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W4221019062 abstract "Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The biogenesis of eukaryotic ribosomes involves the ordered assembly of around 80 ribosomal proteins. Supplying equimolar amounts of assembly-competent ribosomal proteins is complicated by their aggregation propensity and the spatial separation of their location of synthesis and pre-ribosome incorporation. Recent evidence has highlighted that dedicated chaperones protect individual, unassembled ribosomal proteins on their path to the pre-ribosomal assembly site. Here, we show that the co-translational recognition of Rpl3 and Rpl4 by their respective dedicated chaperone, Rrb1 or Acl4, reduces the degradation of the encoding RPL3 and RPL4 mRNAs in the yeast Saccharomyces cerevisiae. In both cases, negative regulation of mRNA levels occurs when the availability of the dedicated chaperone is limited and the nascent ribosomal protein is instead accessible to a regulatory machinery consisting of the nascent-polypeptide-associated complex and the Caf130-associated Ccr4-Not complex. Notably, deregulated expression of Rpl3 and Rpl4 leads to their massive aggregation and a perturbation of overall proteostasis in cells lacking the E3 ubiquitin ligase Tom1. Taken together, we have uncovered an unprecedented regulatory mechanism that adjusts the de novo synthesis of Rpl3 and Rpl4 to their actual consumption during ribosome assembly and, thereby, protects cells from the potentially detrimental effects of their surplus production. Editor's evaluation The work describes an exciting new mechanism for how r-proteins are produced in the correct abundances. Specifically, the authors find that the co-translational recognition of Rpl3/4 by their respective chaperones maintains the stability of RPL3 and RPL4 mRNAs. This mechanism is reminiscent of mechanisms of translation regulation in yeast mitochondria where oxidative phosphorylation complex assembly factors similarly regulate RNA stability and translation to ensure subunits are not produced in excess. https://doi.org/10.7554/eLife.74255.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Living cells are packed full of molecules known as proteins, which perform many vital tasks the cells need to survive and grow. Machines called ribosomes inside the cells use template molecules called messenger RNAs (or mRNAs for short) to produce proteins. The newly-made proteins then have to travel to a specific location in the cell to perform their tasks. Some newly-made proteins are prone to forming clumps, so cells have other proteins known as chaperones that ensure these clumps do not form. The ribosomes themselves are made up of several proteins, some of which are also prone to clumping as they are being produced. To prevent this from happening, cells control how many ribosomal proteins they make, so there are just enough to form the ribosomes the cell needs at any given time. Previous studies found that, in yeast, two ribosomal proteins called Rpl3 and Rpl4 each have their own dedicated chaperone to prevent them from clumping. However, it remained unclear whether these chaperones are also involved in regulating the levels of Rpl3 and Rpl4. To address this question, Pillet et al. studied both of these dedicated chaperones in yeast cells. The experiments showed that the chaperones bound to their target proteins (either units of Rpl3 or Rpl4) as they were being produced on the ribosomes. This protected the template mRNAs the ribosomes were using to produce these proteins from being destroyed, thus allowing further units of Rpl3 and Rpl4 to be produced. When enough Rpl3 and Rpl4 units were made, there were not enough of the chaperones to bind them all, leaving the mRNA templates unprotected. This led to the destruction of the mRNA templates, which decreased the numbers of Rpl3 and Rpl4 units being produced. The work of Pillet et al. reveals a feedback mechanism that allows yeast to tightly control the levels of Rpl3 and Rpl4. In the future, these findings may help us understand diseases caused by defects in ribosomal proteins, such as Diamond-Blackfan anemia, and possibly also neurodegenerative diseases caused by clumps of proteins forming in cells. The next step will