IP6K1 upregulates the formation of processing bodies by influencing protein-protein interactions on the mRNA cap

Inositol hexakisphosphate kinase 1 (IP6K1) is a small molecule kinase that catalyzes the conversion of the inositol phosphate IP6 to 5-IP7. We show that IP6K1 acts independent of its catalytic activity to upregulate the formation of processing bodies (P-bodies), which are cytoplasmic ribonucleoprotein granules that store translationally repressed mRNA. IP6K1 does not localize to P-bodies, but instead binds to ribosomes, where it interacts with the mRNA decapping complex - the scaffold protein EDC4, activator proteins DCP1A/B, decapping enzyme DCP2, and RNA helicase DDX6. Along with its partner 4E-T, DDX6 is known to nucleate protein-protein interactions on the 5’ mRNA cap to facilitate P-body formation. IP6K1 binds the translation initiation complex eIF4F on the mRNA cap, augmenting the interaction of DDX6 with 4E-T and the cap binding protein eIF4E. Cells with reduced IP6K1 show downregulated microRNA-mediated translational suppression and increased stability of DCP2-regulated transcripts. Our findings unveil IP6K1 as a novel facilitator of proteome remodelling on the mRNA cap, tipping the balance in favour of translational repression over initiation, thus leading to P-body assembly.

Characterization of an Atypical eIF4E Ortholog in Leishmania, LeishIF4E-6

Leishmania parasites are digenetic protists that shuffle between sand fly vectors and mammalian hosts, transforming from flagellated extracellular promastigotes that reside within the intestinal tract of female sand flies to the obligatory intracellular and non-motile amastigotes within mammalian macrophages. Stage differentiation is regulated mainly by post-transcriptional mechanisms, including translation regulation. Leishmania parasites encode six different cap-binding proteins, LeishIF4E1-6, that show poor conservation with their counterparts from higher eukaryotes and among themselves. In view of the changing host milieu encountered throughout their life cycle, we propose that each LeishIF4E has a unique role, although these functions may be difficult to determine. Here we characterize LeishIF4E-6, a unique eIF4E ortholog that does not readily associate with m7GTP cap in either of the tested life forms of the parasite. We discuss the potential effect of substituting two essential tryptophan residues in the cap-binding pocket, expected to be involved in the cap-binding activity, as judged from structural studies in the mammalian eIF4E. LeishIF4E-6 binds to LeishIF4G-5, one of the five eIF4G candidates in Leishmania. However, despite this binding, LeishIF4E-6 does not appear to function as a translation factor. Its episomal overexpression causes a general reduction in the global activity of protein synthesis, which was not observed in the hemizygous deletion mutant generated by CRISPR-Cas9. This genetic profile suggests that LeishIF4E-6 has a repressive role. The interactome of LeishIF4E-6 highlights proteins involved in RNA metabolism such as the P-body marker DHH1, PUF1 and an mRNA-decapping enzyme that is homologous to the TbALPH1.

Structure of the poxvirus decapping enzyme D9 reveals its mechanism of cap recognition and catalysis

SUMMARYPoxviruses encode decapping enzymes that remove the protective 5’ cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral dsRNA, a trigger for antiviral responses. Here we present the first high resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a novel domain organization in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5’ mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap binding proteins VP39, eIF4E, and CBP20 and distinct from eukaryotic decapping enzyme Dcp2.

Dcp2 C-terminal Cis-Binding Elements Control Selective Targeting of the Decapping Enzyme by Forming Distinct Decapping Complexes

A single Dcp1-Dcp2 decapping enzyme targets diverse classes of yeast mRNAs for decapping-dependent 5′ to 3′ decay, but the molecular mechanisms controlling selective mRNA targeting by the enzyme remain elusive. Through extensive genetic analyses we uncover cis-regulatory elements in the Dcp2 C-terminal domain that control selective targeting of the decapping enzyme by forming distinct decapping complexes. Two Upf1-binding motifs target the decapping enzyme to NMD substrates, and a single Edc3-binding motif targets both Edc3 and Dhh1 substrates. Pat1-binding leucine-rich motifs target Edc3 and Dhh1 substrates under selective conditions. Although it functions as a unique targeting component of specific complexes, Edc3 is a common component of multiple complexes. Xrn1 also has a specific Dcp2 binding site, allowing it to be directly recruited to decapping complexes. Collectively, our results demonstrate that Upf1, Edc3, and Pat1 function as regulatory subunits of the holo-decapping enzyme, controlling both its targeting specificity and enzymatic activation.

miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation

Non-coding RNAs (ncRNAs) involve in diverse biological processes by post-transcriptional regulation of gene expression. Emerging evidence shows that miRNA-4293 plays a significant role in the development of non-small cell lung cancer. However, the oncogenic functions of miR-4293 have not been studied. Our results demonstrated that miR-4293 expression is markedly enhanced in lung carcinoma tissue and cells. Moreover, miR-4293 promotes tumor cell proliferation and metastasis but suppresses apoptosis. Mechanistic investigations identified mRNA-decapping enzyme 2 (DCP2) as a target of miR-4293 and its expression is suppressed by miR-4293. DCP2 can directly or indirectly bind to WFDC21P and downregulates its expression. Consequently, miR-4293 can further promote WFDC21P expression by regulating DCP2. With a positive correlation to miR-4293 expression, WFDC21P also plays an oncogenic role in lung carcinoma. Furthermore, knockdown of WFDC21P results in functional attenuation of miR-4293 on tumor promotion. In vivo xenograft growth is also promoted by both miR-4293 and WFDC21P. Overall, our results establish oncogenic roles for both miR-4293 and WFDC21P and demonstrate that interactions between miRNAs and lncRNAs through DCP2 are important in the regulation of carcinoma pathogenesis. These results provided a valuable theoretical basis for the discovery of lung carcinoma therapeutic targets and diagnostic markers based on miR-4293 and WFDC21P.

Spontaneous and targeted mutations in the decapping enzyme enhance replication of modified vaccinia virus Ankara (MVA) in monkey cells

Modified vaccinia virus Ankara (MVA) was derived by repeated passaging in chick fibroblasts, during which deletions and mutations rendered the virus unable to replicate in most mammalian cells. Marker rescue experiments demonstrated that the host range defect could be overcome by replacing DNA that had been deleted from near the left end of the genome. One virus isolate, however, recovered the ability to replicate in monkey BS-C-1 cells but not human cells without added DNA suggesting it arose from a spontaneous mutation. Here we showed that variants with enhanced ability to replicate in BS-C-1 cells could be isolated by blind passaging MVA and that in each there was a point mutation leading to an amino acid substitution in the D10 decapping enzyme. The sufficiency of these single mutations to enhance host range was confirmed by constructing recombinant viruses. The D10 mutations occurred at N- or C-terminal locations distal from the active site, suggesting an indirect effect on decapping or on another previously unknown role of D10. Although increased amounts of viral mRNA and proteins were found in BS-C-1 cells infected with the mutants compared to parental MVA, the increase was much less than the one to two logs higher virus yields. Nevertheless, a contributing role for diminished decapping in overcoming the host range defect was consistent with increased replication and viral protein synthesis in BS-C-1 cells infected with an MVA engineered to have active site mutations that abrogate decapping activity entirely. Optimal decapping may vary depending on the biological context. IMPORTANCE Modified vaccinia virus Ankara (MVA) is an attenuated virus that is approved as a smallpox vaccine and is in clinical trials as a vector for other pathogens. The safety of MVA is due in large part to its inability to replicate in mammalian cells. Although, host-range restriction is considered a stable feature of the virus, we describe the occurrence of spontaneous mutations in MVA that increase replication considerably in monkey BS-C-1 cells but only slightly in human cells. The mutants contain single nucleotide changes that lead to amino acid substitutions in one of the two decapping enzymes. Although the spontaneous mutations are distant from the decapping enzyme active site, engineered active site-mutations also increased virus replication in BS-C-1 cells. The effects of these mutations on the immunogenicity of MVA vectors remain to be determined.

Structural insight into molecular inhibitory mechanism of InsP6 on African Swine Fever Virus mRNA-decapping enzyme g5Rp

Removal of 5' cap on cellular mRNAs by the African Swine Fever Virus (ASFV) decapping enzyme g5R protein (g5Rp) is beneficial to viral gene expression during the early stages of infection. As the only nucleoside diphosphate linked moiety X (Nudix) decapping enzyme encoded in the ASFV genome, g5Rp works in both the degradation of cellular mRNA and hydrolyzation of the diphosphoinositol polyphosphates. Here, we report the structures of dimeric g5Rp and its complex with inositol hexakisphosphate (InsP6). The two g5Rp protomers interact head to head to form a dimer, and the dimeric interface is formed by extensive polar and nonpolar interactions. Each protomer composed a unique N terminal helical domain and C terminal classic Nudix domain. As a mRNA decapping enzyme, we identified key residues, including K8, K94, K95, K98, K175, R221, and K243 located on the substrate RNA binding interfaces of g5Rp, are important to RNA binding and decapping enzyme activity. Furthermore, we identified that the g5Rp mediated mRNA decapping was inhibited by the InsP6. The g5Rp/InsP6 complex structure showed that the InsP6 molecules occupy the same regions that primarily mediate g5Rp-RNA interaction, elucidating the roles of InsP6 in the regulation of the viral decapping activity of g5Rp in mRNA degradation. Collectively, these results provide the structural basis of interaction between RNA and g5Rp and highlight the inhibitory mechanism of InsP6 on mRNA decapping by g5Rp.

