptide to 1.61 Å resolution. The peptide binds within a groove that is formed by both the D- and PHAT subdomains. Structural modeling supported by experimental data suggested that an α-helix within the disordered region of Oskar binds to the D-PHAT domain in a mode similar to Smoothened. Together, our data uncover the N-terminal D-PHAT domain of Smaug as peptide-binding domain.

Posttranscriptional regulation of the maternal nanos mRNA is essential for the development of the anterior – posterior axis of the Drosophila embryo. The nanos RNA is regulated by the protein Smaug. Binding to Smaug recognition elements (SREs) in the nanos 3’-UTR, Smaug nucleates the assembly of a larger repressor complex including the eIF4E-T paralog Cup and five additional proteins. The Smaug-dependent complex represses translation of nanos and induces its deadenylation by the CCR4-NOT deadenylase. Here we report an in vitro reconstitution of the Drosophila CCR4-NOT complex and Smaug-dependent deadenylation. We find that Smaug by itself is sufficient to cause deadenylation by the Drosophila or human CCR4-NOT complexes in an SRE-dependent manner. CCR4-NOT subunits NOT10 and NOT11 are dispensable, but the NOT module, consisting of NOT2, NOT3 and the C-terminal part of NOT1, is required. Smaug interacts with the C-terminal domain of NOT3. Both catalytic subunits of CCR4-NOT contribute to Smaug-dependent deadenylation. Whereas the CCR4-NOT complex itself acts distributively, Smaug induces a processive behavior. The cytoplasmic poly(A) binding protein (PABPC) has but a minor effect on Smaug-dependent deadenylation. Among the additional constituents of the Smaug-dependent repressor complex, Cup also facilitates CCR4-NOT-dependent deadenylation, both independently and in cooperation with Smaug.

The LOTUS domain (also known as OST-HTH) is a highly conserved protein domain found in a variety of bacteria and eukaryotes. In animals, the LOTUS domain is present in the proteins Oskar, TDRD5/Tejas, TDRD7/TRAP/Tapas, and MARF1/Limkain B1, all of which play essential roles in animal development, in particular during oogenesis and/or spermatogenesis. This review summarizes the diverse biological as well as molecular functions of LOTUS-domain proteins and discusses their roles as helicase effectors, post-transcriptional regulators, and critical cofactors of piRNA-mediated transcript silencing.

Characterizing protein-protein interactions (PPIs) is fundamental for understanding biochemical processes. Many methods have been established to identify and study direct PPIs; however, screening and investigating PPIs involving large and poorly soluble proteins remain challenges. As a result, we developed ReLo, a simple cell culture-based method to detect and investigate interactions in a cellular context. ReLo allows both the identification of protein domains that mediate the formation of complexes and screening of interfering point mutations. ReLo is sensitive to drugs that mediate or interfere with a specific interaction. Furthermore, protein conformation- and protein arginine methylation-dependent interactions can be studied. Importantly, ReLo can be used for specifically detecting direct but not indirect PPIs and can be applied for describing the binding topology of subunits within multiprotein complexes. Because of these attributes, ReLo is a simple, quick and versatile tool for identifying and studying binary PPIs, as well as for characterizing multisubunit complexes.

DEAD-box RNA helicases play important roles in a wide range of metabolic processes. Regulatory proteins can stimulate or block the activity of DEAD-box helicases. Here, we show that LOTUS (Limkain, Oskar, and Tudor containing proteins 5 and 7) domains present in the germline proteins Oskar, TDRD5 (Tudor domain-containing 5), and TDRD7 bind and stimulate the germline-specific DEAD-box RNA helicase Vasa. Our crystal structure of the LOTUS domain of Oskar in complex with the C-terminal RecA-like domain of Vasa reveals that the LOTUS domain occupies a surface on a DEAD-box helicase not implicated previously in the regulation of the enzyme's activity. We show that, in vivo, the localization of Drosophila Vasa to the nuage and germ plasm depends on its interaction with LOTUS domain proteins. The binding and stimulation of Vasa DEAD-box helicases by LOTUS domains are widely conserved.

Turnover of mRNA releases, in addition to the four regular nucleoside monophosphates, the methylated cap nucleotide in the form of 7-methylguanosine monophosphate (m7GMP) or diphosphate (m7GDP). The existence of pathways to eliminate the modified nucleotide seems likely, as its incorporation into nucleic acids is undesirable. Here we describe a novel 5′ nucleotidase from Drosophila that cleaves m7GMP to 7-methylguanosine and inorganic phosphate. The enzyme, encoded by the predicted gene CG3362, also efficiently dephosphorylates CMP, although with lower apparent affinity; UMP and the purine nucleotides are poor substrates. The enzyme is inhibited by elevated concentrations of AMP and also cleaves m7GDP to the nucleoside and two inorganic phosphates, albeit less efficiently. CG3362 has equivalent sequence similarity to two human enzymes, cytosolic nucleotidase III (cNIII) and the previously uncharacterized cytosolic nucleotidase III-like (cNIII-like). We show that cNIII-like also displays 5′ nucleotidase activity with a high affinity for m7GMP. CMP is a slightly better substrate but again with a higher Km. The activity of cNIII-like is stimulated by phosphate. In contrast to cNIII-like, cNIII and human cytosolic nucleotidase II do not accept m7GMP as a substrate. We suggest that the m7G-specific nucleotidases protect cells against undesired salvage of m7GMP and its incorporation into nucleic acids. Background: mRNA decay releases, in addition to the regular nucleotides, 7-methyl GMP derived from the 5′ cap. Results: We describe new members of the 5′ nucleotidase family degrading 7-methyl GMP to 7-methylguanosine and orthophosphate. Conclusion: Cells have mechanisms to prevent potential salvage of 7-methyl GMP. Significance: 7-Methyl GMP degradation may be important to prevent its incorporation into nucleic acids.

