Rbp95 binds to 25S rRNA helix H95 and cooperates with the Npa1 complex during early pre-60S particle maturation.

Eukaryotic ribosome synthesis involves more than 200 assembly factors, which promote ribosomal RNA (rRNA) processing, modification and folding, and assembly of ribosomal proteins. The formation and maturation of the earliest pre-60S particles requires structural remodeling by the Npa1 complex, but is otherwise still poorly understood. Here, we introduce Rbp95 (Ycr016w), a constituent of early pre-60S particles, as a novel ribosome assembly factor. We show that Rbp95 is both genetically and physically linked to most Npa1 complex members and to ribosomal protein Rpl3. We demonstrate that Rbp95 is an RNA-binding protein containing two independent RNA-interacting domains. In vivo, Rbp95 associates with helix H95 in the 3' region of the 25S rRNA, in close proximity to the binding sites of Npa1 and Rpl3. Additionally, Rbp95 interacts with several snoRNAs. The absence of Rbp95 results in alterations in the protein composition of early pre-60S particles. Moreover, combined mutation of Rbp95 and Npa1 complex members leads to a delay in the maturation of early pre-60S particles. We propose that Rbp95 acts together with the Npa1 complex during early pre-60S maturation, potentially by promoting pre-rRNA folding events within pre-60S particles.

MiRNAs and snoRNAs in Bone Metastasis: Functional Roles and Clinical Potential.

Bone is a frequent site of metastasis. Bone metastasis is associated with a short-term prognosis in cancer patients, and current treatments aim to slow its growth, but are rarely curative. Thus, revealing molecular mechanisms that explain why metastatic cells are attracted to the bone micro-environment, and how they successfully settle in the bone marrow-taking advantage over bone resident cells-and grow into macro-metastasis, is essential to propose new therapeutic approaches. MicroRNAs and snoRNAs are two classes of small non-coding RNAs that post-transcriptionally regulate gene expression. Recently, microRNAs and snoRNAs have been pointed out as important players in bone metastasis by (i) preparing the pre-metastatic niche, directly and indirectly affecting the activities of osteoclasts and osteoblasts, (ii) promoting metastatic properties within cancer cells, and (iii) acting as mediators within cells to support cancer cell growth in bone. This review aims to highlight the importance of microRNAs and snoRNAs in metastasis, specifically in bone, and how their roles can be linked together. We then discuss how microRNAs and snoRNAs are secreted by cancer cells and be found as extracellular vesicle cargo. Finally, we provide evidence of how microRNAs and snoRNAs can be potential therapeutic targets, at least in pre-clinical settings, and how their detection in liquid biopsies can be a useful diagnostic and/or prognostic biomarker to predict the risk of relapse in cancer patients.

2'O-Ribose Methylation of Ribosomal RNAs: Natural Diversity in Living Organisms, Biological Processes, and Diseases.

Recent findings suggest that ribosomes, the translational machineries, can display a distinct composition depending on physio-pathological contexts. Thanks to outstanding technological breakthroughs, many studies have reported that variations of rRNA modifications, and more particularly the most abundant rRNA chemical modification, the rRNA 2'O-ribose methylation (2'Ome), intrinsically occur in many organisms. In the last 5 years, accumulating reports have illustrated that rRNA 2'Ome varies in human cell lines but also in living organisms (yeast, plant, zebrafish, mouse, human) during development and diseases. These rRNA 2'Ome variations occur either within a single cell line, organ, or patient's sample (i.e., intra-variability) or between at least two biological conditions (i.e., inter-variability). Thus, the ribosomes can tolerate the absence of 2'Ome at some specific positions. These observations question whether variations in rRNA 2'Ome could provide ribosomes with particular translational regulatory activities and functional specializations. Here, we compile recent studies supporting the heterogeneity of ribosome composition at rRNA 2'Ome level and provide an overview of the natural diversity in rRNA 2'Ome that has been reported up to now throughout the kingdom of life. Moreover, we discuss the little evidence that suggests that variations of rRNA 2'Ome can effectively impact the ribosome activity and contribute to the etiology of some human diseases.

