Anthony Henras

Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs

The small nucleolar ribonucleoprotein particles containing H/ACA‐type snoRNAs (H/ACA snoRNPs) are crucial trans‐acting factors intervening in eukaryotic ribosome biogenesis. Most of these particles generate the site‐specific pseudouridylation of rRNAs while a subset are required for 18S rRNA synthesis. To understand in detail how these particles carry out these functions, all of their protein components have to be characterized. For that purpose, we have affinity‐purified complexes containing epitope‐tagged Gar1p protein, previously shown to be part of H/ACA snoRNPs. Under the conditions used, three polypeptides of 65, 22 and 10 kDa apparent molecular weight specifically copurify with epitope‐tagged Gar1p. The 22 and 10 kDa polypeptides were identified as Nhp2p and a novel protein we termed Nop10p, respectively. Both proteins are conserved, essential and present in the dense fibrillar component of the nucleolus. Nhp2p and Nop10p are specifically associated with all H/ACA snoRNAs and are essential to the function of H/ACA snoRNPs. Cells lacking Nhp2p or Nop10p are impaired in global rRNA pseudouridylation and in the A1 and A2 cleavage steps of the pre‐rRNA required for the synthesis of mature 18S rRNA. These phenotypes are probably a direct consequence of the instability of H/ACA snoRNAs and Gar1p observed in cells deprived of Nhp2p or Nop10p. Our results suggest that Nhp2p and Nop10p, together with Cbf5p, constitute the core of H/ACA snoRNPs.

Accumulation of H/ACA snoRNPs depends on the integrity of the conserved central domain of the RNA-binding protein Nhp2p

Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. How box H/ACA snoRNPs are assembled remains unknown. Here we show that yeast Nhp2p, a core component of these particles, directly binds RNA. In vitro, Nhp2p interacts with high affinity with RNAs containing irregular stem-loop structures but shows weak affinity for poly(A), poly(C) or for double-stranded RNAs. The central region of Nhp2p is believed to function as an RNA-binding domain, since it is related to motifs found in various RNA-binding proteins. Removal of two amino acids that shortens a putative beta-strand element within Nhp2p central domain impairs the ability of the protein to interact with H/ACA snoRNAs in cell extracts. In vivo, this deletion prevents cell viability and leads to a strong defect in the accumulation of H/ACA snoRNAs and Gar1p. These data suggest that proper direct binding of Nhp2p to H/ACA snoRNAs is required for the assembly of H/ACA snoRNPs and hence for the stability of some of their components. In addition, we show that converting a highly conserved glycine residue (G(59)) within Nhp2p central domain to glutamate significantly reduces cell growth at 30 and 37 degrees C. Remarkably, this modification affects the steady-state levels of H/ACA snoRNAs and the strength of Nhp2p association with these RNAs to varying degrees, depending on the nature of the H/ACA snoRNA. Finally, we show that the modified Nhp2p protein whose interaction with H/ACA snoRNAs is impaired cannot accumulate in the nucleolus, suggesting that only the assembled H/ACA snoRNP particles can be efficiently retained in the nucleolus.

Utp23p is required for dissociation of snR30 small nucleolar RNP from preribosomal particles

Yeast snR30 is an essential box H/ACA small nucleolar RNA (snoRNA) that promotes 18S rRNA processing through forming transient base-pairing interactions with the newly synthesized 35S pre-rRNA. By using a novel tandem RNA affinity selection approach, followed by coimmunoprecipitation and in vivo cross-linking experiments, we demonstrate that in addition to the four H/ACA core proteins, Cbf5p, Nhp2p, Nop10p and Gar1p, a fraction of snR30 specifically associates with the Utp23p and Kri1p nucleolar proteins. Depletion of Utp23p and Kri1p has no effect on the accumulation and recruitment of snR30 to the nascent pre-ribosomes. However, in the absence of Utp23p, the majority of snR30 accumulates in large pre-ribosomal particles. The retained snR30 is not base-paired with the 35S pre-rRNA, indicating that its aberrant tethering to nascent preribosomes is likely mediated by pre-ribosomal protein(s). Thus, Utp23p may promote conformational changes of the pre-ribosome, essential for snR30 release. Neither Utp23p nor Kri1p is required for recruitment of snR30 to the nascent pre-ribosome. On the contrary, depletion of snR30 prevents proper incorporation of both Utp23p and Kri1p into the 90S pre-ribosome containing the 35S pre-rRNA, indicating that snR30 plays a central role in the assembly of functionally active small subunit processome.

Evolutionarily conserved function of RRP36 in early cleavages of the pre-rRNA and production of the 40S ribosomal subunit.

ABSTRACT Ribosome biogenesis in eukaryotes is a major cellular activity mobilizing the products of over 200 transcriptionally coregulated genes referred to as the rRNA and ribosome biosynthesis regulon. We investigated the function of an essential, uncharacterized gene of this regulon, renamed RRP36. We show that the Rrp36p protein is nucleolar and interacts with 90S and pre-40S preribosomal particles. Its depletion affects early cleavages of the 35S pre-rRNA and results in a rapid decrease in mature 18S rRNA levels. Rrp36p is a novel component of the 90S preribosome, the assembly of which has been suggested to result from the stepwise incorporation of several modules, including the tUTP/UTP-A, PWP2/UTP-B, and UTP-C subcomplexes. We show that Rrp36p depletion does not impair the incorporation of these subcomplexes and the U3 small nucleolar RNP into preribosomes. In contrast, depletion of components of the UTP-A or UTP-B modules, but not Rrp5p, prevents Rrp36p recruitment and reduces its accumulation levels. In parallel, we studied the human orthologue of Rrp36p in HeLa cells, and we show that the function of this protein in early cleavages of the pre-rRNA has been conserved through evolution in eukaryotes.

