Mature 18S Research Articles

Potent and selective ‘genetic zipper’ method for DNA-programmable plant protection: innovative oligonucleotide insecticides against Trioza alacris Flor

Chemical insecticides increased the chemical burden on natural ecosystems posing environmental health risk factor. The urgent need for a more sustainable and ecological approach has produced many innovative ideas, including eco-friendly ‘genetic zipper’ method (or CUAD platform) based on contact oligonucleotide insecticides. Oligonucleotide insecticides have enjoyed success recently on many sternorrhynchans showing highly adaptable structure for distinct insect pest species and selective mode of action. In this article, we describe the efficiency of the oligonucleotide insecticides (briefly, olinscides or DNA insecticides) Alacris-11 and Laura-11, as well as their combined use in mixture (1:1), designed for control of bay sucker (Trioza alacris Flor), an important psyllid pest of noble laurel (Laurus nobilis L.). These olinscides are based on short unmodified antisense DNA oligonucleotides that target ITS2 between 5.8S rRNA and 28S rRNA in pre-rRNA (Laura-11) and 28S rRNA region in mature 28S rRNA and pre-rRNA (Alacris-11). The maximum pest mortality, observed on the 14th day of the experiment, comprised 95.01 ± 4.42% for Alacris-11, 97.16 ± 2.48% for Laura-11, and 98.72 ± 1.14% for their mixture (1:1). The control oligonucleotide CTGA-11 did not cause any significant mortality (9.38 ± 0.57%), emphasizing selectivity in the action of oligonucleotide insecticides. The results show potent and specific nature of oligonucleotide insecticides for pest control and open up new frontiers in control of economically important psyllids in agriculture and forestry, including Asian citrus psyllid (Diaphorina citri Kuwayama) and many others. Scientists can easily adopt ‘genetic zipper’ method for plethora of insect pests because DNA is a programmable molecule and provides game-changing characteristics for plant protection.Graphical

Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish

Dnttip2 is one of the components of the small subunit (SSU) processome. In yeast, depletion of dnttip2 leads to an inefficient processing of pre-rRNA and a decrease in synthesis of the mature 18S rRNA. However, the biological roles of Dnttip2 in higher organisms are poorly defined. In this study, we demonstrate that dnttip2 is a maternal gene in zebrafish. Depletion of Dnttip2 leads to embryonic lethal with severe digestive organs hypoplasia. The loss of function of Dnttip2 also leads to partial defects in cleavage at the A0-site and E-site during 18S rRNA processing. In conclusion, Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish.

The Role of Ribosomal Proteins eL15 and eL36 in the Early Steps of Yeast 60S Ribosomal Subunit Assembly

Ribosomal proteins have important roles in maintaining the structure and function of mature ribosomes, but they also drive crucial rearrangement reactions during ribosome biogenesis. The contribution of most, but not all, ribosomal proteins to ribosome synthesis has been previously analyzed in the yeast Saccharomyces cerevisiae. Herein, we characterize the role of yeast eL15 during 60S ribosomal subunit formation. In vivo depletion of eL15 results in a shortage of 60S subunits and the appearance of half-mer polysomes. This is likely due to defective processing of the 27SA3 to the 27SBS pre-rRNA and impaired subsequent processing of both forms of 27SB pre-rRNAs to mature 25S and 5.8S rRNAs. Indeed, eL15 depletion leads to the efficient turnover of the de novo formed 27S pre-rRNAs. Additionally, depletion of eL15 blocks nucleocytoplasmic export of pre-60S particles. Moreover, we have analyzed the impact of depleting either eL15 or eL36 on the composition of early pre-60S particles, thereby revealing that the depletion of eL15 or eL36 not only affects each other's assembly into pre-60S particles but also that of neighboring ribosomal proteins, including eL8. These intermediates also lack most ribosome assembly factors required for 27SA3 and 27SB pre-rRNA processing, named A3- and B-factors, respectively. Importantly, our results recapitulate previous ones obtained upon eL8 depletion. We conclude that assembly of eL15, together with that of eL8 and eL36, is a prerequisite to shape domain I of 5.8S/25S rRNA within early pre-60S particles, through their binding to this rRNA domain and the recruitment of specific groups of assembly factors.

