An exon junction complex-independent function of Barentsz in neuromuscular synapse growth.

The exon junction complex controls the translation, degradation, and localization of spliced mRNAs, and three of its core subunits also play a role in splicing. Here, we show that a fourth subunit, Barentsz, has distinct functions within and separate from the exon junction complex in Drosophila neuromuscular development. The distribution of mitochondria in larval muscles requires Barentsz as well as other exon junction complex subunits and is not rescued by a Barentsz transgene in which residues required for binding to the core subunit eIF4AIII are mutated. In contrast, interactions with the exon junction complex are not required for Barentsz to promote the growth of neuromuscular synapses. We find that the Activin ligand Dawdle shows reduced expression in barentsz mutants and acts downstream of Barentsz to control synapse growth. Both barentsz and dawdle are required in motor neurons, muscles, and glia for normal synapse growth, and exogenous Dawdle can rescue synapse growth in the absence of barentsz. These results identify a biological function for Barentsz that is independent of the exon junction complex.

Hakai is required for stabilization of core components of the m6A mRNA methylation machinery.

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.

Molecular basis for GIGYF-Me31B complex assembly in 4EHP-mediated translational repression.

GIGYF (Grb10-interacting GYF [glycine-tyrosine-phenylalanine domain]) proteins coordinate with 4EHP (eIF4E [eukaryotic initiation factor 4E] homologous protein), the DEAD (Asp-Glu-Ala-Asp)-box helicase Me31B/DDX6, and mRNA-binding proteins to elicit transcript-specific repression. However, the underlying molecular mechanism remains unclear. Here, we report that GIGYF contains a motif necessary and sufficient for direct interaction with Me31B/DDX6. A 2.4 Å crystal structure of the GIGYF-Me31B complex reveals that this motif arranges into a coil connected to a ß hairpin on binding to conserved hydrophobic patches on the Me31B RecA2 domain. Structure-guided mutants indicate that 4EHP-GIGYF-DDX6 complex assembly is required for tristetraprolin-mediated down-regulation of an AU-rich mRNA, thus revealing the molecular principles of translational repression.

Direct role for the Drosophila GIGYF protein in 4EHP-mediated mRNA repression.

The eIF4E-homologous protein (4EHP) is a translational repressor that competes with eIF4E for binding to the 5'-cap structure of specific mRNAs, to which it is recruited by protein factors such as the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins (GIGYF). Several experimental evidences suggest that GIGYF proteins are not merely facilitating 4EHP recruitment to transcripts but are actually required for the repressor activity of the complex. However, the underlying molecular mechanism is unknown. Here, we investigated the role of the uncharacterized Drosophila melanogaster (Dm) GIGYF protein in post-transcriptional mRNA regulation. We show that, when in complex with 4EHP, Dm GIGYF not only elicits translational repression but also promotes target mRNA decay via the recruitment of additional effector proteins. We identified the RNA helicase Me31B/DDX6, the decapping activator HPat and the CCR4-NOT deadenylase complex as binding partners of GIGYF proteins. Recruitment of Me31B and HPat via discrete binding motifs conserved among metazoan GIGYF proteins is required for downregulation of mRNA expression by the 4EHP-GIGYF complex. Our findings are consistent with a model in which GIGYF proteins additionally recruit decapping and deadenylation complexes to 4EHP-containing RNPs to induce translational repression and degradation of mRNA targets.

GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression.

The eIF4E homologous protein (4EHP) is thought to repress translation by competing with eIF4E for binding to the 5' cap structure of specific mRNAs to which it is recruited through interactions with various proteins, including the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins 1 and 2 (GIGYF1/2). Despite its similarity to eIF4E, 4EHP does not interact with eIF4G and therefore fails to initiate translation. In contrast to eIF4G, GIGYF1/2 bind selectively to 4EHP but not eIF4E. Here, we present crystal structures of the 4EHP-binding regions of GIGYF1 and GIGYF2 in complex with 4EHP, which reveal the molecular basis for the selectivity of the GIGYF1/2 proteins for 4EHP. Complementation assays in a GIGYF1/2-null cell line using structure-based mutants indicate that 4EHP requires interactions with GIGYF1/2 to down-regulate target mRNA expression. Our studies provide structural insights into the assembly of 4EHP-GIGYF1/2 repressor complexes and reveal that rather than merely facilitating 4EHP recruitment to transcripts, GIGYF1/2 proteins are required for repressive activity.

