Search

Phosphofurin acidic cluster sorting protein 1 (PACS‐1) is canonically a cytosolic trafficking protein, yet recent reports have described nuclear roles for PACS‐1. Herein, we sought to define the nuclear transport mechanism of PACS‐1. We demonstrate that PACS‐1 nucleocytoplasmic trafficking is dependent on its interaction with the nuclear transport receptors importin alpha 5 and exportin 1. PACS‐1 nuclear entry and exit are defined by a nuclear localization signal (NLS, residues 311‐318) and nuclear export signal (NES3, residues 366‐375). Mutation of the PACS‐1 NLS and NES3 altered the localization of a complex formed between PACS‐1 and an RNA‐binding protein, polypyrimidine tract‐binding protein 1. Overall, we identify the nuclear localization mechanism of PACS‐1 and highlight a potential role for PACS‐1 in RNA‐binding protein trafficking. [ABSTRACT FROM AUTHOR]

Nucleotide repeat expansions in the C9orf72 gene cause frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Transcribed repeat RNA accumulates within RNA foci and is also translated into toxic dipeptide repeat proteins (DPR). The mechanism of repeat RNA accumulation, however, remains unclear. The RNA exosome complex is a multimeric ribonuclease involved in degradation of defective RNA. Here, we uncover the RNA exosome as a major degradation complex for pathogenic C9orf72‐derived repeat RNA. Knockdown of EXOSC10, the catalytic subunit of the complex, enhanced repeat RNA and DPR protein expression levels. RNA degradation assays confirmed that EXOSC10 can degrade both sense and antisense repeats. Furthermore, EXOSC10 reduction increased RNA foci and repeat transcripts in patient‐derived cells. Cells expressing toxic poly‐GR or poly‐PR proteins accumulate a subset of small nucleolar RNA precursors, which are physiological substrates of EXOSC10, as well as excessive repeat RNA, indicating that arginine‐rich DPR proteins impair the intrinsic activity of EXOSC10. Collectively, arginine‐rich DPR‐mediated impairment of EXOSC10 and the RNA exosome complex compromises repeat RNA metabolism and may thus exacerbate C9orf72‐FTLD/ALS pathologies in a vicious cycle. Synopsis: Bidirectional transcription and translation of the FTLD/ALS‐associated repeat expansion in the C9orf72 gene causes accumulation of repeat RNA foci and toxic dipeptide‐repeat proteins. Here, the RNA exosome is found to degrade repeat RNAs, until overwhelmed by inhibitory effects of the toxic repeat proteins. Knockdown of the exosome catalytic subunit EXOSC10 causes accumulation and foci formation of repeat RNA.EXOSC10 can degrade sense and antisense strand‐derived expanded hexanucleotide repeat RNA.EXOSC10 knockdown boosts dipeptide repeat protein expression through RAN translation.Arginine‐rich dipeptide repeat proteins impair the activity of EXOSC10 and thereby accelerate repeat RNA and dipeptide repeat protein accumulation. [ABSTRACT FROM AUTHOR]

Studies of the mTOR pathway have prompted speculation that diminished mTOR complex-1 (mTORC1) function may be involved in controlling the aging process. Our previous studies have shown diminished mTORC1 activity in tissues of three long-lived mutant mice: Snell dwarf mice, growth hormone receptor gene disrupted mice (GHRKO), and in this article, mice deficient in the pregnancy-associated protein-A (PAPPA-KO). The ways in which lower mTOR signals slow aging and age-related diseases are, however, not well characterized. Here, we show that Snell, GHKRO, and PAPPA-KO mice express high levels of two proteins involved in DNA repair, O-6-methylguanine-DNA methyltransferase (MGMT) and N-myc downstream-regulated gene 1 (NDRG1). Furthermore, we report that lowering mTOR enhances MGMT and NDRG1 protein expression via post-transcriptional mechanisms. We show that the CCR4-NOT complex, a post-transcriptional regulator of gene expression, is downstream of the mTORC1 pathway and may be responsible for the upregulation of MGMT and NDRG1 in all three varieties of long-lived mice. Our data thus suggest a novel link between DNA repair and mTOR signaling via post-transcriptional regulation involving specific alteration in the CCR4-NOT complex, whose modulation could control multiple aspects of the aging process. [ABSTRACT FROM AUTHOR]

