Ribosomes Need Straight A's to Sleep.

Like falling asleep and waking up, many biological processes in mammals cycle in a diurnal fashion. Now, Sinturel et al. demonstrate that diurnal size changes in the liver require eating during a mouse's normal awake time and that these size changes are controlled by a nuclear mechanism that modulates ribosome production.

Mapping subcellular localizations of unannotated microproteins and alternative proteins with MicroID.

Proteogenomic identification of translated small open reading frames has revealed thousands of previously unannotated, largely uncharacterized microproteins, or polypeptides of less than 100 amino acids, and alternative proteins (alt-proteins) that are co-encoded with canonical proteins and are often larger. The subcellular localizations of microproteins and alt-proteins are generally unknown but can have significant implications for their functions. Proximity biotinylation is an attractive approach to define the protein composition of subcellular compartments in cells and in animals. Here, we developed a high-throughput technology to map unannotated microproteins and alt-proteins to subcellular localizations by proximity biotinylation with TurboID (MicroID). More than 150 microproteins and alt-proteins are associated with subnuclear organelles. One alt-protein, alt-LAMA3, localizes to the nucleolus and functions in pre-rRNA transcription. We applied MicroID in a mouse model, validating expression of a conserved nuclear microprotein, and establishing MicroID for discovery of microproteins and alt-proteins in vivo.

The cytidine deaminase APOBEC3A regulates nucleolar function to promote cell growth and ribosome biogenesis.

Cancer initiates as a consequence of genomic mutations and its subsequent progression relies in part on increased production of ribosomes to maintain high levels of protein synthesis for unchecked cell growth. Recently, cytidine deaminases have been uncovered as sources of mutagenesis in cancer. In an attempt to form a connection between these 2 cancer driving processes, we interrogated the cytidine deaminase family of proteins for potential roles in human ribosome biogenesis. We identified and validated APOBEC3A and APOBEC4 as novel ribosome biogenesis factors through our laboratory's established screening platform for the discovery of regulators of nucleolar function in MCF10A cells. Through siRNA depletion experiments, we highlight APOBEC3A's requirement in making ribosomes and specific role within the processing and maturation steps that form the large subunit 5.8S and 28S ribosomal (r)RNAs. We demonstrate that a subset of APOBEC3A resides within the nucleolus and associates with critical ribosome biogenesis factors. Mechanistic insight was revealed by transient overexpression of both wild-type and a catalytically dead mutated APOBEC3A, which both increase cell growth and protein synthesis. Through an innovative nuclear RNA sequencing methodology, we identify only modest predicted APOBEC3A C-to-U target sites on the pre-rRNA and pre-mRNAs. Our work reveals a potential direct role for APOBEC3A in ribosome biogenesis likely independent of its editing function. More broadly, we found an additional function of APOBEC3A in cancer pathology through its function in ribosome biogenesis, expanding its relevance as a target for cancer therapeutics.

Discovery of novel microRNA mimic repressors of ribosome biogenesis.

While microRNAs and other non-coding RNAs are the next frontier of novel regulators of mammalian ribosome biogenesis (RB), a systematic exploration of microRNA-mediated RB regulation has not yet been undertaken. We carried out a high-content screen in MCF10A cells for changes in nucleolar number using a library of 2603 mature human microRNA mimics. Following a secondary screen for nucleolar rRNA biogenesis inhibition, we identified 72 novel microRNA negative regulators of RB after stringent hit calling. Hits included 27 well-conserved microRNAs present in MirGeneDB, and were enriched for mRNA targets encoding proteins with nucleolar localization or functions in cell cycle regulation. Rigorous selection and validation of a subset of 15 microRNA hits unexpectedly revealed that most of them caused dysregulated pre-rRNA processing, elucidating a novel role for microRNAs in RB regulation. Almost all hits impaired global protein synthesis and upregulated CDKN1A (p21) levels, while causing diverse effects on RNA Polymerase 1 (RNAP1) transcription and TP53 protein levels. We provide evidence that the MIR-28 siblings, hsa-miR-28-5p and hsa-miR-708-5p, potently target the ribosomal protein mRNA RPS28 via tandem primate-specific 3' UTR binding sites, causing a severe pre-18S pre-rRNA processing defect. Our work illuminates novel microRNA attenuators of RB, forging a promising new path for microRNA mimic chemotherapeutics.