RNA Post-transcriptional Modifications of an Early-Stage Large-Subunit Ribosomal Intermediate.

Protein production by ribosomes is fundamental to life, and proper assembly of the ribosome is required for protein production. The RNA, which is post-transcriptionally modified, provides the platform for ribosome assembly. Thus, a complete understanding of ribosome assembly requires the determination of the RNA post-transcriptional modifications in all of the ribosome assembly intermediates and on each pathway. There are 26 RNA post-transcriptional modifications in 23S RNA of the mature Escherichia coli (E. coli) large ribosomal subunit. The levels of these modifications have been investigated extensively only for a small number of large subunit intermediates and under a limited number of cellular and environmental conditions. In this study, we determined the level of incorporations of 2-methyl adenosine, 3-methyl pseudouridine, 5-hydroxycytosine, and seven pseudouridines in an early-stage E. coli large-subunit assembly intermediate with a sedimentation coefficient of 27S. The 27S intermediate is one of three large subunit intermediates accumulated in E. coli cells lacking the DEAD-box RNA helicase DbpA and expressing the helicase inactive R331A DbpA construct. The majority of the investigated modifications are incorporated into the 27S large subunit intermediate to similar levels to those in the mature 50S large subunit, indicating that these early modifications or the enzymes that incorporate them play important roles in the initial events of large subunit ribosome assembly.

Global profiling of ribosomal protein acetylation reveals essentiality of acetylation homeostasis in maintaining ribosome assembly and function.

Acetylation is a global post-translational modification that regulates various cellular processes. Bacterial acetylomic studies have revealed extensive acetylation of ribosomal proteins. However, the role of acetylation in regulating ribosome function remains poorly understood. In this study, we systematically profiled ribosomal protein acetylation and identified a total of 289 acetylated lysine residues in 52 ribosomal proteins (r-proteins) from Salmonella Typhimurium. The majority of acetylated lysine residues of r-proteins were found to be regulated by both acetyltransferase Pat and metabolic intermediate acetyl phosphate. Our results show that acetylation plays a critical role in the assembly of the mature 70S ribosome complex by modulating r-proteins binding to rRNA. Moreover, appropriate acetylation is important for the interactions between elongation factors and polysomes, as well as regulating ribosome translation efficiency and fidelity. Dysregulation of acetylation could alter bacterial sensitivity to ribosome-targeting antibiotics. Collectively, our data suggest that the acetylation homeostasis of ribosomes is crucial for their assembly and function. Furthermore, this mechanism may represent a universal response to environmental signals across different cell types.

Identification of eight novel proteasome variants in five unrelated cases of proteasome-associated autoinflammatory syndromes (PRAAS).

Mutations in genes coding for proteasome subunits and/or proteasome assembly helpers typically cause recurring autoinflammation referred to as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperatures (CANDLE) or proteasome-associated autoinflammatory syndrome (PRAAS). Patients with CANDLE/PRAAS present with mostly chronically elevated type I interferon scores that emerge as a consequence of increased proteotoxic stress by mechanisms that are not fully understood. Here, we report on five unrelated patients with CANDLE/PRAAS carrying novel inherited proteasome missense and/or nonsense variants. Four patients were compound heterozygous for novel pathogenic variants in the known CANDLE/PRAAS associated genes, PSMB8 and PSMB10, whereas one patient showed additive loss-of-function mutations in PSMB8. Variants in two previously not associated proteasome genes, PSMA5 and PSMC5, were found in a patient who also carried the PSMB8 founder mutation, p.T75M. All newly identified mutations substantially impact the steady-state expression of the affected proteasome subunits and/or their incorporation into mature 26S proteasomes. Our observations expand the spectrum of PRAAS-associated genetic variants and improve a molecular diagnosis and genetic counseling of patients with sterile autoinflammation.

A homozygous POLR1A variant causes leukodystrophy and affects protein homeostasis.