Is mRNA decapping by ApaH like phosphatases present in eukaryotes beyond the Kinetoplastida?

Background ApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and have been identified in all eukaryotic super-groups. Only two of these proteins have been functionally characterised. We have shown that the ApaH like phosphatase ALPH1 from the Kinetoplastid Trypanosoma brucei is the mRNA decapping enzyme of the parasite. In eukaryotes, Dcp2 is the major mRNA decapping enzyme and mRNA decapping by ALPHs is unprecedented, but the bacterial ApaH protein was recently found decapping non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or could be more widespread among eukaryotes. Results We screened 827 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to characterize the phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of this protein family. For most organisms, we found ALPH proteins to be either absent (495/827 organisms) or to have non-cytoplasmic localisation predictions (73% of all ALPHs), excluding a function in mRNA decapping. Although, non-cytoplasmic ALPH proteins had in vitro mRNA decapping activity. Only 71 non-Kinetoplastida have ALPH proteins with predicted cytoplasmic localisations. However, in contrast to Kinetoplastida, these organisms also possess a homologue of Dcp2 and in contrast to ALPH1 of Kinetoplastida, these ALPH proteins are very short and consist of the catalytic domain only. Conclusions ALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme, or use it exclusively outside the cytoplasm. The acceptance of mRNA as a substrate indicates that ALPHs, like bacterial ApaH, have a wide substrate range: the need to protect mRNAs from unregulated degradation is one possible explanation for the selection against the presence of cytoplasmic ALPH proteins in most eukaryotes. Kinetoplastida succeeded to exploit ALPH as their only or major mRNA decapping enzyme. 71 eukaryotic organisms outside the Kinetoplastid lineage have short ALPH proteins with cytoplasmic localisation predictions: whether these proteins are used as decapping enzymes in addition to Dcp2 or else have adapted to not accept mRNAs as a substrate, remains to be explored.

Pbp1, the yeast ortholog of human Ataxin-2, functions in the cell growth on non-fermentable carbon sources

Pbp1, the yeast ortholog of human Ataxin-2, was originally isolated as a poly(A) binding protein (Pab1)-binding protein. Pbp1 regulates the Pan2-Pan3 deadenylase complex, thereby modulating the mRNA stability and translation efficiency. However, the physiological significance of Pbp1 remains unclear since a yeast strain harboring PBP1 deletion grows similarly to wild-type strain on normal glucose-containing medium. In this study, we found that Pbp1 has a role in cell growth on the medium containing non-fermentable carbon sources. While the pbp1Δ mutant showed a similar growth compared to the wild-type cell on a normal glucose-containing medium, the pbp1Δ mutant showed a slower growth on the medium containing glycerol and lactate. Microarray analyses revealed that expressions of the genes involved in gluconeogenesis, such as PCK1 and FBP1, and of the genes involved in mitochondrial function, such as COX10 and COX11, were decreased in the pbp1Δ mutant. Pbp1 regulated the expressions of PCK1 and FBP1 via their promoters, while the expressions of COX10 and COX11 were regulated by Pbp1, not through their promoters. The decreased expressions of COX10 and COX11 in the pbp1Δ mutant were recovered by the loss of Dcp1 decapping enzyme or Xrn1 5’-3’exonuclease. Our results suggest that Pbp1 regulates the expressions of the genes involved in gluconeogenesis and mitochondrial function through multiple mechanisms.

mRNA-decapping associated DcpS enzyme controls critical steps of neuronal development

Homozygous mutations in the gene encoding the scavenger mRNA decapping enzyme, DcpS, have been shown to underlie developmental delay and intellectual disability. Intellectual disability is associated with both abnormal neocortical development and mRNA metabolism. However, the role of DcpS and its scavenger decapping activity in neuronal development is unknown. Here, we show that human neurons derived from patients with a DcpS mutation have compromised differentiation and neurite outgrowth. Moreover, in the developing mouse neocortex, DcpS is required for the radial migration, polarity, neurite outgrowth and identity of developing glutamatergic neurons. Collectively, these findings demonstrate that the scavenger mRNA decapping activity contributes to multiple pivotal roles in neural development, and further corroborate that mRNA metabolism and neocortical pathologies are associated with intellectual disability.