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.

Nascent alt-protein chemoproteomics reveals a pre-60S assembly checkpoint inhibitor.

Many unannotated microproteins and alternative proteins (alt-proteins) are coencoded with canonical proteins, but few of their functions are known. Motivated by the hypothesis that alt-proteins undergoing regulated synthesis could play important cellular roles, we developed a chemoproteomic pipeline to identify nascent alt-proteins in human cells. We identified 22 actively translated alt-proteins or N-terminal extensions, one of which is post-transcriptionally upregulated by DNA damage stress. We further defined a nucleolar, cell-cycle-regulated alt-protein that negatively regulates assembly of the pre-60S ribosomal subunit (MINAS-60). Depletion of MINAS-60 increases the amount of cytoplasmic 60S ribosomal subunit, upregulating global protein synthesis and cell proliferation. Mechanistically, MINAS-60 represses the rate of late-stage pre-60S assembly and export to the cytoplasm. Together, these results implicate MINAS-60 as a potential checkpoint inhibitor of pre-60S assembly and demonstrate that chemoproteomics enables hypothesis generation for uncharacterized alt-proteins.

Biallelic splicing variants in the nucleolar 60S assembly factor RBM28 cause the ribosomopathy ANE syndrome.

Alopecia, neurologic defects, and endocrinopathy (ANE) syndrome is a rare ribosomopathy known to be caused by a p.(Leu351Pro) variant in the essential, conserved, nucleolar large ribosomal subunit (60S) assembly factor RBM28. We report the second family of ANE syndrome to date and a female pediatric ANE syndrome patient. The patient presented with alopecia, craniofacial malformations, hypoplastic pituitary, and hair and skin abnormalities. Unlike the previously reported patients with the p.(Leu351Pro) RBM28 variant, this ANE syndrome patient possesses biallelic precursor messenger RNA (pre-mRNA) splicing variants at the 5' splice sites of exon 5 (ΔE5) and exon 8 (ΔE8) of RBM28 (NM_018077.2:c.[541+1_541+2delinsA]; [946G > T]). In silico analyses and minigene splicing experiments in cells indicate that each splice variant specifically causes skipping of its respective mutant exon. Because the ΔE5 variant results in an in-frame 31 amino acid deletion (p.(Asp150_Lys180del)) in RBM28 while the ΔE8 variant leads to a premature stop codon in exon 9, we predicted that the ΔE5 variant would produce partially functional RBM28 but the ΔE8 variant would not produce functional protein. Using a yeast model, we demonstrate that the ΔE5 variant does indeed lead to reduced overall growth and large subunit ribosomal RNA (rRNA) production and pre-rRNA processing. In contrast, the ΔE8 variant is comparably null, implying that the partially functional ΔE5 RBM28 protein enables survival but precludes correct development. This discovery further defines the underlying molecular pathology of ANE syndrome to include genetic variants that cause aberrant splicing in RBM28 pre-mRNA and highlights the centrality of nucleolar processes in human genetic disease.

Human nucleolar protein 7 (NOL7) is required for early pre-rRNA accumulation and pre-18S rRNA processing.

The main components of the essential cellular process of eukaryotic ribosome biogenesis are highly conserved from yeast to humans. Among these, the U3 Associated Proteins (UTPs) are a small subunit processome subcomplex that coordinate the first two steps of ribosome biogenesis in transcription and pre-18S processing. While we have identified the human counterparts of most of the yeast Utps, the homologs of yeast Utp9 and Bud21 (Utp16) have remained elusive. In this study, we find that NOL7 is the likely ortholog of Bud21. Previously described as a tumour suppressor through regulation of antiangiogenic transcripts, we now show that NOL7 is required for early pre-rRNA accumulation and pre-18S rRNA processing in human cells. These roles lead to decreased protein synthesis and induction of the nucleolar stress response upon NOL7 depletion. Beyond Bud21's nonessential role in yeast, we establish human NOL7 as an essential UTP that is necessary to maintain both early pre-rRNA levels and processing.

