Nol9 Is a Spatial Regulator for the Human ITS2 Pre-rRNA Endonuclease–Kinase Complex

The ribosome plays a universal role in translating the cellular proteome. Defects in the ribosome assembly factor Las1L are associated with congenital lethal motor neuron disease and X-linked intellectual disability disorders, yet its role in processing precursor ribosomal RNA (pre-rRNA) is largely unclear. The Las1L endoribonuclease associates with the Nol9 polynucleotide kinase to form the internal transcribed spacer 2 (ITS2) pre-rRNA endonuclease–kinase machinery. Together, Las1L–Nol9 catalyzes RNA cleavage and phosphorylation to mark the ITS2 for degradation. While ITS2 processing is critical for the production of functional ribosomes, the regulation of mammalian Las1L–Nol9 remains obscure. Here we characterize the human Las1L–Nol9 complex and identify critical molecular features that regulate its assembly and spatial organization. We establish that Las1L and Nol9 form a higher-order complex and identify the regions responsible for orchestrating this intricate architecture. Structural analysis by high-resolution imaging defines the intricate spatial pattern of Las1L–Nol9 within the nucleolar sub-structure linked with late pre-rRNA processing events. Furthermore, we uncover a Nol9-encoded nucleolar localization sequence that is responsible for nucleolar transport of the assembled Las1L–Nol9 complex. Together, these data provide a mechanism for the assembly and nucleolar localization of the human ITS2 pre-rRNA endonuclease–kinase complex.     

BCCIP is required for nucleolar recruitment of eIF6 and 12S pre-rRNA production during 60S ribosome biogenesis

Ribosome biogenesis is a fundamental process required for cell proliferation. Although evolutionally conserved, the mammalian ribosome assembly system is more complex than in yeasts. BCCIP was originally identified as a BRCA2 and p21 interacting protein. A partial loss of BCCIP function was sufficient to trigger genomic instability and tumorigenesis. However, a complete deletion of BCCIP arrested cell growth and was lethal in mice. Here, we report that a fraction of mammalian BCCIP localizes in the nucleolus and regulates 60S ribosome biogenesis. Both abrogation of BCCIP nucleolar localization and impaired BCCIP–eIF6 interaction can compromise eIF6 recruitment to the nucleolus and 60S ribosome biogenesis. BCCIP is vital for a pre-rRNA processing step that produces 12S pre-rRNA, a precursor to the 5.8S rRNA. However, a heterozygous Bccip loss was insufficient to impair 60S biogenesis in mouse embryo fibroblasts, but a profound reduction of BCCIP was required to abrogate its function in 60S biogenesis. These results suggest that BCCIP is a critical factor for mammalian pre-rRNA processing and 60S generation and offer an explanation as to why a subtle dysfunction of BCCIP can be tumorigenic but a complete depletion of BCCIP is lethal.     

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.     

Ribosome biogenesis requires a highly diverged XRN family 5′→3′ exoribonuclease for rRNA processing in Trypanosoma brucei

Although biogenesis of ribosomes is a crucial process in all organisms and is thus well conserved, Trypanosoma brucei ribosome biogenesis, of which maturation of rRNAs is an early step, has multiple points of divergence. Our aim was to determine whether in the processing of the pre-rRNA precursor molecule, 5′→3′ exoribonuclease activity in addition to endonucleolytic cleavage is necessary in T. brucei as in other organisms. Our approach initiated with the bioinformatic identification of a putative 5′→3′ exoribonuclease, XRNE, which is highly diverged from the XRN2/Rat1 enzyme responsible for rRNA processing in other organisms. Tagging this protein in vivo allowed us to classify XRNE as nucleolar by indirect immunofluorescence and identify by copurification interacting proteins, many of which were ribosomal proteins, ribosome biogenesis proteins, and/or RNA processing proteins. To determine whether XRNE plays a role in ribosome biogenesis in procyclic form cells, we inducibly depleted the protein by RNA interference. This resulted in the generation of aberrant preprocessed 18S rRNA and 5′ extended 5.8S rRNA, implicating XRNE in rRNA processing. Polysome profiles of XRNE-depleted cells demonstrated abnormal features including an increase in ribosome small subunit abundance, a decrease in large subunit abundance, and defects in polysome assembly. Furthermore, the 5′ extended 5.8S rRNA in XRNE-depleted cells was observed in the large subunit, monosomes, and polysomes in this gradient. Therefore, the function of XRNE in rRNA processing, presumably due to exonucleolytic activity very early in ribosome biogenesis, has consequences that persist throughout all biogenesis stages.     