be to find out whether the mechanism uncovered by Pillet et al. also exists in human and other mammalian cells. Introduction Ribosomes are the molecular machines that synthesize all cellular proteins from mRNA templates (Melnikov et al., 2012). Eukaryotic 80S ribosomes are made up of two unequal ribosomal subunits (r-subunits): the small 40S and the large 60S r-subunit. In the yeast Saccharomyces cerevisiae, the 40S r-subunit is composed of the 18S ribosomal RNA (rRNA) and 33 ribosomal proteins (r-proteins), while the 60S r-subunit contains 3 rRNA species (25S, 5.8S, and 5S) and 46 r-proteins (Melnikov et al., 2012). Accordingly, the making of ribosomes corresponds to a gigantic molecular jigsaw puzzle, which, when accurately pieced together, results in the formation of translation-competent ribosomes. Our current understanding of ribosome biogenesis is mostly derived from studying this multistep assembly process in the model organism S. cerevisiae. An exponentially growing yeast cell contains ~200,000 ribosomes and, with a generation time of 90 min, needs to produce more than 2000 ribosomes per minute, thus, requiring the synthesis of at least ~160,000 r-proteins per minute (Warner, 1999). Given the enormous complexity of the process, it is not surprising that a plethora (>200) of mostly essential biogenesis factors is involved to ensure its fast and faultless completion (Kressler et al., 2010; Woolford and Baserga, 2013; Kressler et al., 2017; Pena et al., 2017; Bassler and Hurt, 2019; Klinge and Woolford, 2019). While atomic structures of eukaryotic ribosomes have already been obtained 10 years ago (Ben-Shem et al., 2011; Klinge et al., 2011; Rabl et al., 2011), recent advances in cryo-EM have now enabled to solve high-resolution structures of several distinct pre-ribosomal particles, thereby starting to provide a detailed molecular view of ribosome assembly (Greber, 2016; Kressler et al., 2017; Pena et al., 2017; Bassler and Hurt, 2019; Klinge and Woolford, 2019). The early steps of ribosome synthesis take place in the nucleolus where the rDNA genes are transcribed into precursor rRNAs (pre-rRNAs). Three of the four rRNAs (18S, 5.8S, and 25S) are transcribed by RNA polymerase I (RNA Pol I) into a 35S pre-rRNA, which undergoes covalent modifications and endo- and exonucleolytic cleavage reactions (Watkins and Bohnsack, 2012; Fernández-Pevida et al., 2015; Turowski and Tollervey, 2015), whereas the fourth rRNA (5S) is transcribed as a pre-5S rRNA by RNA Pol III. The stepwise association of several biogenesis modules, additional biogenesis factors, and selected small-subunit r-proteins with the nascent 35S pre-rRNA leads to the formation of the 90S pre-ribosome. Then, endonucleolytic cleavage of the pre-rRNA separates the two assembly paths and gives rise to the first pre-40S and pre-60S particles, which are, upon further maturation, exported to the cytoplasm where they are converted into translation-competent 40S and 60S r-subunits (Kressler et al., 2017; Pena et al., 2017; Bassler and Hurt, 2019; Klinge and Woolford, 2019). To sustain optimal rates of ribosome assembly, each of the 79 r-proteins must be produced in an assembly-competent amount that, at least, matches the abundance of the newly synthesized 35S pre-rRNA. This enormous logistic task is complicated by the fact that 59 r-proteins are synthesized from duplicated r-protein genes (RPGs) and that most primary RPG mRNA transcripts (102 of 138) contain introns (Planta and Mager, 1998; Woolford and Baserga, 2013). As a first mechanism to ensure the roughly equimolar supply of each r-protein, RPG transcription is regulated such that the output for each of the 79 RPG mRNAs, regardless of whether derived from a single-copy or duplicated RPG, is within a similar range (Zeevi et al., 2011; Knight et al., 2014). This co-regulation of the three different RPG promoter types is mediated by the complementary action of the two TORC1-controlled transcription factors Ifh1 and Sfp1, which are either mainly required for activation of category I and II (Ifh1) or category III (Sfp1) promoters (Zencir et al., 2020; Shore et al., 2021). Moreover, RPG transcription is also coordinated with RNA Pol I activity via Utp22-dependent sequestration of Ifh1 in the CURI complex (Albert et al., 2016). However, transcriptional harmonization is likely not sufficient because the quantitative and qualitative production of r-proteins is influenced by additional parameters, such as the stability and translatability of the different RPG mRNAs as well as the intrinsic stability and aggregation propensity of each individual r-protein. Despite their difficult structural characteristics and highly basic nature, which make them susceptible for aggregation (Jäkel et al., 2002), r-proteins are nevertheless, as shown in mammalian cells, continuously produced beyond their actual consumption in ribosome assembly (Lam et al., 2007). Apparently, cells can readily cope with a moderate excess of unassembled r-proteins in the nucleus as these are selectively recognized and ubiquitinated by the conserved E3 ubiquitin ligase Tom1 (ERISQ pathway) and subsequently degraded by the proteasome (Sung et al., 2016a; Sung et al., 2016b). However, when orphan r-proteins are more excessively present, owing to a severe perturbation of ribosome assembly, and start to aggregate, a stress response pathway, termed RASTR or RPAS, is activated, which alleviates the proteostatic burden by upregulating Hsf1-dependent target genes and downregulating RPG transcription (Albert et al., 2019; Tye et al., 2019). In order to not unnecessarily strain cellular proteostasis under normal growth conditions, cells have evolved general as well as highly specific mechanisms to protect newly synthesized r-proteins from aggregation and safely guide them to their pre-ribosomal assembly site. For instance, the two ribosome-associated chaperone systems, the RAC-Ssb chaperone triad and the nascent polypeptide-associated complex (NAC) (Zhang et al., 2017; Deuerling et al., 2019), functionally cooperate to promote the soluble expression of many r-proteins (Koplin et al., 2010). However, most r-proteins associate with pre-ribosomal particles in the nucle(ol)us; thus, their risky journey does not end in the cytoplasm. Despite their small size, nuclear import of r-proteins largely depends on active transport mediated by importins (Rout et al., 1997; Bange et al., 2013; de la Cruz et al., 2015), which exhibit, likely by recognizing and shielding the exposed rRNA-binding regions of r-proteins, a dual function as transport receptors and chaperones (Jäkel et al., 2002; Melnikov et al., 2015; Huber and Hoelz, 2017). Besides being assisted by these general mechanisms, some r-proteins also rely on tailor-made solutions. For instance, 9 of the 79 r-proteins are transiently associated with a selective binding partner belonging to the heterogeneous class of dedicated chaperones (Espinar-Marchena et al., 2017; Pena et al., 2017; Pillet et al., 2017). These exert their beneficial effects by, for example, already capturing the nascent r-protein client in a co-translational manner (Pausch et al., 2015; Pillet et al., 2015; Black et al., 2019; Rössler et al., 2019), coupling the co-import of two r-proteins with their ribosomal assembly (Kressler et al., 2012; Calviño et al., 2015), or facilitating the nuclear transfer from an importin to the assembly site (Schütz et al., 2014; Ting et al., 2017). In addition, some r-proteins regulate their own expression levels through autoregulatory feedback loops, for example, by repressing translation, inhibiting splicing, or promoting degradation of their own (pre-)mRNA (Fewell and Woolford, 1999; Gudipati et al., 2012; Johnson and Vilardell, 2012; He et al., 2014; Gabunilas and Chanfreau, 2016; Petibon et al., 2016; Roy et al., 2020). In this study, we show that a common regulatory machinery subjects the RPL3 and RPL4 mRNAs to co-translational downregulation when the dedicated chaperone Rrb1 or Acl4 is not available for binding to nascent Rpl3 or Rpl4, respectively. Central to the here-described regulatory mechanism is the Caf130-mediated connection between the NAC and, via the N-terminal domain of Not1, the Ccr4-Not complex, which is assembled around the essential Not1 scaffold and implicated in many aspects of mRNA metabolism, notably including cytoplasmic mRNA degradation (Parker, 2012; Collart, 2016). The tight regulation of Rpl3 and Rpl4 levels appears to be of physiological relevance as their deregulated expression in cells