Paired Box 9 (PAX9), the RNA polymerase II transcription factor, regulates human ribosome biogenesis and craniofacial development.

Dysregulation of ribosome production can lead to a number of developmental disorders called ribosomopathies. Despite the ubiquitous requirement for these cellular machines used in protein synthesis, ribosomopathies manifest in a tissue-specific manner, with many affecting the development of the face. Here we reveal yet another connection between craniofacial development and making ribosomes through the protein Paired Box 9 (PAX9). PAX9 functions as an RNA Polymerase II transcription factor to regulate the expression of proteins required for craniofacial and tooth development in humans. We now expand this function of PAX9 by demonstrating that PAX9 acts outside of the cell nucleolus to regulate the levels of proteins critical for building the small subunit of the ribosome. This function of PAX9 is conserved to the organism Xenopus tropicalis, an established model for human ribosomopathies. Depletion of pax9 leads to craniofacial defects due to abnormalities in neural crest development, a result consistent with that found for depletion of other ribosome biogenesis factors. This work highlights an unexpected layer of how the making of ribosomes is regulated in human cells and during embryonic development.

A protein interaction map of the LSU processome.

Maturation of the large ribosomal subunit (LSU) in eukaryotes is a complex and highly coordinated process that requires the concerted action of a large, dynamic, ribonucleoprotein complex, the LSU processome. While we know that >80 ribosome biogenesis factors are required throughout the course of LSU assembly, little is known about how these factors interact with each other within the LSU processome. To interrogate its organization and architecture, we took a systems biology approach and performed a semi-high-throughput, array-based, directed yeast two-hybrid assay. Assaying 4800 protein-protein interactions, we identified 232 high-confidence, binary-interacting protein pairs, representing a fourfold increase from current knowledge. The resulting LSU processome interactome map has enhanced our understanding of the organization and function of the biogenesis factors within the LSU processome, revealing both novel and previously identified subcomplexes and hub proteins, including Nop4.

Ribosomopathies: Old Concepts, New Controversies.

Ribosomopathies are a diverse subset of diseases caused by reduced expression of, or mutations in, factors necessary for making ribosomes, the protein translation machinery in the cell. Despite the ubiquitous need for ribosomes in all cell types, ribosomopathies manifest with tissue-specific defects and sometimes increased cancer susceptibility, but few treatments target the underlying cause. By highlighting new research in the field, we review current hypotheses for the basis of this tissue specificity. Based on new work, we broaden our understanding of the role of ribosome biogenesis in diverse tissue types throughout embryonic development. We also pose the question of whether previously described human conditions such as aging can be at least partially attributed to defects in making ribosomes.

Nop9 recognizes structured and single-stranded RNA elements of preribosomal RNA.

Nop9 is an essential factor in the processing of preribosomal RNA. Its absence in yeast is lethal, and defects in the human ortholog are associated with breast cancer, autoimmunity, and learning/language impairment. PUF family RNA-binding proteins are best known for sequence-specific RNA recognition, and most contain eight α-helical repeats that bind to the RNA bases of single-stranded RNA. Nop9 is an unusual member of this family in that it contains eleven repeats and recognizes both RNA structure and sequence. Here we report a crystal structure of Saccharomyces cerevisiae Nop9 in complex with its target RNA within the 20S preribosomal RNA. This structure reveals that Nop9 brings together a carboxy-terminal module recognizing the 5' single-stranded region of the RNA and a bifunctional amino-terminal module recognizing the central double-stranded stem region. We further show that the 3' single-stranded region of the 20S target RNA adds sequence-independent binding energy to the RNA-Nop9 interaction. Both the amino- and carboxy-terminal modules retain the characteristic sequence-specific recognition of PUF proteins, but the amino-terminal module has also evolved a distinct interface, which allows Nop9 to recognize either single-stranded RNA sequences or RNAs with a combination of single-stranded and structured elements.