T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´-UTRs) derived from alternative pre-mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T-cell surface glycoprotein that negatively regulates TCR signaling from the onset of T-cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T-cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´-UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR-204. These mechanisms fine-tune CD5 expression levels and thus ultimately T-cell responses. [ABSTRACT FROM AUTHOR]

mi RNAs associate with Argonaute ( AGO) proteins to silence the expression of mRNA targets by inhibiting translation and promoting deadenylation, decapping, and mRNA degradation. A current model for silencing suggests that AGOs mediate these effects through the sequential recruitment of GW182 proteins, the CCR4- NOT deadenylase complex and the translational repressor and decapping activator DDX6. An alternative model posits that AGOs repress translation by interfering with eIF4A function during 43S ribosomal scanning and that this mechanism is independent of GW182 and the CCR4- NOT complex in Drosophila melanogaster. Here, we show that mi RNAs, AGOs, GW182, the CCR4- NOT complex, and DDX6/Me31B repress and degrade polyadenylated mRNA targets that are translated via scanning-independent mechanisms in both human and Dm cells. This and additional observations indicate a common mechanism used by these proteins and mi RNAs to mediate silencing. This mechanism does not require eIF4A function during ribosomal scanning. [ABSTRACT FROM AUTHOR]

Nanos proteins repress the expression of target mRNAs by recruiting effector complexes through non-conserved N-terminal regions. In vertebrates, Nanos proteins interact with the NOT1 subunit of the CCR4- NOT effector complex through a NOT1 interacting motif ( NIM), which is absent in Nanos orthologs from several invertebrate species. Therefore, it has remained unclear whether the Nanos repressive mechanism is conserved and whether it also involves direct interactions with the CCR4- NOT deadenylase complex in invertebrates. Here, we identify an effector domain ( NED) that is necessary for the Drosophila melanogaster ( Dm) Nanos to repress mRNA targets. The NED recruits the CCR4- NOT complex through multiple and redundant binding sites, including a central region that interacts with the NOT module, which comprises the C-terminal domains of NOT1-3. The crystal structure of the NED central region bound to the NOT module reveals an unanticipated bipartite binding interface that contacts NOT1 and NOT3 and is distinct from the NIM of vertebrate Nanos. Thus, despite the absence of sequence conservation, the N-terminal regions of Nanos proteins recruit CCR4- NOT to assemble analogous repressive complexes. [ABSTRACT FROM AUTHOR]

GW182 family proteins interact with Argonaute proteins and are required for the translational repression, deadenylation and decay of miRNA targets. To elicit these effects, GW182 proteins interact with poly(A)-binding protein (PABP) and the CCR4-NOT deadenylase complex. Although the mechanism of miRNA target deadenylation is relatively well understood, how GW182 proteins repress translation is not known. Here, we demonstrate that GW182 proteins decrease the association of eIF4E, eIF4G and PABP with miRNA targets. eIF4E association is restored in cells in which miRNA targets are deadenylated, but decapping is inhibited. In these cells, eIF4G binding is not restored, indicating that eIF4G dissociates as a consequence of deadenylation. In contrast, PABP dissociates from silenced targets in the absence of deadenylation. PABP dissociation requires the interaction of GW182 proteins with the CCR4-NOT complex. Accordingly, NOT1 and POP2 cause dissociation of PABP from bound mRNAs in the absence of deadenylation. Our findings indicate that the recruitment of the CCR4-NOT complex by GW182 proteins releases PABP from the mRNA poly(A) tail, thereby disrupting mRNA circularization and facilitating translational repression and deadenylation. [ABSTRACT FROM AUTHOR]