lacking Tom1 leads to their massive aggregation and cell inviability. Taken together, our data indicate that this novel, co-translational regulatory mechanism specifically operates to continuously adjust the expression levels of Rpl3 and Rpl4 to their actual consumption during ribosome assembly, thereby avoiding that their surplus production might negatively affect cellular proteostasis. Results The growth defect of ∆acl4 cells is suppressed by the absence of Caf130, Cal4, and the nascent polypeptide-associated complex We and others have previously shown that the dedicated chaperone Acl4 associates with the r-protein Rpl4 in a co-translational manner and protects Rpl4 from aggregation and degradation on its path to its assembly site on nucleolar pre-60S particles (Pillet et al., 2015; Stelter et al., 2015; Sung et al., 2016a; Huber and Hoelz, 2017). While growing ∆acl4 null mutant cells on YPD plates, we observed that spontaneous suppressors of the severe slow-growth (sg) phenotype arose at a relatively high frequency (Figure 1—figure supplement 1A). Since mild overexpression of Rpl4a from a centromeric plasmid almost completely restored the ∆acl4 growth defect (Pillet et al., 2015), we hypothesized that the ∆acl4 suppressor mutations might either increase the expression level or stability of Rpl4 or facilitate the incorporation of Rpl4 into pre-60S particles. To unravel the reason for this observation, we isolated a large number of ∆acl4 and ∆acl4/∆rpl4a suppressors and identified causative candidate mutations by whole-genome sequencing (see Materials and methods). Bioinformatics analysis of the sequenced genomes revealed that the 48 independent suppressors harbored 47 different candidate mutations, which mapped to only four different genes: CAF130 (35 different mutations), YJR011C/CAL4 (7), NOT1 (4), and RPL4A (1) (see Supplementary file 3). Notably, Caf130 is a sub-stoichiometric subunit of the Ccr4-Not complex (Chen et al., 2001; Nasertorabi et al., 2011) and, as shown below, interacts directly with the previously uncharacterized Yjr011c, which we have accordingly named Cal4 (Caf130-associated regulator of Rpl4). Given that the suppressor screen yielded early frameshift mutations in both CAF130 and CAL4, we first tested whether their complete deletion would restore the severe growth defect of ∆acl4 cells. As shown in Figure 1A and B, this was indeed the case; however, while both the absence of Caf130 and Cal4 restored growth of ∆acl4 cells virtually to the wild-type extent at 16, 23, and 30°C, only the ∆cal4/∆acl4 double mutant combination grew well at 37°C as the single ∆caf130 mutant already exhibited a temperature-sensitive (ts) phenotype (Figure 1A and B). Figure 1 with 2 supplements see all Download asset Open asset Absence of Caf130, Cal4, or the nascent polypeptide-associated complex (NAC) suppresses the ∆acl4 growth defect by increasing RPL4 mRNA levels. (A–F) Suppression of the ∆acl4 growth defect. The indicated wild-type (WT), single, double, and triple deletion strains, all derived from tetratype tetrads, were spotted in 10-fold serial dilution steps onto YPD plates, which were incubated for the indicated times at 16, 23, 30, or 37°C. (G) Cells lacking Caf130, Cal4, or the NAC exhibit increased RPL4 mRNA levels. Cells of the indicated genotype were grown in YPD medium at 30°C to an OD600 of around 0.6, and relative changes in mRNA levels were determined by qRT-PCR (see Materials and methods). The data shown were obtained from three independent strains of the same genotype (biological triplicates), in each case consisting of a technical triplicate. The darker-colored boxes highlight the quartiles of each dataset, while the whiskers indicate the minimal and maximal limits of the distribution; outliers are shown as diamonds. The horizontal line in the quartile box represents the median log2 fold change of each dataset. (H) Christmas tree representation of differential gene expression analysis between ∆caf130 (left panel) or ∆cal4 (right panel) and WT cells. The RNA-seq data were generated from the same total RNA samples used for the above qRT-PCRs. Genes exhibiting statistically significant differential mRNA levels are colored in dark gray (adjusted p-value, padj<0.05). The adjusted p-values for the