Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing

Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3′-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.     

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.     

植物核糖体应激响应机制研究进展

核仁是真核细胞中重要的核结构,核糖体发生最初在核仁中进行,该过程涉及一系列复杂的反应,需要许多核仁相关因子参与。核糖体生物发生出现异常通常引起核仁结构紊乱,并导致细胞周期阻滞、细胞衰老甚至凋亡。核糖体应激响应机制在哺乳动物细胞中研究得较为深入,但在植物细胞中尚不明晰。尽管如此,人们逐渐发现某些植物特有的NAC转录因子家族成员在植物细胞中可能参与包括核糖体应激在内的多种胞内应激响应过程。此外,前期研究发现生长素系统与核糖体生物合成之间存在一种相互协调机制来调控植物发育。该文结合哺乳动物细胞中已知的核糖体应激响应通路,探讨植物细胞潜在的核糖体应激机制。     

Active regulator of SIRT1 is required for ribosome biogenesis and function

Active regulator of SIRT1 (AROS) binds and upregulates SIRT1, an NAD⁺-dependent deacetylase. In addition, AROS binds RPS19, a structural ribosomal protein, which also functions in ribosome biogenesis and is implicated in multiple disease states. The significance of AROS in relation to ribosome biogenesis and function is unknown. Using human cells, we now show that AROS localizes to (i) the nucleolus and (ii) cytoplasmic ribosomes. Co-localization with nucleolar proteins was verified by confocal immunofluorescence of endogenous protein and confirmed by AROS depletion using RNAi. AROS association with cytoplasmic ribosomes was analysed by sucrose density fractionation and immunoprecipitation, revealing that AROS selectively associates with 40S ribosomal subunits and also with polysomes. RNAi-mediated depletion of AROS leads to deficient ribosome biogenesis with aberrant precursor ribosomal RNA processing, reduced 40S subunit ribosomal RNA and 40S ribosomal proteins (including RPS19). Together, this results in a reduction in 40S subunits and translating polysomes, correlating with reduced overall cellular protein synthesis. Interestingly, knockdown of AROS also results in a functionally significant increase in eIF2α phosphorylation. Overall, our results identify AROS as a factor with a role in both ribosome biogenesis and ribosomal function.     

The function of RH22, a DEAD RNA helicase, in the biogenesis of the 50S ribosomal subunits of Arabidopsis chloroplasts

The chloroplast ribosome is a large and dynamic ribonucleoprotein machine that is composed of the 30S and 50S subunits. Although the components of the chloroplast ribosome have been identified in the last decade, the molecular mechanisms driving chloroplast ribosome biogenesis remain largely elusive. Here, we show that RNA helicase 22 (RH22), a putative DEAD RNA helicase, is involved in chloroplast ribosome assembly in Arabidopsis (Arabidopsis thaliana). A loss of RH22 was lethal, whereas a knockdown of RH22 expression resulted in virescent seedlings with clear defects in chloroplast ribosomal RNA (rRNA) accumulation. The precursors of 23S and 4.5S, but not 16S, rRNA accumulated in rh22 mutants. Further analysis showed that RH22 was associated with the precursors of 50S ribosomal subunits. These results suggest that RH22 may function in the assembly of 50S ribosomal subunits in chloroplasts. In addition, RH22 interacted with the 50S ribosomal protein RPL24 through yeast two-hybrid and pull-down assays, and it was also bound to a small 23S rRNA fragment encompassing RPL24-binding sites. This action of RH22 may be similar to, but distinct from, that of SrmB, a DEAD RNA helicase that is involved in the ribosomal assembly in Escherichia coli, which suggests that DEAD RNA helicases and rRNA structures may have coevolved with respect to ribosomal assembly and function.     

Coordination of Ribosomal Protein and Ribosomal RNA Gene Expression in Response to TOR Signaling

Cells grow in response to nutrients or growth factors, whose presence is detected and communicated by elaborate signaling pathways. Protein kinases play crucial roles in processes such as cell cycle progression and gene expression, and misregulation of such pathways has been correlated with various diseased states. Signals intended to promote cell growth converge on ribosome biogenesis, as the ability to produce cellular proteins is intimately tied to cell growth. Part of the response to growth signals is therefore the coordinate expression of genes encoding ribosomal RNA (rRNA) and ribosomal proteins (RP). A key player in regulating cell growth is the Target of Rapamycin (TOR) kinase, one of the gatekeepers that prevent cell cycle progression from G1 to S under conditions of nutritional stress. TOR is structurally and functionally conserved in all eukaryotes. Under favorable growth conditions, TOR is active and cells maintain a robust rate of ribosome biogenesis, translation initiation and nutrient import. Under stress conditions, TOR signaling is suppressed, leading to cell cycle arrest, while the failure of TOR to respond appropriately to environmental or nutritional signals leads to uncontrolled cell growth. Emerging evidence from Saccharomyces cerevisiae indicates that High Mobility Group (HMGB) proteins, non-sequence-specific chromosomal proteins, participate in mediating responses to growth signals. As HMGB proteins are distinguished by their ability to alter DNA topology, they frequently function in the assembly of higher-order nucleoprotein complexes. We review here recent evidence, which suggests that HMGB proteins may function to coordinate TOR-dependent regulation of rRNA and RP gene expression.Key Words: Rapamycin, TORC1, HMO1, high mobility group, yeast, RP gene, rDNA.     