Regulated alternative polyadenylation is an important feature of gene expression, but how gene transcription rate affects this process remains to be investigated. polo is a cell-cycle gene that uses two poly(A) signals in the 3? untranslated region (UTR) to produce alternative messenger RNAs that differ in their 3?UTR length. Using a mutant Drosophila strain that has a lower transcriptional elongation rate, we show that transcription kinetics can determine alternative poly(A) site selection. The physiological consequences of incorrect polo poly(A) site choice are of vital importance; transgenic flies lacking the distal poly(A) signal cannot produce the longer transcript and die at the pupa stage due to a failure in the proliferation of the precursor cells of the abdomen, the histoblasts. This is due to the low translation efficiency of the shorter transcript produced by proximal poly(A) site usage. Our results show that correct polo poly(A) site selection functions to provide the correct levels of protein expression necessary for histoblast proliferation, and that the kinetics of RNA polymerase II have an important role in the mechanism of alternative polyadenylation. [ABSTRACT FROM AUTHOR]

We have previously demonstrated how to transform the conventional method of hybridoma production and screening into a fast, high-throughput technology. Nevertheless, there were still open questions related to automated procedures and immunization protocols that we address now by comparing the hybridoma production work-flow in automated and manually executed processes. In addition, since the animals' antibody responses to single or multiple antigen challenge affect monoclonal antibody throughput, different immunization and fusion strategies were tested. Specifically, the results obtained with multiplexing (multiple target antigens injected into a single animal) and single antigen immunization followed by splenocyte pooling immediately before fusion were compared with conventional methods. The results presented here demonstrate that the optimal protocol consists of automated somatic-cell fusion and hybridoma dilution followed by manual plating of hybridoma cells. Additionally, more specific and productive hybridoma clones were obtained with multiplexed immunization in a single animal with respect to the splenocyte pooling from single antigen immunized animals. However, in terms of overall antibody yield, the conventional method consisting of single immunization for each single animal assured ten times more specific hybridoma cell lines than the strategy based on the multiple antigen immunization followed by separate fusion step. In conclusion, the most productive approach for recovering a large number of suitable antibodies relies on single antigen immunization followed by automated fusion and dilution steps and manual plating. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011 [ABSTRACT FROM AUTHOR]

Importin13 (Imp13) is an unusual β-karyopherin that is able to both import and export cargoes in and out of the nucleus. In the cytoplasm, Imp13 associates with different cargoes such as Mago-Y14 and Ubc9, and facilitates their import into the nucleus where RanGTP binding promotes the release of the cargo. In this study, we present the 2.8 Å resolution crystal structure of Imp13 in complex with the SUMO E2-conjugating enzyme, Ubc9. The structure shows an uncommon mode of cargo-karyopherin recognition with Ubc9 binding at the N-terminal portion of Imp13, occupying the entire RanGTP-binding site. Comparison of the Imp13-Ubc9 complex with Imp13-Mago-Y14 shows the remarkable plasticity of Imp13, whose conformation changes from a closed ring to an open superhelix when bound to the two different cargoes. The structure also shows that the binding mode is compatible with the sumoylated states of Ubc9. Indeed, we find that Imp13 is able to bind sumoylated Ubc9 in vitro and suppresses autosumoylation activity in the complex. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

