Ribosome biogenesis factors-from names to functions.

The assembly of ribosomal subunits is a highly orchestrated process that involves a huge cohort of accessory factors. Most eukaryotic ribosome biogenesis factors were first identified by genetic screens and proteomic approaches of pre-ribosomal particles in Saccharomyces cerevisiae. Later, research on human ribosome synthesis not only demonstrated that the requirement for many of these factors is conserved in evolution, but also revealed the involvement of additional players, reflecting a more complex assembly pathway in mammalian cells.

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.

Structural basis for the final steps of human 40S ribosome maturation.

Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins. Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB1.

USP16 counteracts mono-ubiquitination of RPS27a and promotes maturation of the 40S ribosomal subunit.

Establishment of translational competence represents a decisive cytoplasmic step in the biogenesis of 40S ribosomal subunits. This involves final 18S rRNA processing and release of residual biogenesis factors, including the protein kinase RIOK1. To identify novel proteins promoting the final maturation of human 40S subunits, we characterized pre-ribosomal subunits trapped on RIOK1 by mass spectrometry, and identified the deubiquitinase USP16 among the captured factors.

Genome-wide RNAi Screening Identifies Protein Modules Required for 40S Subunit Synthesis in Human Cells.

Ribosome biogenesis is a highly complex process requiring many assisting factors. Studies in yeast have yielded comprehensive knowledge of the cellular machinery involved in this process. However, many aspects of ribosome synthesis are different in higher eukaryotes, and the global set of mammalian ribosome biogenesis factors remains unexplored.

The NF45/NF90 Heterodimer Contributes to the Biogenesis of 60S Ribosomal Subunits and Influences Nucleolar Morphology.

The interleukin enhancer binding factors ILF2 (NF45) and ILF3 (NF90/NF110) have been implicated in various cellular pathways, such as transcription, microRNA (miRNA) processing, DNA repair, and translation, in mammalian cells. Using tandem affinity purification, we identified human NF45 and NF90 as components of precursors to 60S (pre-60S) ribosomal subunits. NF45 and NF90 are enriched in nucleoli and cosediment with pre-60S ribosomal particles in density gradient analysis.

The shelterin component TPP1 is a binding partner and substrate for the deubiquitinating enzyme USP7.

The telomeric shelterin component TPP1 has critical functions in telomeric protein complex assembly and telomerase recruitment and regulation. Here we identify USP7 as a novel interacting protein of the oligonucleotide/oligosaccharide-binding fold of TPP1, which was previously known to recruit telomerase to telomeres. We identify amino acids in TPP1 and USP7 that are critical for their interaction and multiple lysines within TPP1 that are oligo-ubiquitinated and deubiquitinated by USP7.

CK1δ and CK1ε are components of human 40S subunit precursors required for cytoplasmic 40S maturation.

Biogenesis of 40S pre-ribosomal subunits requires many trans-acting factors, among them several protein kinases. In this study, we show that the human casein kinase 1 (CK1) isoforms δ and ε are required for cytoplasmic maturation steps of 40S subunit precursors. We show that both CK1δ and CK1ε isoforms are components of pre-40S subunits, on which they phosphorylate the ribosome biogenesis factors ENP1/BYSL and LTV1.

A three-state model for the regulation of telomerase by TERRA and hnRNPA1.

Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeat-containing RNA (TERRA), a large non-coding RNA, which forms an integral part of telomeric heterochromatin. In vitro, naked TERRA molecules are efficient inhibitors of human telomerase, base-pairing via their 5'-UUAGGG-3' repeats with the template sequence of telomerase RNA, in addition to contacting the telomerase reverse transcriptase protein subunit. In vivo, however, TERRA-mediated inhibition of telomerase can be prevented by unknown mechanisms.

The kinase activity of human Rio1 is required for final steps of cytoplasmic maturation of 40S subunits.

RIO proteins form a conserved family of atypical protein kinases. Humans possess three distinct RIO kinases-hRio1, hRio2, and hRio3, of which only hRio2 has been characterized with respect to its role in ribosomal biogenesis. Here we show that both hRio1 and hRio3, like hRio2, are associated with precursors of 40S ribosomal subunits in human cells.

Tandem affinity purification combined with inducible shRNA expression as a tool to study the maturation of macromolecular assemblies.

Tandem affinity purification (TAP) is an efficient method for the purification and characterization of large macromolecular complexes. To elucidate the role of specific components of such complexes, it is important to address the question of how loss of a specific factor affects complex composition. Here, we introduce a method that combines TAP of large macromolecular assemblies with inducible shRNA-mediated protein depletion in human somatic cells.

A protein inventory of human ribosome biogenesis reveals an essential function of exportin 5 in 60S subunit export.

The assembly of ribosomal subunits in eukaryotes is a complex, multistep process so far mostly studied in yeast. In S. cerevisiae, more than 200 factors including ribosomal proteins and trans-acting factors are required for the ordered assembly of 40S and 60S ribosomal subunits.

Anguinomycins and derivatives: total syntheses, modeling, and biological evaluation of the inhibition of nucleocytoplasmic transport.

The preparation of the polyketide natural products anguinomycin C and D is reported based on key steps such as Negishi stereoinversion cross coupling, Jacobsen Cr(III)-catalyzed Hetero Diels-Alder reaction, Evans B-mediated syn-aldol chemistry, and B-alkyl Suzuki-Miyaura cross coupling. The configuration of both natural products was established as (5R,10R,16R,18S,19R,20S). Biological evaluation demonstrated that these natural products are inhibitors of the nuclear export receptor CRM1, leading to shutdown of CRM1-mediated nuclear protein export at concentrations above 10 nM.

Distinct cytoplasmic maturation steps of 40S ribosomal subunit precursors require hRio2.

During their biogenesis, 40S ribosomal subunit precursors are exported from the nucleus to the cytoplasm, where final maturation occurs. In this study, we show that the protein kinase human Rio2 (hRio2) is part of a late 40S preribosomal particle in human cells. Using a novel 40S biogenesis and export assay, we analyzed the contribution of hRio2 to late 40S maturation.

Nuclear export and cytoplasmic maturation of ribosomal subunits.

Based on the characterization of ribosome precursor particles and associated trans-acting factors, a biogenesis pathway for the 40S and 60S subunits has emerged. After nuclear synthesis and assembly steps, pre-ribosomal subunits are exported through the nuclear pore complex in a Crm1- and RanGTP-dependent manner. Subsequent cytoplasmic biogenesis steps of pre-60S particles include the facilitated release of several non-ribosomal proteins, yielding fully functional 60S subunits.