selected mRNAs are indicated in parentheses. Categories of genes or specific genes, regardless of the adjusted p-value, are colored as indicated. Considering that both Caf130 and Cal4 have been suggested to be physically connected with Btt1, the minor β-subunit of NAC, and the NAC α-subunit Egd2 by previous studies (Ito et al., 2001; Krogan et al., 2006; Cui et al., 2008), we next explored this potential link to the co-translational sensing of nascent polypeptides by assessing whether the absence of either of the two NAC subunits would restore the growth defect of ∆acl4 cells. While absence of Btt1 (∆btt1) resulted in a modest growth amelioration of ∆acl4 cells at 23 and 30°C, full suppression could be observed at 37°C; however, no restoration of the growth defect could be discerned at 16°C (Figure 1C). Given that there was no suppression at any of the tested temperatures when ∆acl4 cells were lacking the major NAC β-subunit Egd1 (∆egd1) (Figure 1D), we tested whether the complete absence of NAC-β (∆egd1, ∆btt1) would enhance the extent of suppression. Indeed, a very robust suppression of the ∆acl4 growth defect could be witnessed from 16 to 30°C (Figure 1E); but, in line with the ts phenotype of ∆egd1/∆btt1 double mutant cells (Figure 1—figure supplement 1B), there was no mutual suppression of the respective growth defects at 37°C in ∆egd1/∆btt1/∆acl4 triple mutant cells. Similarly, absence of NAC-α (∆egd2), which conferred a ts phenotype, also rescued the ∆acl4 growth defect to the wild-type extent at temperatures up to 30°C (Figure 1F). In support of a specific role of NAC, deletion of Zuo1 (∆zuo1), a component of the ribosome-associated RAC-Ssb chaperone triad, did not enable suppression of the ∆acl4 growth defect (Figure 1—figure supplement 1C). We conclude that the absence of either the accessory Ccr4-Not component Caf130, the previously uncharacterized Cal4, or the NAC compensates for the lack of Acl4, suggesting that these factors may be part of a regulatory network controlling the expression levels of Rpl4. Moreover, with respect to NAC’s two paralogous β-subunits, the suppression analyses indicate that the Btt1-containing NAC heterodimer provides the main contribution, especially at elevated temperature, although Egd1-containing NAC appears to operate in a partially redundant manner, as evidenced by the finding that full ∆acl4 suppression at temperatures below 37°C can only be observed when both NAC β-subunits are simultaneously absent. RPL4 mRNA levels are increased in the absence of Caf130, Cal4, and the nascent polypeptide-associated complex To obtain insight into how the above-described components might regulate Rpl4 expression, we first compared the total RPL4 mRNA levels between wild-type and mutant cells, grown in YPD medium at 30°C, by quantitative reverse transcription PCR (qRT-PCR). In good correlation with the suppression efficiency, we observed an about twofold relative increase of the RPL4 mRNA levels in ∆caf130, ∆cal4, ∆egd2, and ∆btt1/∆egd1 mutant cells but no increase in ∆egd1 and ∆btt1 cells (Figure 1G). Given that mild overexpression of Rpl4a efficiently restores the ∆acl4 growth defect (Pillet et al., 2015), the moderate rise in RPL4 mRNA levels likely accounts for the observed suppression. To evaluate the specificity of this upregulation, we next determined the levels of the RPL3, RPL5, and RPS3 mRNA. While there were only minor changes in the abundance of the RPL5 and RPS3 mRNAs, the RPL3 mRNA exhibited a similar increase as the RPL4 mRNA in ∆caf130, ∆egd2, and ∆btt1/∆egd1 mutant cells; conspicuously, however, the absence of Cal4 did not augment RPL3 mRNA levels, indicating that Cal4 may be specifically required for the regulation of the RPL4 mRNA. Notably, the inverse effect was observed in ∆acl4 cells, which exclusively displayed decreased levels of the RPL4 and, to a lesser extent, the RPL3 mRNA, suggesting that co-translational capturing of nascent Rpl4 by Acl4 may have a positive impact on the abundance of the RPL4 mRNA (see below). To discern whether altered transcription initiation or mRNA stability could be the reason for the observed changes in RPL3 and/or RPL4 mRNA abundance, we assessed RNA Pol II occupancy around the transcription start site (TSS) of several RPGs by chromatin