miRNA-mediated gene silencing requires the GW182 proteins, which are characterized by an N-terminal domain that interacts with Argonaute proteins (AGOs), and a C-terminal silencing domain (SD). In Drosophila melanogaster (Dm) GW182 and a human (Hs) orthologue, TNRC6C, the SD was previously shown to interact with the cytoplasmic poly(A)-binding protein (PABPC1). Here, we show that two regions of GW182 proteins interact with PABPC1: the first contains a PABP-interacting motif 2 (PAM2; as shown before for TNRC6C) and the second contains the M2 and C-terminal sequences in the SD. The latter mediates indirect binding to the PABPC1 N-terminal domain. In D. melanogaster cells, the second binding site dominates; however, in HsTNRC6A-C the PAM2 motif is essential for binding to both Hs and DmPABPC1. Accordingly, a single amino acid substitution in the TNRC6A-C PAM2 motif abolishes the interaction with PABPC1. This mutation also impairs TNRC6s silencing activity. Our findings reveal that despite species-specific differences in the relative strength of the PABPC1-binding sites, the interaction between GW182 proteins and PABPC1 is critical for miRNA-mediated silencing in animal cells. [ABSTRACT FROM AUTHOR]

Pat proteins regulate the transition of mRNAs from a state that is translationally active to one that is repressed, committing targeted mRNAs to degradation. Pat proteins contain a conserved N-terminal sequence, a proline-rich region, a Mid domain and a C-terminal domain (Pat-C). We show that Pat-C is essential for the interaction with mRNA decapping factors (i.e. DCP2, EDC4 and LSm1–7), whereas the P-rich region and Mid domain have distinct functions in modulating these interactions. DCP2 and EDC4 binding is enhanced by the P-rich region and does not require LSm1–7. LSm1–7 binding is assisted by the Mid domain and is reduced by the P-rich region. Structural analysis revealed that Pat-C folds into an α–α superhelix, exposing conserved and basic residues on one side of the domain. This conserved and basic surface is required for RNA, DCP2, EDC4 and LSm1–7 binding. The multiplicity of interactions mediated by Pat-C suggests that certain of these interactions are mutually exclusive and, therefore, that Pat proteins switch decapping partners allowing transitions between sequential steps in the mRNA decapping pathway. [ABSTRACT FROM AUTHOR]

This article focuses on EMBO Workshop "Cell Biology of Virus Infection," held in September 25-29, 2004, EMBL, Heidelberg, Germany. The sessions covered virus entry, intracellular transport, cell tropism, signalling, nuclear events, assembly at cellular membranes and the analysis of viral structures using electron tomography. Before viruses can replicate, amplify their genome and assemble new particles, they must gain access to the host cytosol. Chlamydia uptake also involves a rearrangement of the actin cytoskeleton at the entry site that is controlled by the small GTPase Rac-1, and for some strains also by Cdc42.

The TREX complex is involved in both transcription elongation and mRNA export and is recruited to nascent transcription complexes. We have examined Yra1p, Sub2p and Hpr1p recruitment to nine genes of varying lengths and transcription frequencies. All three proteins increase from the 5' to the 3' ends of the four intronless genes examined. A modified chromatin immunoprecipitation assay that includes an RNase step indicates that Sub2p is bound to nascent RNA, Yra1p is associated with both RNA and DNA, and Hpr1p is associated with DNA. Although Hpr1p is recruited similarly to both intronless and intron-containing genes, low Yra1p and Sub2p levels are present on a subset of intron-containing genes. The residual Yra1p and Sub2p recruitment is less RNA-associated, and this correlates with high levels of U1 SnRNP on these genes. These experiments support a model in which TREX is recruited via the transcription machinery and then Yra1p and Sub2p are transferred to the nascent RNA. On some intron-containing genes, retention and/or transfer of Yra1p and Sub2p to nascent RNA are inhibited. [ABSTRACT FROM AUTHOR]

Importin a is well known as an adaptor that functions with Importin ß in the nuclear import of proteins containing specific nuclear localization signals (NLSs). We show here that either an excess or a lack of Importin a blocks nuclear envelope (NE) assembly in vitro, and our data suggest that soluble Importin a functions in NE assembly in conjunction with NLS-containing partner proteins. Surprisingly, a significant proportion of Importin a is found to fractionate with Xenopus egg membranes. We demonstrate that membrane association of Importin a is regulated by phosphorylation. Using mutant forms of Importin a that either do not bind membranes or are not released from them by phosphorylation, we provide evidence that membrane-associated Importin a is required for NE formation. Unlike other functions of Importin a, this membrane-associated activity does not require interaction with NLS proteins. [ABSTRACT FROM AUTHOR]