RNA polymerase I transcribes ribosomal DNA to produce precursor 47S rRNA. Post-transcriptional processing of this rRNA generates mature 28S, 18S and 5.8S rRNAs, which form the ribosomes, together with 5S rRNA, assembly factors, and ribosomal proteins. We previously reported a homozygous variant in the catalytic subunit of RNA polymerase I, POLR1A, in two brothers with leukodystrophy and progressive course. However, the disease mechanism remained unknown. In this report, we describe another missense variant POLR1A NM_015425.3:c.1925C > A; p.(Thr642Asn) in homozygosity in two unrelated patients. Patient 1 was a 16-year-old male and patient 2 was a 2-year-old female. Both patients manifested neurological deficits, with brain MRIs showing hypomyelinating leukodystrophy, and cerebellar atrophy; and in patient 1 additionally with hypointensity of globi pallidi and small volume of the basal ganglia. Patient 1 had progressive disease course, leading to death at the age of 16.5 years. Extensive in vitro experiments in fibroblasts from patient 1 documented that the mutated POLR1A led to aberrant rRNA processing and degradation, and abnormal nucleolar homeostasis. Proteomics data analyses and further in vitro experiments documented abnormal protein homeostasis, and endoplasmic reticulum stress responses. We confirm that POLR1A biallelic variants cause neurodegenerative disease, expand the knowledge of the clinical phenotype of the disorder, and provide evidence for possible pathological mechanisms leading to POLR1A-related leukodystrophy.

Altered conformational landscape upon sensing guanine nucleotides in a disease mutant of Elongation Factor-like 1 (EFL1) GTPase.

Ribosome biogenesis is a highly regulated process that requires the participation of several accessory proteins that participates in the cytoplasmic last maturation step before entering the pool of translating ribosomes. One of these accessory proteins is the GTPase Elongation Factor-like 1 (EFL1), a homologue of the elongation factor 2 (EF-2). EFL1, together with SBDS (yeast Sdo1), evict the anti-association factor eIF6 (yeast Tif6) from the surface of the 60S subunit, allowing mature ribosome assembly. Defects in the function of any of these two proteins lead to a disease called Shwachman-Diamond Syndrome (SDS). Mutations prevent the release of eIF6 from the 60S subunit, with the consequent imbalance of mature 60S subunits entering the pool of active translating 80S ribosomes and a decrease in global translation. EFL1 is a molecular motor that adopts at least four distinct conformations in solution regulated by SBDS and guanine nucleotides whose malfunction in the SDS has been scarcely studied. The malfunction of the non-synonymous mutations in EFL1 has been scarcely studied, with little effect on its catalytic activity or fold. We used Isothermal Titration Calorimetry to gain further insights into the malfunction of Efl1 mutations. We characterised the binding energetics of S. cerevisiae Efl1 R1086Q equivalent to EFL1 R1095Q reported in patients with SDS. The data presented exhibits a different structural and energetic response in the yeast mutant Efl1 R1086Q compared to that previously reported Efl1 wild type. The heat capacity change (ΔCp) and conformational binding entropy of the complexes formed between the mutant and the wild type proteins showed opposite values suggesting they adopt different conformations. We conclude that mutation R1086Q profoundly disrupts the internal rearrangements necessary to elicit a similar conformational change as that in native EFL1.

Transcription Factor Profiling Reveals That CEBPA Promotes Ribosomal RNA Synthesis in Myeloid Progenitors

