Co-translational assembly and localized translation of nucleoporins in nuclear pore complex biogenesis.

mRNA translation is coupled to multiprotein complex assembly in the cytoplasm or to protein delivery into intracellular compartments. Here, by combining systematic RNA immunoprecipitation and single-molecule RNA imaging in yeast, we have provided a complete depiction of the co-translational events involved in the biogenesis of a large multiprotein assembly, the nuclear pore complex (NPC). We report that binary interactions between NPC subunits can be established during translation, in the cytoplasm. Strikingly, the nucleoporins Nup1/Nup2, together with a number of nuclear proteins, are instead translated at nuclear pores, through a mechanism involving interactions between their nascent N-termini and nuclear transport receptors. Uncoupling this co-translational recruitment further triggers the formation of cytoplasmic foci of unassembled polypeptides. Altogether, our data reveal that distinct, spatially segregated modes of co-translational interactions foster the ordered assembly of NPC subunits and that localized translation can ensure the proper delivery of proteins to the pore and the nucleus.

Phase-separated ribosome-nascent chain complexes in genotoxic stress response.

Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in yeast and human cells for the proteasome subunit Rpt1, and the disordered amino-terminal part of the nascent chain was found to be indispensable for the accumulation of the Rpt1-RNP into assemblysomes. Motivated by this, to find other assemblysome-associated RNPs we used bioinformatics to rank subunits of Saccharomyces cerevisiae protein complexes according to their amino-terminal disorder propensity. The results revealed that gene products involved in DNA repair are enriched among the top candidates. The Sgs1 DNA helicase was chosen for experimental validation. We found that indeed nascent chains of Sgs1 form EDTA-resistant RNP condensates, assemblysomes by definition. Moreover, upon exposure to UV, SGS1 mRNA shifted from assemblysomes to polysomes, suggesting that external stimuli are regulators of assemblysome dynamics. We extended our studies to human cell lines. The BLM helicase, ortholog of yeast Sgs1, was identified upon sequencing assemblysome-associated RNAs from the MCF7 human breast cancer cell line, and mRNAs encoding DNA repair proteins were overall enriched. Using the radiation-resistant A549 cell line, we observed by transmission electron microscopy that 1,6-hexanediol, an agent known to disrupt phase-separated condensates, depletes ring ribosome structures compatible with assemblysomes from the cytoplasm of cells and makes the cells more sensitive to X-ray treatment. Taken together, these findings suggest that assemblysomes may be a component of the DNA damage response from yeast to human.

Not4-dependent targeting of MMF1 mRNA to mitochondria limits its expression via ribosome pausing, Egd1 ubiquitination, Caf130, no-go-decay and autophagy.

The Ccr4-Not complex is a conserved multi protein complex with diverse roles in the mRNA life cycle. Recently we determined that the Not1 and Not4 subunits of Ccr4-Not inversely regulate mRNA solubility and thereby impact dynamics of co-translation events. One mRNA whose solubility is limited by Not4 is MMF1 encoding a mitochondrial matrix protein. In this work we uncover a mechanism that limits MMF1 overexpression and depends upon its co-translational targeting to the mitochondria. We have named this mechanism Mito-ENCay. This mechanism relies on Not4 promoting ribosome pausing during MMF1 translation, and hence the co-translational docking of the MMF1 mRNA to mitochondria via the mitochondrial targeting sequence of the Mmf1 nascent chain, the Egd1 chaperone, the Om14 mitochondrial outer membrane protein and the co-translational import machinery. Besides co-translational Mitochondrial targeting, Mito-ENCay depends upon Egd1 ubiquitination by Not4, the Caf130 subunit of the Ccr4-Not complex, the mitochondrial outer membrane protein Cis1, autophagy and no-go-decay.

Ribosome pausing, a dangerous necessity for co-translational events.