U8 variants on the brain: a small nucleolar RNA and human disease.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs vital for ribosomal RNA (rRNA) maturation. The U8 snoRNA, encoded by the SNORD118 gene in humans, is an atypical C/D box snoRNA as it promotes rRNA cleavage rather than 2'-O-methylation and is unique to vertebrates. The U8 snoRNA is critical for cleavage events that produce the mature 5.8S and 28S rRNAs of the large ribosomal subunit. Unexpectedly, single nucleotide polymorphisms (SNPs) in the SNORD118 gene were recently found causal to the neurodegenerative disease leukoencephalopathy, brain calcifications, and cysts (LCC; aka Labrune syndrome), but its molecular pathogenesis is unclear. Here, we will review current knowledge on the function of the U8 snoRNA in ribosome biogenesis, and connect it to the preservation of brain function in humans as well as to its dysregulation in inherited white matter disease.

Fanconi anemia protein FANCI functions in ribosome biogenesis.

Fanconi anemia (FA) is a disease of DNA repair characterized by bone marrow failure and a reduced ability to remove DNA interstrand cross-links. Here, we provide evidence that the FA protein FANCI also functions in ribosome biogenesis, the process of making ribosomes that initiates in the nucleolus. We show that FANCI localizes to the nucleolus and is functionally and physically tied to the transcription of pre-ribosomal RNA (pre-rRNA) and to large ribosomal subunit (LSU) pre-rRNA processing independent of FANCD2. While FANCI is known to be monoubiquitinated when activated for DNA repair, we find that it is predominantly in the deubiquitinated state in the nucleolus, requiring the nucleoplasmic deubiquitinase (DUB) USP1 and the nucleolar DUB USP36. Our model suggests a possible dual pathophysiology for FA that includes defects in DNA repair and in ribosome biogenesis.

Driving nucleolar assembly.

In this issue of Genes & Development, Grob and colleagues (pp. 220-230) identify the minimal molecular requirements to assemble a fully functional nucleolus in human cells and demonstrate the importance of the nucleolar transcription factor upstream binding factor (UBF) as a mitotic bookmark at the ribosomal DNA (rDNA).

RPSA, a candidate gene for isolated congenital asplenia, is required for pre-rRNA processing and spleen formation in Xenopus.

A growing number of tissue-specific inherited disorders are associated with impaired ribosome production, despite the universal requirement for ribosome function. Recently, mutations in RPSA, a protein component of the small ribosomal subunit, were discovered to underlie approximately half of all isolated congenital asplenia cases. However, the mechanisms by which mutations in this ribosome biogenesis factor lead specifically to spleen agenesis remain unknown, in part due to the lack of a suitable animal model for study. Here we reveal that RPSA is required for normal spleen development in the frog, Xenopus tropicalis Depletion of Rpsa in early embryonic development disrupts pre-rRNA processing and ribosome biogenesis, and impairs expression of the key spleen patterning genes nkx2-5, bapx1 and pod1 in the spleen anlage. Importantly, we also show that whereas injection of human RPSA mRNA can rescue both pre-rRNA processing and spleen patterning, injection of human mRNA bearing a common disease-associated mutation cannot. Together, we present the first animal model of RPSA-mediated asplenia and reveal a crucial requirement for RPSA in pre-rRNA processing and molecular patterning during early Xenopus development.

Mutations in KDSR Cause Recessive Progressive Symmetric Erythrokeratoderma.