Hematopoiesis involves orchestrated differentiation of hematopoietic stem cells (HSCs) and lineage-restricted progenitors to mature blood cells. Different populations in this hierarchy vary in function, size, proliferation, and protein synthesis rates. It is also known that different normal hematopoietic cell types differ in the rates of ribosomal RNA (rRNA) transcription, the key rate-limiting step in ribosome biogenesis that occurs in the nucleolus. Leukemic blast cells have long been known to have enlarged nucleoli, indicating aberrantly increased rRNA transcription and ribosome biogenesis. Ribosome biogenesis is an energy-intensive process, beginning with the transcription of multi-copy rDNA genes by RNA polymerase I (Pol I) to produce 47S precursor rRNA (pre-rRNA), which is further processed into mature 18S, 5.8S, and 28S rRNA and assembled with 5S rRNA and 80 different ribosomal proteins to form mature ribosomes (Fig A). Because rRNA transcription is often considered a housekeeping process, the variations in its cell-type and disease-specific rates have not been studied, leading to a lack of understanding of the mechanisms underlying its regulation. To identify cell-type-specific regulators of rRNA transcription in hematopoiesis, we compiled a list of 249 hematopoietic transcription factors (TFs) and epigenetic factors, and mapped 2200 publicly available ChIP-Seq datasets for these factors to rDNA, creating the first atlas of hematopoietic TF-rDNA binding. Through this atlas, we identified that CEBPA shows consistent and abundant binding to human and mouse rDNA at a conserved, previously unknown motif (Fig B). CEBPA is an essential myeloid lineage-specific TF whose knockout leads to complete loss of granulocyte-monocyte progenitors (GMPs) and all downstream myeloid lineage cells. CEBPA also shows hotspot mutations in 10% of AML patients. To characterize the role of CEBPA in rRNA transcription, we used a mouse HoxA9-ER cell line (which closely resembles GMPs), and biallelically fused FKBP degron into endogenous loci of the Cebpa gene, enabling rapid degradation of CEBPA protein using small molecule ligand dTAGV-1 (Fig C, D). To quantify effects on rRNA transcription, we developed a novel assay named '47S-FISH-Flow’ that involves flowcytometric quantification of fluorescent probes hybridized to nascent rRNA in the nucleolus (Fig E, F). We found that depleting CEBPA caused a rapid reduction in 47S rRNA level, plateauing at 35% reduction within hours (Fig G). Decrease in rRNA production was followed by reduction in nucleolar size, reduction in mature ribosome subunit abundance, and reduced growth (Fig H, I). To understand the molecular function of CEBPA on rDNA machinery, we performed time course ChIP-Seq on key rDNA transcriptional machinery, and found that CEBPA degradation specifically reduced occupancy of Pol I along with its initiation factor RRN3 on rDNA (Fig J). In summary, we have found a novel role for the extensively studied myeloid lineage-specific TF CEBPA in directly binding rDNA and regulating rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by lineage-specific hematopoietic TFs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Human pre-60S assembly factors link rRNA transcription to pre-rRNA processing.

In eukaryotes, the nucleolus is the site of ribosome biosynthesis, an essential process in all cells. While human ribosome assembly is largely evolutionarily conserved, many of the regulatory details underlying its control and function have not yet been well-defined. The nucleolar protein RSL24D1 was originally identified as a factor important for 60S ribosomal subunit biogenesis. In addition, the PeBoW (BOP1-PES1-WDR12) complex has been well-defined as required for pre-28S rRNA processing and cell proliferation. In this study, we show that RSL24D1 depletion impairs both pre-ribosomal RNA (pre-rRNA) transcription and mature 28S rRNA production, leading to decreased protein synthesis and p53 stabilization in human cells. Surprisingly, each of the PeBoW complex members is also required for pre-rRNA transcription. We demonstrate that RSL24D1 and WDR12 co-immunoprecipitate with the RNA polymerase I subunit, RPA194, and regulate its steady state levels. These results uncover the dual role of RSL24D1 and the PeBoW complex in multiple steps of ribosome biogenesis, and provide evidence implicating large ribosomal subunit biogenesis factors in pre-rRNA transcription control.

In situ single particle classification reveals distinct 60S maturation intermediates in cells.

Previously, we showed that high-resolution template matching can localize ribosomes in two-dimensional electron cryo-microscopy (cryo-EM) images of untilted Mycoplasma pneumoniae cells with high precision (Lucas et al., 2021). Here, we show that comparing the signal-to-noise ratio (SNR) observed with 2DTM using different templates relative to the same cellular target can correct for local variation in noise and differentiate related complexes in focused ion beam (FIB)-milled cell sections. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the Saccharomyces cerevisiae cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. 2DTM can distinguish related molecular populations without the need to generate 3D reconstructions from the data to be classified, permitting classification even when only a few target particles exist in a cell.