Effects of ribosomal proteins S1, S2 and the DeaD/CsdA DEAD-box helicase on translation of leaderless and canonical mRNAs in Escherichia coli

Leaderless mRNAs beginning with the AUG initiating codon occur in all kingdoms of life. It has been previously reported that translation of the leaderless cI mRNA is stimulated in an Escherichia coli rpsB mutant deficient in ribosomal protein S2. Here, we have studied this phenomenon at the molecular level by making use of an E. coli rpsB(ts) mutant. The analysis of the ribosomes isolated under the non-permissive conditions revealed that in addition to ribosomal protein S2, ribosomal protein S1 was absent, demonstrating that S2 is essential for binding of S1 to the 30S ribosomal subunit. In vitro translation assays and the selective translation of a leaderless mRNA in vivo at the non-permissive temperature corroborate and extend previous in vitro ribosome binding studies in that S1 is indeed dispensable for translation of leaderless mRNAs. The deaD/csdA gene, encoding the "DeaD/CsdA" DEAD-box helicase, has been isolated as a multicopy suppressor of rpsB(ts) mutations. Here, we show that expression of a plasmid-borne DeaD/CsdA gene restores both S1 and S2 on the ribosome at the non-permissive temperature in the rpsB(ts) strain, which in turn leads to suppression of the translational defect affecting canonical mRNSa. These data are discussed in terms of a model, wherein DeaD/CsdA is involved in ribosome biogenesis rather than acting directly on mRNA.     

Yeast and human RNA helicases involved in ribosome biogenesis: Current status and perspectives

Ribosome biogenesis is a fundamental process that is conserved in eukaryotes. Although spectacular progress has been made in understanding mammalian ribosome synthesis in recent years, by far, this process has still been best characterised in the yeast Saccharomyces cerevisiae. In yeast, besides the rRNAs, the ribosomal proteins and the 75 small nucleolar RNAs, more than 250 non-ribosomal proteins, generally referred to as trans-acting factors, are involved in ribosome biogenesis. These factors include nucleases, RNA modifying enzymes, ATPases, GTPases, kinases and RNA helicases. Altogether, they likely confer speed, accuracy and directionality to the ribosome synthesis process, however, the precise functions for most of them are still largely unknown. This review summarises our current knowledge on eukaryotic RNA helicases involved in ribosome biogenesis, particularly focusing on the most recent advances with respect to the molecular roles of these enzymes and their co-factors in yeast and human cells. This article is part of a Special Issue entitled: The Biology of RNA helicases—Modulation for life.     

Alopecia, neurological defects, and endocrinopathy syndrome caused by decreased expression of RBM28, a nucleolar protein associated with ribosome biogenesis

Single-gene disorders offer unique opportunities to shed light upon fundamental physiological processes in humans. We investigated an autosomal-recessive phenotype characterized by alopecia, progressive neurological defects, and endocrinopathy (ANE syndrome). By using homozygosity mapping and candidate-gene analysis, we identified a loss-of-function mutation in RBM28, encoding a nucleolar protein. RBM28 yeast ortholog, Nop4p, was previously found to regulate ribosome biogenesis. Accordingly, electron microscopy revealed marked ribosome depletion and structural abnormalities of the rough endoplasmic reticulum in patient cells, ascribing ANE syndrome to the restricted group of inherited disorders associated with ribosomal dysfunction.     

Functional link between ribosome formation and biogenesis of iron-sulfur proteins

In genetic screens for ribosomal export mutants, we identified CFD1, NBP35 and NAR1 as factors involved in ribosome biogenesis. Notably, these components were recently reported to function in extramitochondrial iron-sulfur (Fe-S) cluster biosynthesis. In particular, Nar1 was implicated to generate the Fe-S clusters within Rli1, a potential substrate protein of unknown function. We tested whether the Fe-S protein Rli1 functions in ribosome formation. We report that rli1 mutants are impaired in pre-rRNA processing and defective in the export of both ribosomal subunits. In addition, Rli1p is associated with both pre-40S particles and mature 40S subunits, and with the eIF3 translation initiation factor complex. Our data reveal an unexpected link between ribosome biogenesis and the biosynthetic pathway of cytoplasmic Fe-S proteins.