The exon junction complex (EJC) is deposited on mRNAs as a consequence of splicing and influences postsplicing mRNA metabolism. The Mago-Y14 heterodimer is a core component of the EJC. Recently, the protein PYM has been identified as an interacting partner of Mago-Y14. Here we show that PYM is a cytoplasmic RNA-binding protein that is excluded from the nucleus by Crm1. PYM interacts directly with Mago-Y14 by means of its N-terminal domain. The crystal structure of the Drosophila ternary complex at 1.9 Å resolution reveals that PYM binds Mago and Y14 simultaneously, capping their heterodimerization interface at conserved surface residues. Formation of this ternary complex is also observed with the human proteins. Mago residues involved in the interaction with PYM have been implicated in nonsense-mediated mRNA decay (NMD). Consistently, human PYM is active in NMD tethering assays. Together, these data suggest a role for PYM in NMD. [ABSTRACT FROM AUTHOR]

Most vertebrate mRNAs with premature termination codons (PTC5) are specifically recognized and degraded by a process referred to as nonsense-mediated mRNA decay (NMD) while still associated with the nucleus. However, it is still a mailer of debate whether PTCs can be identified by intranuclear scanning or only by ribosomes on the cytoplasmic side of the nuclear envelope. Here we show that inhibition of mRNA export by two independent approaches does not affect the downregulation of PTC-containing T-cell receptor β transcripts in the nuclear fraction of mammalian cells, providing strong evidence for intranuclear NMD. Our results are fully consistent with recently reported evidence for nuclear translation and suggest that an important biological role for nuclear ribosomes is the early elimination of nonsense mRNA during a pioneer round of translation. [ABSTRACT FROM AUTHOR]

We investigated the role of RanBP3, a nuclear member of the Ran-binding protein 1 family, in CRM1-mediated protein export in higher eukaryotes. RanBP3 interacts directly with CRM1 and also forms a trimeric complex with CRM1 and RanGTP. However, RanBP3 does not bind to CRM1 like an export substrate. Instead, it can stabilize CRM1-export substrate interaction. Nuclear RanBP3 stimulates CRM1- dependent protein export in permeabilized cells. These data indicate that RanBP3 functions by a novel mechanism as a cofactor in recognition and export of certain CRM1 substrates. In vitro, RanBP3 binding to CRM1 affects the relative affinity of CRM1 for different substrates. [ABSTRACT FROM AUTHOR]

Human TAP is implicated in mRNA nuclear export and is used by simian type D retroviruses to export their unspliced genomic RNA to the cytoplasm of the host cell. We have determined the crystal structure of the minimal TAP fragment that binds the constitutive transport element (CTE) of retroviral RNAs. Unexpectedly, we find the fragment consists of a ribonucleoprotein (RNP) domain, which is not identifiable by its sequence, and a leucine-rich repeat (LRR) domain. The non-canonical RNP domain functions as the general RNA-binding portion of the fragment. The LRR domain is required in cis to the RNP domain for CTE RNA binding. The structural and biochemical properties of the domains point to a remarkable similarity with the U2B″ (RNP)-U2A′(LRR) spliceosomal heterodimer. Our in vitro and in vivo functional studies using strurture­based mutants suggest that a phylogenetically conserved surface of the LRR domain of TAP may have different roles in the export of viral and cellular RNAs. [ABSTRACT FROM AUTHOR]