Impaired Transcription Elongation by RNA Polymerase I Results in Defective Ribosomal RNA Processing

RNA polymerase I (Pol I) synthesizes the majority of the ribosomal RNA (rRNA) in a eukaryotic cell, which is then assembled into the mature ribosome with ribosomal proteins. Pol I transcribes a single gene, the 35S gene in yeast, containing three gene elements (18S, 5.8S, and 25S) and four spacer regions. Previous literature demonstrates that processing events occur both co‐ and post‐transcriptionally so that the spacer regions are removed and degraded, resulting in mature 18S, 5.8S, and 25S rRNAs. While it has been established that rRNA processing begins during transcription, the relationship between Pol I transcription elongation properties and processing events have not been well defined. The objective of this project was to determine whether transcription elongation kinetics have a regulatory role in rRNA processing, and we hypothesized that if transcription elongation was impaired, this would result in defects in processing. To test this hypothesis, we generated a yeast strain containing a point mutation in RPA190, the largest subunit of Pol I. We found that there was a modest decrease in transcription elongation rate and an increase in pausing in the mutant compared to wild‐type Pol I in vitro. To analyze elongation effects of the mutant Pol I in vivo, we used native elongating transcript sequencing, which probes for Pol I occupancy on the ribosomal DNA template at single‐nucleotide resolution. Our in vivo results corroborated the in vitro findings, indicating that this mutation introduced an increase in pausing across the template during transcription elongation. Furthermore, Northern blots and polysome profiling indicated that rRNA processing and ribosome assembly were impaired when this mutation was introduced. These three independent in vitroand in vivo experimental strategies converge to demonstrate that transcription elongation and Pol I pausing may play a regulatory role in downstream processing steps.

Maize RNA 3'-terminal phosphate cyclase-like protein promotes 18S pre-rRNA cleavage and is important for kernel development.

Plant ribosomes contain four specialized ribonucleic acids, the 5S, 5.8S, 18S, and 25S ribosomal RNAs (rRNAs). Maturation of the latter three rRNAs requires cooperative processing of a single transcript by several endonucleases and exonucleases at specific sites. In maize (Zea mays), the exact nucleases and components required for rRNA processing remain poorly understood. Here, we characterized a conserved RNA 3′-terminal phosphate cyclase (RCL)-like protein, RCL1, that functions in 18S rRNA maturation. RCL1 is highly expressed in the embryo and endosperm during early seed development. Loss of RCL1 function resulted in lethality due to aborted embryo cell differentiation. We also observed pleiotropic defects in the rcl1 endosperm, including abnormal basal transfer cell layer growth and aleurone cell identity, and reduced storage reserve accumulation. The rcl1 seeds had lower levels of mature 18S rRNA and the related precursors were altered in abundance compared with wild type. Analysis of transcript levels and protein accumulation in rcl1 revealed that the observed lower levels of zein and starch synthesis enzymes mainly resulted from effects at the transcriptional and translational levels, respectively. These results demonstrate that RCL1-mediated 18S pre-rRNA processing is essential for ribosome function and messenger RNA translation during maize seed development.

CEBPA Directly Binds Ribosomal DNA and Promotes Ribosomal RNA Transcription in Myeloid Progenitors