Crucial role of the Rcl1p–Bms1p interaction for yeast pre-ribosomal RNA processing

The essential Rcl1p and Bms1p proteins form a complex required for 40S ribosomal subunit maturation. Bms1p is a GTPase and Rcl1p has been proposed to catalyse the endonucleolytic cleavage at site A2 separating the pre-40S and pre-60S maturation pathways. We determined the 2.0 Å crystal structure of Bms1p associated with Rcl1p. We demonstrate that Rcl1p nuclear import depends on Bms1p and that the two proteins are loaded into pre-ribosomes at a similar stage of the maturation pathway and remain present within pre-ribosomes after cleavage at A2. Importantly, GTP binding to Bms1p is not required for the import in the nucleus nor for the incorporation of Rcl1p into pre-ribosomes, but is essential for early pre-rRNA processing. We propose that GTP binding to Bms1p and/or GTP hydrolysis may induce conformational rearrangements within the Bms1p-Rcl1p complex allowing the interaction of Rcl1p with its RNA substrate.

Mammalian HCA66 protein is required for both ribosome synthesis and centriole duplication

Ribosome production, one of the most energy-consuming biosynthetic activities in living cells, is adjusted to growth conditions and coordinated with the cell cycle. Connections between ribosome synthesis and cell cycle progression have been described, but the underlying mechanisms remain only partially understood. The human HCA66 protein was recently characterized as a component of the centrosome, the major microtubule-organizing center (MTOC) in mammalian cells, and was shown to be required for centriole duplication and assembly of the mitotic spindle. We show here that HCA66 is also required for nucleolar steps of the maturation of the 40S ribosomal subunit and therefore displays a dual function. Overexpression of a dominant negative version of HCA66, accumulating at the centrosome but absent from the nucleoli, alters centrosome function but has no effect on pre-rRNA processing, suggesting that HCA66 acts independently in each process. In yeast and HeLa cells, depletion of MTOC components does not impair ribosome synthesis. Hence our results suggest that both in yeast and human cells, assembly of a functional MTOC and ribosome synthesis are not closely connected processes.

Crosstalk Between Ribosome Synthesis and Cell Cycle Progression and Its Potential Implications in Human Diseases

The connections between ribosome synthesis and cell cycle progression have been the focus of numerous studies and two conserved features emerge from the reported data. The first one is that ribosome synthesis is monitored during the G1 phase of the cell cycle through a surveillance mechanism that communicates with and regulates the G1–S transition machinery. In mammalian cells, this mechanism implicates p53 and the current model proposes that in response to defects in ribosome biogenesis, several ribosomal proteins and ribosome synthesis factors inhibit the p53 ubiquitin ligase Mdm2 and thereby stabilize p53, which results in cell cycle arrest in G1 and/or apoptosis. The second emerging feature, the biological significance of which remains unclear, is that several factors involved in ribosome synthesis are also directly required for specific stages of cell cycle progression, in particular S phase or mitosis. In the past decade, mutations in genes encoding ribosome components or ribosome synthesis factors have been associated with several human diseases, collectively referred to as “ribosomopathies,” characterized by recurrent symptoms including hematopoietic defects, developmental anomalies, and cancer predisposition. One current hypothesis is that these symptoms result from impaired proliferation due to p53 activation in response to ribosome synthesis defects.

A synthetic microbial consortium to detect and kill Vibrio cholerae

Vibrio cholerae is nowadays still problematic in several countries which are exposed to recurrent disease outbreaks. The current disease detection and treatment methods are efficient so this approach focused on the prevention of the disease. Indeed, current solutions are not efficient enough to deal with this situation. As V. cholerae which infects more than a million people each year is usually found in water, a synthetic microbial consortium was designed to detect and kill efficiently the bacteria in water. This work shows that Vibrio harveyi, a non-pathogenic strain to human, can be an efficient detector of V. cholerae. Moreover, it proves that Pichia pastoris, a yeast, can efficiently produce a novel antimicrobial peptide coming from the crocodile Crocodylus siamensis (i.e D-NY15) and that this peptide has a killing action towards V. cholerae. This study also shows that a communication between a prokaryote (Vibrio harveyi) and an eukaryote (Pichia pastoris) may be possible.

Vasohibin1, a new IRES trans-acting factor for sequential induction of angiogenic factors in hypoxia

Hypoxia, a major inducer of angiogenesis, is known to trigger major changes of gene expression at the transcriptional level. Furthermore, global protein synthesis is blocked while internal ribosome entry sites (IRES) allow specific mRNAs to be translated. Here we report the transcriptome and translatome signatures of (lymph)angiogenic genes in hypoxic HL-1 cardiomyocytes: most genes are not induced at the transcriptome-, but at the translatome level, including all IRES-containing mRNAs. Our data reveal sequential activation of (lymph)angiogenic mRNA IRESs in early hypoxia. We identify vasohibin1 (VASH1) as an IRES trans-acting factor (ITAF) able to activate the earliest induced FGF1 IRES while it inhibits the other IRESs. Thus this new ITAF may have opposite effects on IRES activities in the steps of early hypoxia. These data suggest that IRESome composition varies at different stages of hypoxia, leading to sequential induction of (lymph)angiogenic factors required to form new functional vessels in ischemic heart.