The nanos (nos) mRNA encodes the posterior determinant of the Drosophila embryo. Translation of the RNA is repressed throughout most of the embryo by the protein Smaug binding to Smaug recognition elements (SREs) in the 3′ UTR. Translation is locally activated at the posterior pole by Oskar. This paper reports that the SREs govern the time- and ATP-dependent assembly of an exceedingly stable repressed ribonucleoprotein particle (RNP) in embryo extract. Repression can be virtually complete. Smaug and its co-repressor Cup as well as Trailer hitch and the DEAD box protein Me31B are part of the repressed RNP. The initiation factor eIF4G is specifically displaced, and 48S pre-initiation complex formation is inhibited. However, later steps in translation initiation are also sensitive to SRE-dependent inhibition. These data confirm several previously untested predictions of a current model for Cup-dependent repression but also suggest that the Cup model by itself is insufficient to explain translational repression of the nos RNA. In the embryo extract, recombinant Oskar relieves translational repression and deadenylation by preventing Smaug's binding to the SREs.

Deadenylation is the removal of poly(A) tails from mRNA. This chapter presents two methods to assay deadenylation in vitro. The first is a quick and quantitative assay for the degradation of radiolabeled poly(A) that can easily be adapted to be used for many different enzymes. The second method uses an extract from Drosophila embryos to catalyze the deadenylation of an RNA dependent on a specific sequence that also directs deadenylation in vivo.

Deadenylation is the removal of poly(A) tails from mRNA. This chapter presents two methods to assay deadenylation in vitro. The first is a quick and quantitative assay for the degradation of radiolabeled poly(A) that can easily be adapted to be used for many different enzymes. The second method uses an extract from Drosophila embryos to catalyze the deadenylation of an RNA dependent on a specific sequence that also directs deadenylation in vivo.

The nanos (nos) mRNA encodes the posterior determinant of the Drosophila embryo. Translation of the RNA is repressed throughout most of the embryo by the protein Smaug binding to Smaug recognition elements (SREs) in the 3' UTR. Translation is locally activated at the posterior pole by Oskar. This paper reports that the SREs govern the time- and ATP-dependent assembly of an exceedingly stable repressed ribonucleoprotein particle (RNP) in embryo extract. Repression can be virtually complete. Smaug and its co-repressor Cup as well as Trailer hitch and the DEAD box protein Me31B are part of the repressed RNP. The initiation factor eIF4G is specifically displaced, and 48S pre-initiation complex formation is inhibited. However, later steps in translation initiation are also sensitive to SRE-dependent inhibition. These data confirm several previously untested predictions of a current model for Cup-dependent repression but also suggest that the Cup model by itself is insufficient to explain translational repression of the nos RNA. In the embryo extract, recombinant Oskar relieves translational repression and deadenylation by preventing Smaug's binding to the SREs.

Deadenylation initiates degradation of most mRNAs in eukaryotes. Regulated deadenylation of an mRNA plays an important role in translation control as well, especially during animal oogenesis and early embryonic development. To investigate the mechanism of sequence-dependent deadenylation, we established an in vitro system derived from 0- to 2-h-old Drosophila embryos. These extracts faithfully reproduce several aspects of the regulation of nanos mRNA: They display translation repression and deadenylation both mediated by the same sequences within the nanos 3' UTR. Here, we describe detailed protocols for preparing Drosophila embryo extracts, and their use in deadenylation assays exemplified with exogenous RNA substrates containing the nanos 3' UTR.

Deadenylation initiates degradation of most mRNAs in eukaryotes. Regulated deadenylation of an mRNA plays an important role in translation control as well, especially during animal oogenesis and early embryonic development. To investigate the mechanism of sequence‐dependent deadenylation, we established an in vitro system derived from 0‐ to 2‐h‐old Drosophila embryos. These extracts faithfully reproduce several aspects of the regulation of nanos mRNA: They display translation repression and deadenylation both mediated by the same sequences within the nanos 3′ UTR. Here, we describe detailed protocols for preparing Drosophila embryo extracts, and their use in deadenylation assays exemplified with exogenous RNA substrates containing the nanos 3′ UTR.