immunoprecipitation (ChIP) and qPCR in wild-type and ∆caf130 cells (see Materials and methods). In support of mRNA stability being the responsible feature, absence of Caf130 did not lead to an increased association of initiating RNA Pol II on the RPL3 and RPL4A/B promoters (Figure 1—figure supplement 2A). Moreover, while inactivation of TORC1 by rapamycin treatment similarly reduced transcription of all tested RPGs both in wild-type and ∆caf130 cells (Figure 1—figure supplement 2A), RPL3 and RPL4 mRNA levels nevertheless remained around twofold higher in cells lacking Caf130, suggesting that the RPL3 and RPL4 mRNAs, even when present at lower abundance, are still subjected to negative regulation under these conditions in wild-type cells (Figure 1—figure supplement 2B). Since the above results indicated that the regulation mediated by Caf130, Cal4, and NAC may only operate on a limited number of common mRNAs, we wished to obtain a global overview of the regulated transcripts. To this end, we assessed, using the same total RNA extracts as for the above qRT-PCRs, the relative abundance of individual mRNAs within the entire transcriptome by RNA-seq (see Materials and methods). Strikingly, when compared to the levels in wild-type cells, the RPL3 mRNA and both RPL4 mRNAs, transcribed from the paralogous RPL4A and RPL4B genes, were amongst the most prominently upregulated transcripts in ∆caf130 cells (Figure 1H; see also Supplementary file 4). In line with the above qRT-PCR data, only the RPL4A and RPL4B transcripts, but not the RPL3 mRNA, belonged to the markedly upregulated transcripts in ∆cal4 cells (Figure 1H). Individual deletion of NAC-α (∆egd2) or NAC-β (∆egd1, ∆btt1) also resulted in an observable, albeit less outstanding, upregulation of the RPL3, RPL4A, and RPL4B mRNAs (Figure 1—figure supplement 1D), presumably due to more pronounced global changes in their transcriptomes. A common feature of all four mutant transcriptomes, although to a lesser extent in the one of ∆cal4 cells, appears to be the upregulation of transcripts encoding components of stress response pathways, including, for example, proteins of the ubiquitin-proteasome system (UPS), the transcription factor Yap1, which is known to mediate oxidative stress tolerance, or proteins involved in iron uptake and homeostasis. On the other hand, the downregulated transcripts are more diverse, but often belong to different anabolic processes that mediate cell growth, such as translation (e.g., genes coding for r-proteins and biogenesis factors), the provisioning of building blocks (e.g., genes coding for permeases and enzymes involved in amino acid synthesis), and mitochondrial metabolism. Importantly, no other RPG transcripts were found to be upregulated in the same manner as the RPL3, RPL4A, and RPL4B mRNAs, suggesting that these three are specific common targets of Caf130 and the NAC, while Cal4 only contributes to the negative regulation of the two RPL4 mRNAs. The full-length translational isoform of Not1 enables negative regulation of RPL3 and RPL4 mRNA levels Encouraged by the above results, we next examined the involvement of Not1, the largest subunit and scaffold protein of the Ccr4-Not complex (Collart, 2016), in the regulatory process. Intriguingly, the four identified ∆acl4 suppressor mutations, even though NOT1 is an essential gene (Collart and Struhl, 1993), either change the start codon (M1L), introduce a premature stop codon (L112*), or result in early frameshifts (K21fs and I128fs) (Figure 2A); they are therefore predicted to interfere with the synthesis of a functional Not1 protein. Moreover, Western analysis of C-terminally TAP-tagged Not1, expressed from the genomic locus, consistently resulted in the detection of two Not1-TAP bands (Figure 2B); hence, the shorter, major Not1 isoform must correspond, as also previously suggested (Liu et al., 1998), to an N-terminally truncated Not1 protein, which could either be generated from different mRNA isoforms or by an alternative translation initiation event. In support of the second possibility, only a single NOT1 mRNA species was detected in a previous study (Collart and Struhl, 1993). Notably, the NOT1 sequence does not contain any out-of-frame ATG trinucleotides between the start codon and