In recent years translation elongation has emerged as an important contributor to the regulation of gene expression. There are multiple quality control checkpoints along the way of producing mature proteins and targeting them to the right cellular compartment, or associating them correctly with their partners. Ribosomes pause to allow co-translational protein folding, protein targeting or protein interactions, and the pausing is dictated by a combination of the mRNA sequence and structure, the tRNA availability and the nascent peptide. However, ribosome pausing can also lead to ribosome collisions and co-translational degradation of both mRNA and nascent chain. Understanding how the translating ribosome tunes the different maturation steps that nascent proteins must undergo, what the timing of these maturation events is, and how degradation can be avoided when pausing is needed, is now possible by the emergence of methods to follow ribosome dynamics in vivo. This review summarizes some of the recent studies that have advanced our knowledge about co-translational events using the power of ribosome profiling, and some of the questions that have emerged from these studies.

Not5-dependent co-translational assembly of Ada2 and Spt20 is essential for functional integrity of SAGA.

Acetylation of histones regulates gene expression in eukaryotes. In the yeast Saccharomyces cerevisiae it depends mainly upon the ADA and SAGA histone acetyltransferase complexes for which Gcn5 is the catalytic subunit. Previous screens have determined that global acetylation is reduced in cells lacking subunits of the Ccr4­Not complex, a global regulator of eukaryotic gene expression. In this study we have characterized the functional connection between the Ccr4­Not complex and SAGA. We show that SAGA mRNAs encoding a core set of SAGA subunits are tethered together for co-translational assembly of the encoded proteins. Ccr4­Not subunits bind SAGA mRNAs and promote the co-translational assembly of these subunits. This is needed for integrity of SAGA. In addition, we determine that a glycolytic enzyme, the glyceraldehyde-3-phosphate dehydrogenase Tdh3, a prototypical moonlighting protein, is tethered at this site of Ccr4­Not-dependent co-translational SAGA assembly and functions as a chaperone.

Building on the Ccr4-Not architecture.

In a recent issue of Nature Communications Ukleja and co-workers reported a cryo-EM 3D reconstruction of the Ccr4-Not complex from Schizosaccharomyces pombe with an immunolocalization of the different subunits. The newly gained architectural knowledge provides cues to apprehend the functional diversity of this major eukaryotic regulator. Indeed, in the cytoplasm alone, Ccr4-Not regulates translational repression, decapping and deadenylation, and the Not module additionally plays a positive role in translation. The spatial distribution of the subunits within the structure is compatible with a model proposing that the Ccr4-Not complex interacts with the 5' and 3' ends of target mRNAs, allowing different functional modules of the complex to act at different stages of the translation process, possibly within a circular constellation of the mRNA. This work opens new avenues, and reveals important gaps in our understanding regarding structure and mode of function of the Ccr4-Not complex that need to be addressed in the future.

The Ccr4-Not Complex: Architecture and Structural Insights.

The Ccr4-Not complex is an essential multi-subunit protein complex that plays a fundamental role in eukaryotic mRNA metabolism and has a multitude of different roles that impact eukaryotic gene expression . It has a conserved core of three Not proteins, the Ccr4 protein, and two Ccr4 associated factors, Caf1 and Caf40. A fourth Not protein, Not4, is conserved, but is only a stable subunit of the complex in yeast. Certain subunits have been duplicated during evolution, with functional divergence, such as Not3 in yeast, and Ccr4 or Caf1 in human. However the complex includes only one homolog for each protein. In addition, species-specific subunits are part of the complex, such as Caf130 in yeast or Not10 and Not11 in human. Two conserved catalytic functions are associated with the complex, deadenylation and ubiquitination . The complex adopts an L-shaped structure, in which different modules are bound to a large Not1 scaffold protein. In this chapter we will summarize our current knowledge of the architecture of the complex and of the structure of its constituents.

The Not5 subunit of the ccr4-not complex connects transcription and translation.

Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription.

Ccr4-Not is at the core of the eukaryotic gene expression circuitry.