Coupling Between Production of Ribosomal RNA and Maturation: Just at the Beginning.

Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35S/47S in yeast/human) is achieved by up to hundreds of RNA polymerase I (Pol I) enzymes simultaneously transcribing a single rRNA gene. In this review, we present recent advances in understanding the coupling between rRNA production and nascent rRNA folding. Mapping of the distribution of Pol I along ribosomal DNA at nucleotide resolution, using either native elongating transcript sequencing (NET-Seq) or crosslinking and analysis of cDNAs (CRAC), revealed frequent Pol I pausing, and CRAC results revealed a direct coupling between pausing and nascent RNA folding. High density of Pol I per gene imposes topological constraints that establish a defined pattern of polymerase distribution along the gene, with a persistent spacing between transcribing enzymes. RNA folding during transcription directly acts as an anti-pausing mechanism, implying that proper folding of the nascent rRNA favors elongation in vivo. Defects in co-transcriptional folding of rRNA are likely to induce Pol I pausing. We propose that premature termination of transcription, at defined positions, can control rRNA production in vivo.

The RNA helicase Dbp7 promotes domain V/VI compaction and stabilization of inter-domain interactions during early 60S assembly.

Early pre-60S ribosomal particles are poorly characterized, highly dynamic complexes that undergo extensive rRNA folding and compaction concomitant with assembly of ribosomal proteins and exchange of assembly factors. Pre-60S particles contain numerous RNA helicases, which are likely regulators of accurate and efficient formation of appropriate rRNA structures. Here we reveal binding of the RNA helicase Dbp7 to domain V/VI of early pre-60S particles in yeast and show that in the absence of this protein, dissociation of the Npa1 scaffolding complex, release of the snR190 folding chaperone, recruitment of the A3 cluster factors and binding of the ribosomal protein uL3 are impaired. uL3 is critical for formation of the peptidyltransferase center (PTC) and is responsible for stabilizing interactions between the 5' and 3' ends of the 25S, an essential pre-requisite for subsequent pre-60S maturation events. Highlighting the importance of pre-ribosome remodeling by Dbp7, our data suggest that in the absence of Dbp7 or its catalytic activity, early pre-ribosomal particles are targeted for degradation.

Association of snR190 snoRNA chaperone with early pre-60S particles is regulated by the RNA helicase Dbp7 in yeast.

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit.

Antitumor activity of the synthetic retinoid ST1926 on primary effusion lymphoma in vitro and in vivo models.

Primary effusion lymphoma (PEL) is a rare B-cell neoplasm, associated with Kaposi sarcoma-associated herpes virus/human herpes virus-8 (KSHV/HHV-8), arising as malignant effusions in body cavities. PEL cells do not harbor conventional genetic cancer mutations; however, their oncogenesis is mainly attributed to HHV-8 latent genes. Treatment strategies are inefficient resulting in poor prognosis of PEL patients, stressing the need for new effective therapy. ST1926 is a synthetic retinoid with favorable antitumor properties and no cross-resistance with the natural retinoid, all-trans retinoic acid. ST1926 has shown potent apoptotic activities on a variety of solid tumors and hematologic malignancies in in vitro and in vivo models. In the present study we elucidated the antitumor activities and underlying molecular mechanism of ST1926 using in vitro, ex vivo, and in vivo PEL preclinical models. ST1926, at sub­micromolar concentrations, displayed potent antiproliferative effects on PEL cell lines and malignant ascites. Furthermore, ST1926 treatment of PEL cells and ascites resulted in their accumulation in the sub-G1 region, S phase cell cycle arrest, early DNA damage, PARP cleavage and p53 activation including the upregulation of its target genes p21 and Bax. However, ST1926 did not significantly modulate HHV-8 latent viral transcripts. Importantly, ST1926 delayed formation of ascites and enhanced survival of PEL mice. These results highlight the therapeutic potential of ST1926 in combination with drugs that target HHV-8 in PEL patients.