Hematopoietic stem cells (HSCs) form a hierarchy of lineage restricted progenitor cells to produce mature hematopoietic cells that vary in function, size, proliferation, and protein synthesis rates. Different hematopoietic cells also vary in the rate of ribosomal RNA (rRNA) transcription, the key rate-limiting step in ribosome biogenesis that occurs in the nucleolus. Leukemic blast cells have long been identified by their prominent nucleoli, indicating high ribosome biogenesis rates (Fig A). Ribosome biogenesis is an extremely energy intensive process begins with transcription of multi-copy rDNA genes by RNA polymerase I (Pol I) to produce 47S precursor rRNA (pre-rRNA) which further processed into the generation of mature 18S, 5.8S, and 28S rRNA and assembled with 5S rRNA and 80 different ribosomal proteins to form mature ribosomes (Fig B). This process is highly dynamic and regulated at the level of rRNA transcription. Despite cell-type and disease-specific variations, rRNA transcription has long been considered a housekeeping process. Hence, cell or tissue type-specific regulation of rRNA transcription has rarely been explored.To identify cell-type-specific regulators of rRNA transcription in hematopoiesis, we mapped 2200 publicly available ChIP-Seq datasets representing 249 hematopoietic transcription factors (TFs) and epigenetic factors to create an atlas of hematopoietic TF-rDNA binding. We identified CEBPA that shows consistent and abundant binding to rDNA at a conserved, previously unknown motif in both species (Fig C). CEBPA is a myeloid lineage specific TF whose knockout leads to complete loss of all myeloid lineage cells. It is also frequently mutated (10%) in AML patients. So we picked CEBPA to further characterize its role in rRNA transcription.Since CEBPA deletion causes loss of granulocyte-monocyte progenitors (GMPs), we used the mouse HoxA9-ER cell line (which closely resembles GMPs). To study the immediate consequences of CEBPA loss, We generated a stable degron cell line by biallelically fusing FKBP degron into endogenous loci of Cebpa, enabling to rapidly degrade endogenous CEBPA protein on treatment with dTagV ligand (Fig D, E). To precisely quantify the rate of rRNA transcription, we developed a novel assay called ‘47S-FISH-Flow’ that involves hybridizing fluorescent oligos unique to 5' end of 47S pre-rRNA, which only marks newly synthesized nascent rRNA in the nucleolus, and quantify using flow cytometry (Fig F, G). We found that depleting CEBPA caused rapid decrease in 47S rRNA level and occupancy of Pol I on rDNA (Fig H, I).In summary, we found that myeloid lineage specific TF CEBPA abundantly binds to a conserved motif in rDNA and the depletion of CEBPA rapidly reduces nascent 47S rRNA, indicating that it directly promotes rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by lineage-specific hematopoietic TFs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

HSF1 induces RNA polymerase II synthesis of ribosomal RNA in S. cerevisiae during nitrogen deprivation

The resource intensive process of accurate ribosome synthesis is essential for cell viability in all organisms. Ribosome synthesis regulation centers on RNA polymerase I (pol I) transcription of a 35S rRNA precursor that is processed into the mature 18S, 5.8S and 25S rRNAs. During nutrient deprivation or stress, pol I synthesis of rRNA is dramatically reduced. Conversely, chronic stress such as mitochondrial dysfunction induces RNA polymerase II (pol II) to transcribe functional rRNA using an evolutionarily conserved cryptic pol II rDNA promoter suggesting a universal phenomenon. However, this polymerase switches and its role in regulation of rRNA synthesis remain unclear. In this paper, we demonstrate that extended nitrogen deprivation induces the polymerase switch via components of the environmental stress response. We further show that the switch is repressed by Sch9 and activated by the stress kinase Rim15. Like stress-induced genes, the switch requires not only pol II transcription machinery, including the mediator, but also requires the HDAC, Rpd3 and stress transcription factor Hsf1. The current work shows that the constitutive allele, Hsf1PO4* displays elevated levels of induction in non-stress conditions while binding to a conserved site in the pol II rDNA promoter upstream of the pol I promoter. Whether the polymerase switch serves to provide rRNA when pol I transcription is inhibited or fine-tunes pol I initiation via RNA interactions is yet to be determined. Identifying the underlying mechanism for this evolutionary conserved phenomenon will help understand the mechanism of pol II rRNA synthesis and its role in stress adaptation.

Processing of the ribosomal ubiquitin-like fusion protein FUBI-eS30/FAU is required for 40S maturation and depends on USP36.

In humans and other holozoan organisms, the ribosomal protein eS30 is synthesized as a fusion protein with the ubiquitin-like protein FUBI. However, FUBI is not part of the mature 40S ribosomal subunit and cleaved off by an as-of-yet unidentified protease. How FUBI-eS30 processing is coordinated with 40S subunit maturation is unknown. To study the mechanism and importance of FUBI-eS30 processing, we expressed non-cleavable mutants in human cells, which affected late steps of cytoplasmic 40S maturation, including the maturation of 18S rRNA and recycling of late-acting ribosome biogenesis factors. Differential affinity purification of wild-type and non-cleavable FUBI-eS30 mutants identified the deubiquitinase USP36 as a candidate FUBI-eS30 processing enzyme. Depletion of USP36 by RNAi or CRISPRi indeed impaired FUBI-eS30 processing and moreover, purified USP36 cut FUBI-eS30 in vitro. Together, these data demonstrate the functional importance of FUBI-eS30 cleavage and identify USP36 as a novel protease involved in this process.