In this mini-review, we summarize our current knowledge about the cross-talk between the different levels of gene expression. We introduce the Ccr4 (carbon catabolite repressed 4)-Not (negative on TATA-less) complex as a candidate to be a master regulator that orchestrates between the different levels of gene expression. An integrated view of the findings about the Ccr4-Not complex suggests that it is involved in gene expression co-ordination. Since the discovery of the Not proteins in a selection for transcription regulators in yeast [Collart and Struhl (1994) Genes Dev. 8: , 525-537], the Ccr4-Not complex has been connected to every step of the mRNA lifecycle. Moreover, it has been found to be relevant for appropriate protein folding and quaternary protein structure by being involved in co-translational protein complex assembly.

Gene expression as a circular process: cross-talk between transcription and mRNA degradation in eukaryotes; International University of Andalusia (UNIA) Baeza, Spain.

Studies on the regulation of gene expression in eukaryotes in the past 20 years have consistently revealed increasing levels of complexity. Thirty years ago it seemed that we had understood the basic principles of gene regulation in eukaryotes. It was thought that regulation of transcription was the first and most important stage at which gene expression was regulated, and transcriptional regulation was considered to be very simple, with DNA-binding activators and repressors talking to the basic transcription machinery. This simple model was overthrown when it became clear that other stages of gene expression are also highly regulated. More recently, other dogmas have started to collapse. In particular, the idea that a linkage between the different steps in gene expression is restricted to processes ongoing in the same compartment has fallen out of favor. It is now evident that functional and physical linkage occurs in eukaryotes. We know that factors contributing to transcription in the nucleus can be found in the cytoplasm, and that RNA binding proteins that contribute to RNA decay in the cytoplasm are present in the nucleus. However, shuttling of such factors between nucleus and cytoplasm has traditionally been thought to serve a simple regulatory purpose, for instance, to avoid untimely activation of a transcription factor in the nucleus. Alternatively, it was thought to be necessary to recruit RNA binding proteins to the relevant RNAs. The notion that is now emerging is that factors thought to have evolved to specialize in regulating a single step of gene regulation in one cellular compartment may be contributing to the regulation of mRNAs at multiple steps along the lifecycle of an mRNA.

Membrane-associated mRNAs: A Post-transcriptional Pathway for Fine-turning Gene Expression.

Gene expression is a fundamental and highly regulated process involving a series of tightly coordinated steps, including transcription, post-transcriptional processing, translation, and post-translational modifications. A growing number of studies have revealed an additional layer of complexity in gene expression through the phenomenon of mRNA subcellular localization. mRNAs can be organized into membraneless subcellular structures within both the cytoplasm and the nucleus, but they can also targeted to membranes. In this review, we will summarize in particular our knowledge on localization of mRNAs to organelles, focusing on important regulators and available techniques for studying organellar localization, and significance of this localization in the broader context of gene expression regulation.

Presence of Not5 and ubiquitinated Rps7A in polysome fractions depends upon the Not4 E3 ligase.

In this study, we determine that Saccharomyces cerevisiae Not4 E3 ligase ubiquitinates Rps7A in vivo and in vitro, but not its paralogue, Rps7B. Ubiquitinated Rps7A is detectable only in 80S and polysomes, but not in free 40S fractions. A different role of the Rps7 paralogues in vivo is supported by the observation that the deletion of Rps7A but not Rps7B is sensitive to translational inhibitors and leads to an accumulation of aggregated proteins. An important accumulation of aggregated proteins that include ribosomal proteins and ribosome-associated chaperones is also observed in cells lacking Not4. A contribution of Not4 to ribosomal function extending beyond Rps7A ubiquitination is supported by the observation that the deletion of Not4 displays a synthetic slow growth phenotype when combined with the deletion of either one of the two Rps7 paralogues. Not4 is detectable in polysome fractions, as are other subunits of the Ccr4-Not complex such as Not5. The optimal presence of Not5 in polysomes is dependent upon Not4 and the deletion of Not5 leads to a dramatic reduction of polysomes. These results lead us to suggest that Not4 contributes to normal polysome levels and is important for cellular protein solubility maybe in part by ubiquitination of Rps7A.