Gle1p is an essential, nuclear pore complex (NPC)-associated RNA export factor. In a screen for high copy suppressors of a GLE1 mutant strain, we identified the FG-nucleoporin Rip1p and the DEAD-box protein Rat8p/Dbp5p, both of which have roles in RNA export; we also found Ymr255p/Gfd1p, a novel inessential protein. All three high copy suppressors interact with the C-terminal domain of Gle1p; immunoelectron microscopy localizations indicate that Gle1p, Rip1p and Rat8p/Dbp5p are present on the NPC cytoplasmic fibrils; Rip1p was also found within the nucleoplasm and on the nuclear baskets, In vivo localizations support the hypothesis that Rip1p contributes to the association of Gle1p with the pore and that Gle1p, in turn, provides a binding site for Rat8p/Dbp5p at the NPC. These data are consistent with the view that Gle1p, Rip1p, Rat8p/ Dbp5p and Ymr255p/Gfd1p associate on the cytoplasmic side of the NPC to act in a terminal step of RNA export. We also describe a human functional homologue of Rip1p, called hCG1, which rescues Rip1p function in yeast, consistent with the evolutionary conservation of this NPC-associated protein. [ABSTRACT FROM AUTHOR]

The cap structure that is characteristic of all polymerase-II-transcribed RNAs has been shown to play an important role in many aspects of RNA metabolism including RNA processing, RNA nuclear transport, and translation initiation. The effects of the cap structure on these different processes is mediated by proteins that recognise and bind to it, and are therefore generically called cap-binding proteins. For example, the cap-binding protein eIF4E, in a complex with other proteins, mediates the effect of the cap on the initiation of translation. EIF-4E is predominantly localised in the cytoplasm. In the last five years, it has been demonstrated that a second cap-binding protein complex, which is mainly localised in the nucleus, mediates the stimulatory effects of the cap in nuclear processes such as pre-mRNA splicing, RNA 3'-end formation, and RNA nuclear export. The purpose of this review is to summarise our current knowledge on the role of the cap structure and of the cap-binding protein complex in nuclear RNA metabolism and present evidence that at least some processes may be coupled in vivo. [ABSTRACT FROM AUTHOR]

The assembly of eukaryotic ribosomal subunits takes place in the nucleolus and requires nuclear import of ribosomal proteins. We have studied this import in a mammalian system and found that the classical nuclear import pathway using the importin α/β heterodimer apparently plays only a minor role. Instead, at least four importin β-like transport receptors, namely importin β itself, transportin, RanBP5 and RanBP7, directly bind and import ribosomal proteins. We found that the ribosomal proteins L23a, S7 and L5 can each be imported alternatively by any of the four receptors. We have studied rpL23a in detail and identified a very basic region to which each of the four import receptors bind avidly. This domain might be considered as an archetypal import signal that evolved before import receptors diverged in evolution. The presence of distinct binding sites for rpL23a and the M9 import signal in transportin, and for rpL23a and importin α in importin β might explain how a single receptor can recognize very different import signals. [ABSTRACT FROM AUTHOR]

Examines the transport into and out of the cell nucleus. Penetration of the nuclear envelope by nuclear pore complexes; Composition of nucleocytoplasmic transport; Identification of the nuclear transport factor.

Macromolecules that are imported into the nucleus can be divided into classes according to their nuclear import signals. The best characterized class consists of proteins which carry a basic nuclear localization signal (NLS), whose transport requires the importin α/β heterodimer. U snRNP import depends on both the trimethylguanosine cap of the snRNA and a signal formed when the Sm core proteins bind the RNA. Here, factor requirements for U snRNP nuclear import are studied using an in vitro system. Depletion of importin α, the importin subunit that binds the NLS, is found to stimulate rather than inhibit U snRNP import. This stimulation is shown to be due to a common requirement for importin β in both U snRNP and NLS protein import. Saturation of importin β-mediated transport with the importin b-binding domain of importin α blocks U snRNP import both in vitro and in vivo. Immunodepletion of importin β inhibits both NLS-mediated and U snRNP import. While the former requires re-addition of both importin α and importin β, re-addition of importin β alone to immunodepleted extracts was sufficient to restore efficient U snRNP import. Thus importin β is required for U snRNP import, and it functions in this process without the NLS-specific importin α. [ABSTRACT FROM AUTHOR]