Proteomic analysis of human Nop56p-associated pre-ribosomal ribonucleoprotein complexes. Possible link between Nop56p and the nucleolar protein treacle responsible for Treacher Collins syndrome

Nop56p is a component of the box C/D small nucleolar ribonucleoprotein complexes that direct 2'-O-methylation of pre-rRNA during its maturation. Genetic analyses in yeast have shown that Nop56p plays important roles in the early steps of pre-rRNA processing. However, its precise function remains elusive, especially in higher eukaryotes. Here we describe the proteomic characterization of human Nop56p (hNop56p)-associated pre-ribosomal ribonucleoprotein complexes. Mass spectrometric analysis of purified pre-ribosomal ribonucleoprotein complexes identified 61 ribosomal proteins, 16 trans-acting factors probably involved in ribosome biogenesis, and 29 proteins whose function in ribosome biogenesis is unknown. Identification of pre-rRNA species within hNop56p-associated pre-ribosomal ribonucleoprotein complexes, coupled with the known functions of yeast orthologs of the probable trans-acting factors identified in human, demonstrated that hNop56p functions in the early to middle stages of 60 S subunit synthesis in human cells. Interestingly, the nucleolar phosphoprotein treacle, which is responsible for the craniofacial disorder associated with Treacher Collins syndrome, was found to be a constituent of hNop56p-associated pre-rRNP complexes. The association of hNop56p and treacle within the complexes was independent of rRNA integrity, indicating a direct interaction. In addition, the protein compositions of the treacle-associated and hNop56p-associated pre-ribosomal ribonucleoprotein complexes were very similar, suggesting functional similarities between these two complexes with respect to ribosome biogenesis in human cells.     

The carboxy-terminal extension of yeast ribosomal protein S14 is necessary for maturation of 43S preribosomes

Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants. These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins. These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway-the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3' end of 18S rRNA, consistent with a role for rpS14 in 3' end processing of 20S pre-rRNA.     

Dbp10p, a putative RNA helicase from Saccharomyces cerevisiae, is required for ribosome biogenesis

Ribosome biogenesis requires, in addition to rRNA molecules and ribosomal proteins, a multitude of trans-acting factors. Recently it has become clear that in the yeast Saccharomyces cerevisiae many RNA helicases of the DEAD-box and related families are involved in ribosome biogenesis. Here we show that the previously uncharacterised open reading frame YDL031w (renamed DBP10 for DEAD-box protein 10) encodes an essential putative RNA helicase that is required for accurate ribosome biogenesis. Genetic depletion of Dbp10p results in a deficit in 60S ribosomal subunits and an accumulation of half-mer polysomes. Furthermore, pulse-chase analyses of pre-rRNA processing reveal a strong delay in the maturation of 27SB pre-rRNA intermediates into 25S rRNA and 7S pre-rRNA. Northern blot analyses indicate that this delay leads to higher steady-state levels of 27SB species and reduced steady-state levels of 7S pre-rRNA and 25S/5.8S mature rRNAs, thus explaining the final deficit in 60S subunit and the formation of half-mer polysomes. Consistent with a direct role in ribosome biogenesis, Dbp10p was found to be located predominantly in the nucleolus.