The discovery of new genetic determinants of inherited skin disorders has been instrumental to the understanding of epidermal function, differentiation, and renewal. Here, we show that mutations in KDSR (3-ketodihydrosphingosine reductase), encoding an enzyme in the ceramide synthesis pathway, lead to a previously undescribed recessive Mendelian disorder in the progressive symmetric erythrokeratoderma spectrum. This disorder is characterized by severe lesions of thick scaly skin on the face and genitals and thickened, red, and scaly skin on the hands and feet. Although exome sequencing revealed several of the KDSR mutations, we employed genome sequencing to discover a pathogenic 346 kb inversion in multiple probands, and cDNA sequencing and a splicing assay established that two mutations, including a recurrent silent third base change, cause exon skipping. Immunohistochemistry and yeast complementation studies demonstrated that the mutations cause defects in KDSR function. Systemic isotretinoin therapy has achieved nearly complete resolution in the two probands in whom it has been applied, consistent with the effects of retinoic acid on alternative pathways for ceramide generation.

A Ribosomopathy Reveals Decoding Defective Ribosomes Driving Human Dysmorphism.

Ribosomal protein (RP) gene mutations, mostly associated with inherited or acquired bone marrow failure, are believed to drive disease by slowing the rate of protein synthesis. Here de novo missense mutations in the RPS23 gene, which codes for uS12, are reported in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. One individual additionally presents with intellectual disability and autism spectrum disorder. The amino acid substitutions lie in two highly conserved loop regions of uS12 with known roles in maintaining the accuracy of mRNA codon translation. Primary cells revealed one substitution severely impaired OGFOD1-dependent hydroxylation of a neighboring proline residue resulting in 40S ribosomal subunits that were blocked from polysome formation. The other disrupted a predicted pi-pi stacking interaction between two phenylalanine residues leading to a destabilized uS12 that was poorly tolerated in 40S subunit biogenesis. Despite no evidence of a reduction in the rate of mRNA translation, these uS12 variants impaired the accuracy of mRNA translation and rendered cells highly sensitive to oxidative stress. These discoveries describe a ribosomopathy linked to uS12 and reveal mechanistic distinctions between RP gene mutations driving hematopoietic disease and those resulting in developmental disorders.

The SSU processome interactome in Saccharomyces cerevisiae reveals novel protein subcomplexes.

Ribosome assembly is an evolutionarily conserved and energy intensive process required for cellular growth, proliferation, and maintenance. In yeast, assembly of the small ribosomal subunit (SSU) requires approximately 75 assembly factors that act in coordination to form the SSU processome, a 6 MDa ribonucleoprotein complex. The SSU processome is required for processing, modifying, and folding the preribosomal RNA (rRNA) to prepare it for incorporation into the mature SSU. Although the protein composition of the SSU processome has been known for some time, the interaction network of the proteins required for its assembly has remained poorly defined. Here, we have used a semi-high-throughput yeast two-hybrid (Y2H) assay and coimmunoprecipitation validation method to produce a high-confidence interactome of SSU processome assembly factors (SPAFs), providing essential insight into SSU assembly and ribosome biogenesis. Further, we used glycerol density-gradient sedimentation to reveal the presence of protein subcomplexes that have not previously been observed. Our work not only provides essential insight into SSU assembly and ribosome biogenesis, but also serves as an important resource for future investigations into how defects in biogenesis and assembly cause congenital disorders of ribosomes known as ribosomopathies.

MicroRNAs and long non-coding RNAs as novel regulators of ribosome biogenesis.

Ribosome biogenesis is the fine-tuned, essential process that generates mature ribosomal subunits and ultimately enables all protein synthesis within a cell. Novel regulators of ribosome biogenesis continue to be discovered in higher eukaryotes. While many known regulatory factors are proteins or small nucleolar ribonucleoproteins, microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) are emerging as a novel modulatory layer controlling ribosome production. Here, we summarize work uncovering non-coding RNAs (ncRNAs) as novel regulators of ribosome biogenesis and highlight their links to diseases of defective ribosome biogenesis. It is still unclear how many miRNAs or lncRNAs are involved in phenotypic or pathological disease outcomes caused by impaired ribosome production, as in the ribosomopathies, or by increased ribosome production, as in cancer. In time, we hypothesize that many more ncRNA regulators of ribosome biogenesis will be discovered, which will be followed by an effort to establish connections between disease pathologies and the molecular mechanisms of this additional layer of ribosome biogenesis control.