The IκB kinase (IKK) is considered to control gene expression primarily through activation of the transcription factor NF‐κB. However, we show here that IKK additionally regulates gene expression on post‐transcriptional level. IKK interacted with several mRNA‐binding proteins, including a Processing (P) body scaffold protein, termed enhancer of decapping 4 (EDC4). IKK bound to and phosphorylated EDC4 in a stimulus‐sensitive manner, leading to co‐recruitment of P body components, mRNA decapping proteins 1a and 2 (DCP1a and DCP2) and to an increase in P body numbers. Using RNA sequencing, we identified scores of transcripts whose stability was regulated via the IKK‐EDC4 axis. Strikingly, in the absence of stimulus, IKK‐EDC4 promoted destabilization of pro‐inflammatory cytokines and regulators of apoptosis. Our findings expand the reach of IKK beyond its canonical role as a regulator of transcription. Synopsis: The IκB kinase – best known to control transcription via NF‐κB activation – can bind mRNA decay factors and affect the formation of P bodies, revealing a new post‐transcriptional axis in inflammatory signalling. DNA damage, TNFα or IL‐1β stimulate an interaction between the IKK complex and mRNA decapping activator EDC4.IKK phosphorylates EDC4 to drive recruitment of decapping factors and an increase in P body numbers.The IKK‐EDC4 axis regulates stability of hundreds of mRNAs, including NF‐κB‐dependent and ‐independent transcripts.In the absence of external stimuli, IKK‐EDC4 promotes degradation of mRNAs encoding of pro‐inflammatory cytokines and regulators of apoptosis. The IκB kinase binds mRNA decay factors and affects the formation of P bodies, extending its function beyond the canonical role as a regulator of NF‐kB‐dependent transcription. [ABSTRACT FROM AUTHOR]

Focal deletions occur frequently in the cancer genome. However, the putative tumor‐suppressive genes residing within these regions have been difficult to pinpoint. To robustly identify these genes, we implemented a computational approach based on non‐negative matrix factorization, NMF, and interrogated the TCGA dataset. This analysis revealed a metagene signature including a small subset of genes showing pervasive hemizygous deletions, reduced expression in cancer patient samples, and nucleolar function. Amid the genes belonging to this signature, we have identified PNRC1, a nuclear receptor coactivator. We found that PNRC1 interacts with the cytoplasmic DCP1α/DCP2 decapping machinery and hauls it inside the nucleolus. PNRC1‐dependent nucleolar translocation of the decapping complex is associated with a decrease in the 5′‐capped U3 and U8 snoRNA fractions, hampering ribosomal RNA maturation. As a result, PNRC1 ablates the enhanced proliferation triggered by established oncogenes such as RAS and MYC. These observations uncover a previously undescribed mechanism of tumor suppression, whereby the cytoplasmic decapping machinery is hauled within nucleoli, tightly regulating ribosomal RNA maturation. Synopsis: Computational analysis of frequent cancer genome deletions reveals that PNRC1‐dependent nucleolar recruitment of the cytoplasmic mRNA decapping complex blocks ribosomal maturation and ablates oncogene‐induced cell proliferation. Nuclear receptor co‐activator PNRC1 is frequently deleted in cancer cells.PNRC1 interacts with the DCP1α/DCP2 decapping complex and stimulates its translocation into the nucleolus.PNRC1 expression blocks ribosomal RNA processing in cancer cells.The nucleolar PNRC1‐DCP1α/DCP2 complex targets the U3 and U8 snoRNAs for decapping.PNRC1 expression ablates oncogene‐induced proliferation, suggesting a tumor suppressive role. Computational analysis of frequent cancer genome deletions reveals that mRNA decapping activity blocks ribosomal maturation and ablates oncogene‐induced proliferation. [ABSTRACT FROM AUTHOR]