Kathleen L. McCann

Genetics. Mysterious ribosomopathies.

Ribosomes are essential in all cell types, yet mutations to ribosomal proteins or assembly factors cause tissue-specific disease. Ribosomes are absolutely essential for life, generating all cellular proteins required for growth. The prevailing thought for many years was that mutations in ribosomal proteins or ribosome assembly factors would be lethal to developing embryos. Complete loss of any single ribosomal protein often leads to embryonic lethality in mice (1). Yet, mutations in ribosomal proteins or ribosome assembly factors result in a puzzling phenomenon—a specific mutation can affect a specific cell type and cause a tissue-specific human disease. What accounts for this tissue proclivity has been a mystery. Why do defects in a macromolecule as ubiquitous and essential as the ribosome cause diseases—ribosomopathies—only in select tissues?

NOL11, implicated in the pathogenesis of North American Indian Childhood Cirrhosis, is required for pre-rRNA transcription and processing

The fundamental process of ribosome biogenesis requires hundreds of factors and takes place in the nucleolus. This process has been most thoroughly characterized in bakers yeast and is generally well conserved from yeast to humans. However, some of the required proteins in yeast are not found in humans, raising the possibility that they have been replaced by functional analogs. Our objective was to identify non-conserved interaction partners for the human ribosome biogenesis factor, hUTP4/Cirhin, since the R565W mutation in the C-terminus of hUTP4/Cirhin was reported to cause North American Indian childhood cirrhosis (NAIC). By screening a yeast two-hybrid cDNA library derived from human liver, and through affinity purification followed by mass spectrometry, we identified an uncharacterized nucleolar protein, NOL11, as an interaction partner for hUTP4/Cirhin. Bioinformatic analysis revealed that NOL11 is conserved throughout metazoans and their immediate ancestors but is not found in any other phylogenetic groups. Co-immunoprecipitation experiments show that NOL11 is a component of the human ribosomal small subunit (SSU) processome. siRNA knockdown of NOL11 revealed that it is involved in the cleavage steps required to generate the mature 18S rRNA and is required for optimal rDNA transcription. Furthermore, abnormal nucleolar morphology results from the absence of NOL11. Finally, yeast two-hybrid analysis shows that NOL11 interacts with the C-terminus of hUTP4/Cirhin and that the R565W mutation partially disrupts this interaction. We have therefore identified NOL11 as a novel protein required for the early stages of ribosome biogenesis in humans. Our results further implicate a role for NOL11 in the pathogenesis of NAIC.

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.

Tissue specific roles for the ribosome biogenesis factor Wdr43 in zebrafish development.

During vertebrate craniofacial development, neural crest cells (NCCs) contribute to most of the craniofacial pharyngeal skeleton. Defects in NCC specification, migration and differentiation resulting in malformations in the craniofacial complex are associated with human craniofacial disorders including Treacher-Collins Syndrome, caused by mutations in TCOF1. It has been hypothesized that perturbed ribosome biogenesis and resulting p53 mediated neuroepithelial apoptosis results in NCC hypoplasia in mouse Tcof1 mutants. However, the underlying mechanisms linking ribosome biogenesis and NCC development remain poorly understood. Here we report a new zebrafish mutant, fantome (fan), which harbors a point mutation and predicted premature stop codon in zebrafish wdr43, the ortholog to yeast UTP5. Although wdr43 mRNA is widely expressed during early zebrafish development, and its deficiency triggers early neural, eye, heart and pharyngeal arch defects, later defects appear fairly restricted to NCC derived craniofacial cartilages. Here we show that the C-terminus of Wdr43, which is absent in fan mutant protein, is both necessary and sufficient to mediate its nucleolar localization and protein interactions in metazoans. We demonstrate that Wdr43 functions in ribosome biogenesis, and that defects observed in fan mutants are mediated by a p53 dependent pathway. Finally, we show that proper localization of a variety of nucleolar proteins, including TCOF1, is dependent on that of WDR43. Together, our findings provide new insight into roles for Wdr43 in development, ribosome biogenesis, and also ribosomopathy-induced craniofacial phenotypes including Treacher-Collins Syndrome.