Roles of the CCR4-Not complex in translation and dynamics of co-translation events.

The Ccr4-Not complex is a global regulator of mRNA metabolism in eukaryotic cells that is most well-known to repress gene expression. Delivery of the complex to mRNAs through a multitude of distinct mechanisms accelerates their decay, yet Ccr4-Not also plays an important role in co-translational processes, such as co-translational association of proteins and delivery of translating mRNAs to organelles. The recent structure of Not5 interacting with the translated ribosome has brought to light that embedded information within the codon sequence can be monitored by recruitment of the Ccr4-Not complex to elongating ribosomes. Thereby, the Ccr4-Not complex is empowered with regulatory decisions determining the fate of proteins being synthesized and their encoding mRNAs. This review will focus on the roles of the complex in translation and dynamics of co-translation events. This article is categorized under: Translation > Mechanisms Translation > Regulation.

Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events.

BACKGROUND: The Ccr4-Not complex is mostly known as the major eukaryotic deadenylase. However, several studies have uncovered roles of the complex, in particular of the Not subunits, unrelated to deadenylation and relevant for translation. In particular, the existence of Not condensates that regulate translation elongation dynamics has been reported. Typical studies that evaluate translation efficiency rely on soluble extracts obtained after the disruption of cells and ribosome profiling. Yet cellular mRNAs in condensates can be actively translated and may not be present in such extracts. RESULTS: In this work, by analyzing soluble and insoluble mRNA decay intermediates in yeast, we determine that insoluble mRNAs are enriched for ribosomes dwelling at non-optimal codons compared to soluble mRNAs. mRNA decay is higher for soluble RNAs, but the proportion of co-translational degradation relative to the overall mRNA decay is higher for insoluble mRNAs. We show that depletion of Not1 and Not4 inversely impacts mRNA solubilities and, for soluble mRNAs, ribosome dwelling according to codon optimality. Depletion of Not4 solubilizes mRNAs with lower non-optimal codon content and higher expression that are rendered insoluble by Not1 depletion. By contrast, depletion of Not1 solubilizes mitochondrial mRNAs, which are rendered insoluble upon Not4 depletion. CONCLUSIONS: Our results reveal that mRNA solubility defines the dynamics of co-translation events and is oppositely regulated by Not1 and Not4, a mechanism that we additionally determine may already be set by Not1 promoter association in the nucleus.

TIA1 Loss Exacerbates Fatty Liver Disease but Exerts a Dual Role in Hepatocarcinogenesis.

Alterations in specific RNA-binding protein expression/activity importantly contribute to the development of fatty liver disease (FLD) and hepatocellular carcinoma (HCC). In particular, adenylate-uridylate-rich element binding proteins (AUBPs) were reported to control the post-transcriptional regulation of genes involved in both metabolic and cancerous processes. Herein, we investigated the pathophysiological functions of the AUBP, T-cell-restricted intracellular antigen-1 (TIA1) in the development of FLD and HCC. Analysis of TIA1 expression in mouse and human models of FLD and HCC indicated that TIA1 is downregulated in human HCC. In vivo silencing of TIA1 using AAV8-delivered shRNAs in mice worsens hepatic steatosis and fibrosis induced by a methionine and choline-deficient diet and increases the hepatic tumor burden in liver-specific PTEN knockout (LPTENKO) mice. In contrast, our in vitro data indicated that TIA1 expression promoted proliferation and migration in HCC cell lines, thus suggesting a dual and context-dependent role for TIA1 in tumor initiation versus progression. Consistent with a dual function of TIA1 in tumorigenesis, translatome analysis revealed that TIA1 appears to control the expression of both pro- and anti-tumorigenic factors in hepatic cancer cells. This duality of TIA1's function in hepatocarcinogenesis calls for cautiousness when considering TIA1 as a therapeutic target or biomarker in HCC.