A CAF40-binding motif facilitates recruitment of the CCR4-NOT complex to mRNAs targeted by Drosophila Roquin.

Human (Hs) Roquin1 and Roquin2 are RNA-binding proteins that promote mRNA target degradation through the recruitment of the CCR4-NOT deadenylase complex and are implicated in the prevention of autoimmunity. Roquin1 recruits CCR4-NOT via a C-terminal region that is not conserved in Roquin2 or in invertebrate Roquin. Here we show that Roquin2 and Drosophila melanogaster (Dm) Roquin also interact with the CCR4-NOT complex through their C-terminal regions. The C-terminal region of Dm Roquin contains multiple motifs that mediate CCR4-NOT binding. One motif binds to the CAF40 subunit of the CCR4-NOT complex. The crystal structure of the Dm Roquin CAF40-binding motif (CBM) bound to CAF40 reveals that the CBM adopts an α-helical conformation upon binding to a conserved surface of CAF40. Thus, despite the lack of sequence conservation, the C-terminal regions of Roquin proteins act as an effector domain that represses the expression of mRNA targets via recruitment of the CCR4-NOT complex.

A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing.

CCR4-NOT is a major effector complex in miRNA-mediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)-containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. Here, we elucidate the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. We show that the conserved CNOT9 subunit attaches to a domain of unknown function (DUF3819) in the CNOT1 scaffold. The resulting complex provides binding sites for TNRC6/GW182, and its crystal structure reveals tandem W-binding pockets located in CNOT9. We further show that the CNOT1 MIF4G domain interacts with the C-terminal RecA domain of DDX6, a translational repressor and decapping activator. The crystal structure of this complex demonstrates striking similarity to the eIF4G-eIF4A complex. Together, our data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping.

NOT10 and C2orf29/NOT11 form a conserved module of the CCR4-NOT complex that docks onto the NOT1 N-terminal domain.

The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotes. This complex catalyzes the removal of mRNA poly(A) tails, thereby repressing translation and committing an mRNA to degradation. The conserved core of the complex is assembled by the interaction of at least two modules: the NOT module, which minimally consists of NOT1, NOT2 and NOT3, and a catalytic module comprising two deadenylases, CCR4 and POP2/CAF1. Additional complex subunits include CAF40 and two newly identified human subunits, NOT10 and C2orf29. The role of the NOT10 and C2orf29 subunits and how they are integrated into the complex are unknown. Here, we show that the Drosophila melanogaster NOT10 and C2orf29 orthologs form a complex that interacts with the N-terminal domain of NOT1 through C2orf29. These interactions are conserved in human cells, indicating that NOT10 and C2orf29 define a conserved module of the CCR4-NOT complex. We further investigated the assembly of the D. melanogaster CCR4-NOT complex, and demonstrate that the conserved armadillo repeat domain of CAF40 interacts with a region of NOT1, comprising a domain of unknown function, DUF3819. Using tethering assays, we show that each subunit of the CCR4-NOT complex causes translational repression of an unadenylated mRNA reporter and deadenylation and degradation of a polyadenylated reporter. Therefore, the recruitment of a single subunit of the complex to an mRNA target induces the assembly of the complete CCR4-NOT complex, resulting in a similar regulatory outcome.

The structural basis for the interaction between the CAF1 nuclease and the NOT1 scaffold of the human CCR4-NOT deadenylase complex.