A divergent Pumilio repeat protein family for pre-rRNA processing and mRNA localization

Significance RNA regulation occurs at many levels including processing to mature forms, subcellular localization, and translation. RNA-binding proteins are crucial to direct and regulate these processes. Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins are RNA-binding proteins formed from eight α-helical repeats [Pumilio (PUM) repeats] that recognize specific mRNA sequences. Previous structural studies revealed characteristic curved structures and sequence specificity unique to these classical PUF proteins. We show here that PUM repeats also form different folds with 11 PUM repeats. Moreover, these proteins, exemplified by human Puf-A and yeast Puf6 proteins, recognize double-stranded RNA or DNA without sequence specificity. Interestingly, Puf-A and Puf6 PUM repeats lack specificity for RNA bases yet use residues at conserved positions on topologically equivalent protein surfaces for new nucleic acid recognition modes. Pumilio/feminization of XX and XO animals (fem)-3 mRNA-binding factor (PUF) proteins bind sequence specifically to mRNA targets using a single-stranded RNA-binding domain comprising eight Pumilio (PUM) repeats. PUM repeats have now been identified in proteins that function in pre-rRNA processing, including human Puf-A and yeast Puf6. This is a role not previously ascribed to PUF proteins. Here we present crystal structures of human Puf-A that reveal a class of nucleic acid-binding proteins with 11 PUM repeats arranged in an “L”-like shape. In contrast to classical PUF proteins, Puf-A forms sequence-independent interactions with DNA or RNA, mediated by conserved basic residues. We demonstrate that equivalent basic residues in yeast Puf6 are important for RNA binding, pre-rRNA processing, and mRNA localization. Thus, PUM repeats can be assembled into alternative folds that bind to structured nucleic acids in addition to forming canonical eight-repeat crescent-shaped RNA-binding domains found in classical PUF proteins.

Long noncoding RNAs as sinks in Prader-Willi syndrome.

In this issue of Molecular Cell, Yin et al. (2012) identify a class of long noncoding RNAs (lncRNAs) and propose a new mechanism as to how they contribute to the pathogenesis of Prader-Willi syndrome.

The molecular basis for ANE syndrome revealed by the large ribosomal subunit processome interactome

ANE syndrome is a ribosomopathy caused by a mutation in an RNA recognition motif of RBM28, a nucleolar protein conserved to yeast (Nop4). While patients with ANE syndrome have fewer mature ribosomes, it is unclear how this mutation disrupts ribosome assembly. Here we use yeast as a model system and show that the mutation confers growth and pre-rRNA processing defects. Recently, we found that Nop4 is a hub protein in the nucleolar large subunit (LSU) processome interactome. Here we demonstrate that the ANE syndrome mutation disrupts Nop4’s hub function by abrogating several of Nop4’s protein-protein interactions. Circular dichroism and NMR demonstrate that the ANE syndrome mutation in RRM3 of human RBM28 disrupts domain folding. We conclude that the ANE syndrome mutation generates defective protein folding which abrogates protein-protein interactions and causes faulty pre-LSU rRNA processing, thus revealing one aspect of the molecular basis of this human disease. DOI: http://dx.doi.org/10.7554/eLife.16381.001

Making Ribosomes: Pre-rRNA Transcription and Processing

Ribosomes are essential, intricate cellular machines that translate mRNA into protein in all cells. The process of building a ribosome is highly complex. The ribosomal RNA (rRNA) has to be properly synthesized, processed, and assembled with ribosomal proteins in the cell nucleolus. Studies carried out in yeast have significantly increased our understanding of the mechanisms driving ribosome assembly in eukaryotes. This chapter will briefly review ribosome assembly in Saccharomyces cerevisiae and discuss what is known about ribosome assembly in other yeast species.

Diverse Regulators of Human Ribosome Biogenesis Discovered by Changes in Nucleolar Number

SUMMARY Ribosome biogenesis is a highly regulated, essential cellular process. Although studies in yeast have established some of the biological principles of ribosome biogenesis, many of the intricacies of its regulation in higher eukaryotes remain unknown. To understand how ribosome biogenesis is globally integrated in human cells, we conducted a genome-wide siRNA screen for regulators of nucleolar number. We found 139 proteins whose depletion changed the number of nucleoli per nucleus from 2–3 to only 1 in human MCF10A cells. Follow-up analyses on 20 hits found many (90%) to be essential for the nucleolar functions of rDNA transcription (7), pre-ribosomal RNA (pre-rRNA) processing (16), and/or global protein synthesis (14). This genome-wide analysis exploits the relationship between nucleolar number and function to discover diverse cellular pathways that regulate the making of ribosomes and paves the way for further exploration of the links between ribosome biogenesis and human disease.

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).