Specific roles for the Ccr4-Not complex subunits in expression of the genome.

In this work we used micro-array experiments to determine the role of each nonessential subunit of the conserved Ccr4-Not complex in the control of gene expression in the yeast Saccharomyces cerevisiae. The study was performed with cells growing exponentially in high glucose and with cells grown to glucose depletion. Specific patterns of gene deregulation were observed upon deletion of any given subunit, revealing the specificity of each subunit's function. Consistently, the purification of the Ccr4-Not complex through Caf40p by tandem affinity purification from wild-type cells or cells lacking individual subunits of the Ccr4-Not complex revealed that each subunit had a particular impact on complex integrity. Furthermore, the micro-arrays revealed that the role of each subunit was specific to the growth conditions. From the study of only two different growth conditions, revealing an impact of the Ccr4-Not complex on more than 85% of all studied genes, we can infer that the Ccr4-Not complex is important for expression of most of the yeast genome.

Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A.

In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1. Not5 resides in punctate loci, co-purifies with ribosomes and Rli1, but not with eIF5A, and limits mRNA solubility. Overexpression of wild-type or non-complementing Rli1 and loss of Rps7A ubiquitination enable Not4 E3 ligase-dependent translation of polyarginine stretches. We propose that Not4 and Not5 modulate translation elongation dynamics to produce a soluble proteome by Rps7A ubiquitination, dynamic condensates that limit mRNA solubility and exclude eIF5A, and a moonlighting function of Rli1.

Ribosome association and stability of the nascent polypeptide-associated complex is dependent upon its own ubiquitination.

In this work we addressed the role of ubiquitination in the function of the nascent polypeptide-associated complex (NAC), named EGD in the yeast Saccharomyces cerevisiae. To this end, we first identified the lysines residues required for ubiquitination of EGD/NAC. While simultaneous mutation of many lysines in the alpha-subunit of NAC (Egd2p) was required to abolish its ubiquitination, for the beta-subunit of NAC (Egd1p), mutation of K29 and K30 was sufficient. We determined that the ubiquitination of the two EGD subunits was coordinated, occurring during growth first on Egd1p and then on Egd2p. Egd2p was ubiquitinated earlier during growth if Egd1p could not be ubiquitinated. The use of mutants revealed the importance of EGD ubiqutination for its ribosome association and stability. Finally, our study demonstrated an interaction of EGD/NAC with the proteasome and revealed the importance of the Not4p E3 ligase, responsible for EGD/NAC ubiquitination, in this association.

The dual control of TFIIB recruitment by NC2 is gene specific.

Negative co-factor 2 (NC2) is a conserved eukaryotic complex composed of two subunits, NC2alpha (Drap1) and NC2beta (Dr1) that associate through a histone-fold motif. In this work, we generated mutants of NC2, characterized target genes for these mutants and studied the assembly of NC2 and general transcription factors on target promoters. We determined that the two NC2 subunits mostly function together to be recruited to DNA and regulate gene expression. We found that NC2 strongly controls promoter association of TFIIB, both negatively and positively. We could attribute the gene-specific repressor effect of NC2 on TFIIB to the C-terminal domain of NC2beta, and define that it requires ORF sequences of the target gene. In contrast, the positive function of NC2 on TFIIB targets is more general and requires adequate levels of the NC2 histone-fold heterodimer on promoters. Finally, we determined that NC2 becomes limiting for TATA-binding protein (TBP) association with a heat inducible promoter under heat stress. This study demonstrates an important positive role of NC2 for formation of the pre-initiation complex on promoters, under normal conditions through control of TFIIB, or upon activation by stress via control of TBP.