The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotic cells. It catalyzes the removal of mRNA poly(A) tails, thereby repressing translation and committing mRNAs to decay. The conserved core of the complex consists of a catalytic module comprising two deadenylases (CAF1/POP2 and CCR4a/b) and the NOT module, which contains at least NOT1, NOT2 and NOT3. NOT1 bridges the interaction between the two modules and therefore, acts as a scaffold protein for the assembly of the complex. Here, we present the crystal structures of the CAF1-binding domain of human NOT1 alone and in complex with CAF1. The NOT1 domain comprises five helical hairpins that adopt an MIF4G (middle portion of eIF4G) fold. This NOT1 MIF4G domain binds CAF1 through a pre-formed interface and leaves the CAF1 catalytic site fully accessible to RNA substrates. The conservation of critical structural and interface residues suggests that the NOT1 MIF4G domain adopts a similar fold and interacts with CAF1 in a similar manner in all eukaryotes. Our findings shed light on the assembly of the CCR4-NOT complex and provide the basis for dissecting the role of the NOT module in mRNA deadenylation.

In Silico Prediction of Evolutionarily Conserved GC-Rich Elements Associated with Antigenic Proteins of Plasmodium falciparum.

The Plasmodium falciparum genome being AT-rich, the presence of GC-rich regions suggests functional significance. Evolution imposes selection pressure to retain functionally important coding and regulatory elements. Hence searching for evolutionarily conserved GC-rich, intergenic regions in an AT-rich genome will help in discovering new coding regions and regulatory elements. We have used elevated GC content in intergenic regions coupled with sequence conservation against P. reichenowi, which is evolutionarily closely related to P. falciparum to identify potential sequences of functional importance. Interestingly, ~30% of the GC-rich, conserved sequences were associated with antigenic proteins encoded by var and rifin genes. The majority of sequences identified in the 5' UTR of var genes are represented by short expressed sequence tags (ESTs) in cDNA libraries signifying that they are transcribed in the parasite. Additionally, 19 sequences were located in the 3' UTR of rifins and 4 also have overlapping ESTs. Further analysis showed that several sequences associated with var genes have the capacity to encode small peptides. A previous report has shown that upstream peptides can regulate the expression of var genes hence we propose that these conserved GC-rich sequences may play roles in regulation of gene expression.

5' and 3' end modifications of spliceosomal RNAs in Plasmodium falciparum.

5' caps provide recognition sequences for the nuclear import of snRNAs. The 5' and 3' ends of snRNAs were studied in Plasmodium falciparum with a modified adapter ligation method, which showed that 5' ends of U1, U2, U4, U5 and U6 snRNAs are capped. In P. falciparum, the 3' ends of U1, U2, U4 and U5 snRNAs have free hydroxyl groups whereas U6 snRNA has a blocked 3' end. An immunoprecipitation assay for trimethyl guanosine caps shows that the cap structures of parasite U1-U5 snRNAs are hypermethylated while U6 snRNA may be gamma-mono-methylated. Bioinformatics analysis of proteins involved in hypermethylation and trafficking of snRNAs indicates that the methyltransferase TGS1 is present in the P. falciparum genome. PfTGS1 is larger than its orthologs and may have transmembrane domains in the C-terminus. Surprisingly, the snRNA trafficking protein Snurportin is absent from the P. falciparum genome suggesting that reminiscent of yeast, parasite snRNAs may be retained in the nucleus.

A screen for conserved sequences with biased base composition identifies noncoding RNAs in the A-T rich genome of Plasmodium falciparum.

Noncoding RNAs (ncRNAs) such as snRNAs, snoRNAs and microRNAs play important roles in transcription and translation control. These ncRNAs have yet to be discovered in the malarial parasite Plasmodium falciparum, an organism in which these basic biological processes are poorly understood. Inspired by a report by Klein et al., we initiated a bioinformatics screen to uncover several candidate ncRNAs from the parasite genome using two simple criteria: first, elevated GC content in the highly A-T rich intergenic regions of the P. falciparum genome and second, conservation of sequence homology between malaria parasite species. We show that all the annotated tRNAs can be successfully identified in our screen as well as several new candidates that show homology to snRNAs and snoRNAs, and ten candidate ncRNAs of unknown function. Three of the candidate snRNAs, a predicted selenocysteine tRNA and two candidates of unknown function are expressed in asexual stage parasites, further validating the screen. With these results, the biological processes underlying RNA-mediated regulation of transcription, translation and splicing can be studied in an important human pathogen.