the second in-frame ATG coding for M163, strongly suggesting that a leaky scanning mechanism enables the synthesis of the N-terminally truncated Not1 variant. To experimentally corroborate this plausible conjecture, we mutated the NOT1 coding sequence by either changing codon 163 such that it codes for another amino acid (construct M163L and M163A) or introducing an out-of-frame ATG trinucleotide at two different positions by silent mutagenesis of codons 40 and 156 (construct N40(oofATG) and N156(oofATG)). These plasmid-borne constructs, expressing the four C-terminally TAP-tagged Not1 variants from the NOT1 promoter, were transformed into a NOT1 shuffle strain. Then, upon plasmid shuffling on 5-fluoroorotic acid (5-FOA)-containing plates, complementation was assessed by growth assays on YPD plates. Importantly, all four Not1 variants sustained growth in the absence of endogenous Not1 equally well as wild-type Not1-TAP (Figure 2—figure supplement 1A). Western analysis of total protein extracts prepared by an alkaline lysis protocol, using antibodies recognizing the protein A moiety of the TAP tag, revealed that Not1-TAP was expressed at higher levels from plasmid than from the genomic locus. Despite the slightly changed start context owing to the introduction of an NdeI site (tac-ATG versus cat-ATG), expression of Not1-TAP from plasmid still resulted in the detection of a full-length and an N-terminally truncated isoform at similar ratios as when expressed from the native context (Figure 2B). In line with ATG codon 163 being the second translation initiation site, only the upper band corresponding to full-length Not1-TAP persisted in the M163L and M163A mutant variants. Concerning the two variants containing out-of-frame ATG trinucleotides upstream of the M163 codon, the N40(oofATG) and, to a lesser extent, the N156(oofATG) construct suppressed the synthesis of the major, N-terminally truncated Not1 isoform, presumably reflecting the relative strength of the two ATG contexts as translation initiation signals. We conclude that in S. cerevisiae Not1 is naturally synthesized as two distinct protein isoforms, which differ, due to a leaky scanning mechanism enabling the utilization of a downstream translation initiation site, by the presence (less abundant, full-length isoform) or absence (major isoform, starting with M163) of the N-terminal 162 amino acids. Figure 2 with 2 supplements see all Download asset Open asset Absence of Not1’s N-terminal domain suppresses the ∆acl4 growth defect and increases RPL3 and RPL4 mRNA levels. (A) Schematic representation of Not1 highlighting its domain organization and known binding sites of Ccr4-Not core components as revealed by diverse (co-)crystal structures (PDB: 4B8B and 4B8A [Basquin et al., 2012], 4CV5 [Mathys et al., 2014], 5AJD [Bhaskar et al., 2015], and 4BY6 [Bhaskar et al., 2013]). As shown in Figure 3H, the N-terminal Not1 segment encompassing amino acids 21–153 corresponds to the minimal Caf130-interacting domain (CaInD). Note that Ccr4 does not directly bind to Not1, it is recruited via its interaction with Caf1. The position and nature of the ∆acl4 suppressor mutations are indicated: M1L (ATG start codon changed to cTG), K21fs (AAA codon with deletion of one A, resulting in a frameshift), L112* (TTG codon changed to TaG stop codon), and I128fs (ATT codon with A deleted, resulting in a frameshift). M163 denotes the second methionine within Not1, it is encoded by the first occurring ATG trinucleotide after the start codon. (B) The shorter, major isoform of Not1 is generated by utilization of the ATG coding for M163 as the start codon. Total protein extracts, derived from cells expressing Not1-TAP, either from the genomic locus or from plasmid in a ∆not1 strain, and the indicated variants, were analyzed by Western blotting using anti-protA and anti-Adh1 (loading control) antibodies. The N40(oofATG) and N156(oofATG) constructs contain an out-of-frame ATG (oofATG) owin" @default.
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W4221019062 title "Author response: Dedicated chaperones coordinate co-translational regulation of ribosomal protein production with ribosome assembly to preserve proteostasis" @default.
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