Cockayne Syndrome-Associated CSA and CSB Mutations Impair Ribosome Biogenesis, Ribosomal Protein Stability, and Global Protein Folding.

Cockayne syndrome (CS) is a developmental disorder with symptoms that are typical for the aging body, including subcutaneous fat loss, alopecia, and cataracts. Here, we show that in the cells of CS patients, RNA polymerase I transcription and the processing of the pre-rRNA are disturbed, leading to an accumulation of the 18S-E intermediate. The mature 18S rRNA level is reduced, and isolated ribosomes lack specific ribosomal proteins of the small 40S subunit. Ribosomal proteins are susceptible to unfolding and the CS cell proteome is heat-sensitive, indicating misfolded proteins and an error-prone translation process in CS cells. Pharmaceutical chaperones restored impaired cellular proliferation. Therefore, we provide evidence for severe protein synthesis malfunction, which together with a loss of proteostasis constitutes the underlying pathophysiology in CS.

A Novel Model for the RNase MRP-Induced Switch between the Formation of Different Forms of 5.8S rRNA.

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the “spacer” sequences. The canonical pathway for the removal of the ITS1 spacer involves cleavages at the 3′ end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5′ end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5′ end by exonuclease Xrn1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA. Traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. However, results from this work show that the MRP-induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the link between RNase MRP and 5.8S 5′ end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.

RbfA Is Involved in Two Important Stages of 30S Subunit Assembly: Formation of the Central Pseudoknot and Docking of Helix 44 to the Decoding Center.

Ribosome biogenesis is a highly coordinated and complex process that requires numerous assembly factors that ensure prompt and flawless maturation of ribosomal subunits. Despite the increasing amount of data collected, the exact role of most assembly factors and mechanistic details of their operation remain unclear, mainly due to the shortage of high-resolution structural information. Here, using cryo-electron microscopy, we characterized 30S ribosomal particles isolated from an Escherichia coli strain with a deleted gene for the RbfA factor. The cryo-EM maps for pre-30S subunits were divided into six classes corresponding to consecutive assembly intermediates: from the particles with a completely unresolved head domain and unfolded central pseudoknot to almost mature 30S subunits with well-resolved body, platform, and head domains and partially distorted helix 44. The structures of two predominant 30S intermediates belonging to most populated classes obtained at 2.7 Å resolutions indicate that RbfA acts at two distinctive 30S assembly stages: early formation of the central pseudoknot including folding of the head, and positioning of helix 44 in the decoding center at a later stage. Additionally, it was shown that the formation of the central pseudoknot may promote stabilization of the head domain, likely through the RbfA-dependent maturation of the neck helix 28. An update to the model of factor-dependent 30S maturation is proposed, suggesting that RfbA is involved in most of the subunit assembly process.

Structural basis of successive adenosine modifications by the conserved ribosomal methyltransferase KsgA.

Biogenesis of ribosomal subunits involves enzymatic modifications of rRNA that fine-tune functionally important regions. The universally conserved prokaryotic dimethyltransferase KsgA sequentially modifies two universally conserved adenosine residues in helix 45 of the small ribosomal subunit rRNA, which is in proximity of the decoding site. Here we present the cryo-EM structure of Escherichia coli KsgA bound to an E. coli 30S at a resolution of 3.1 Å. The high-resolution structure reveals how KsgA recognizes immature rRNA and binds helix 45 in a conformation where one of the substrate nucleotides is flipped-out into the active site. We suggest that successive processing of two adjacent nucleotides involves base-flipping of the rRNA, which allows modification of the second substrate nucleotide without dissociation of the enzyme. Since KsgA is homologous to the essential eukaryotic methyltransferase Dim1 involved in 40S maturation, these results have also implications for understanding eukaryotic ribosome maturation.