The DEAD-box RNA helicase-like Utp25 is an SSU processome component

The SSU processome is a large ribonucleoprotein complex consisting of the U3 snoRNA and at least 43 proteins. A database search, initiated in an effort to discover additional SSU processome components, identified the uncharacterized, conserved and essential yeast nucleolar protein YIL091C/UTP25 as one such candidate. The C-terminal DUF1253 motif, a domain of unknown function, displays limited sequence similarity to DEAD-box RNA helicases. In the absence of the conserved DEAD-box sequence, motif Ia is the only clearly identifiable helicase element. Since the yeast homolog is nucleolar and interacts with components of the SSU processome, we examined its role in pre-rRNA processing. Genetic depletion of Utp25 resulted in slowed growth. Northern analysis of pre-rRNA revealed an 18S rRNA maturation defect at sites A₀, A₁, and A₂. Coimmunoprecipitation confirmed association with U3 snoRNA and with Mpp10, and with components of the t-Utp/UtpA, UtpB, and U3 snoRNP subcomplexes. Mutation of the conserved motif Ia residues resulted in no discernable temperature-sensitive or cold-sensitive growth defects, implying that this motif is dispensable for Utp25 function. A yeast two-hybrid screen of Utp25 against other SSU processome components revealed several interacting proteins, including Mpp10, Utp3, and Utp21, thereby identifying the first interactions among the different subcomplexes of the SSU processome. Furthermore, the DUF1253 domain is required and sufficient for the interaction of Utp25 with Utp3. Thus, Utp25 is a novel SSU processome component that, along with Utp3, forms the first identified interactions among the different SSU processome subcomplexes.     

Loss of rRNA modifications in the decoding center of the ribosome impairs translation and strongly delays pre-rRNA processing

The ribosome decoding center is rich in modified rRNA nucleotides and little is known about their effects. Here, we examine the consequences of systematically deleting eight pseudouridine and 2′-O-methylation modifications in the yeast decoding center. Loss of most modifications individually has no apparent effect on cell growth. However, deletions of 2–3 modifications in the A- and P-site regions can cause (1) reduced growth rates (∼15%–50% slower); (2) reduced amino acid incorporation rates (14%–24% slower); and (3) a significant deficiency in free small subunits. Negative and positive interference effects were observed, as well as strong positional influences. Notably, blocking formation of a hypermodified pseudouridine in the P region delays the onset of the final cleavage event in 18S rRNA formation (∼60% slower), suggesting that modification at this site could have an important role in modulating ribosome synthesis.     

豌豆细胞核仁中U3 snoRNA的分布和转运

rRNA前体剪切是发生在核仁中的重要生物学事件.U3 snoRNA作为rRNA的一个剪切因子被认为是rRNA前体剪切第一步,即5′ ETS剪切所必需的.鉴定U3能够为确定rRNA前体剪切位点和剪切产物转运提供间接证据.本文利用原位杂交技术研究了豌豆(Pisum sativum L.)核仁中U3 snoRNA的分布和转运.结果表明, U3 snoRNA分布在致密纤维组分(dense fibrillar component, DFC)和颗粒组分(granular component, GC)中,在纤维中心(fibrillar center, FC)没有分布.当用放线菌素D (actinomycin D, AMD)处理豌豆根端分生细胞时,rDNA转录受到抑制,标记信号减弱.随着AMD处理时间的延长,标记信号逐渐变弱并出现在DFC远轴区域和GC区域.本文结果提示,rRNA前体剪切发生在DFC和GC区域,剪切产物从围绕FC的区域向周边转运.     

豌豆细胞核仁中U3 snoRNA的分布和转运

rRNA前体剪切是发生在核仁中重要生物学事件。U3 snoRNA作为rRNA的一个剪切因子被认为是rRNA前体剪切第一步,即5′ETS剪切所必需的,鉴定U3能够为确定rRNA前体剪切位点和剪切产物转运提供间接证据。,本文利用原位杂交技术研究了豌豆（Pisum sativum L.)核仁中U3 snoRNA的分布和转运。结果表明,U3 snoRNA分布在致密纤维组分（dense fibrillar component,DFC)和颗粒组分（granular component,GC)中,在纤维中心（fibrillar center,FC)没有分布 ,当用放线菌素D（actinomycin,D,AMD)处理豌豆根端分生细胞时,rDNA转录受到抑制,标记信号减弱,随着AMD处理时间的延长,标记信号逐渐变弱并出现在DFC远轴区域和GC区域。本文结果提示,rRNA前体剪切发生在DFC和GC区域,剪切产物从围绕FC的区域向周边转运。     

The rRNA methyltransferase Bud23 shows functional interaction with components of the SSU processome and RNase MRP

Bud23 is responsible for the conserved methylation of G1575 of 18S rRNA, in the P-site of the small subunit of the ribosome. bud23Δ mutants have severely reduced small subunit levels and show a general failure in cleavage at site A2 during rRNA processing. Site A2 is the primary cleavage site for separating the precursors of 18S and 25S rRNAs. Here, we have taken a genetic approach to identify the functional environment of BUD23. We found mutations in UTP2 and UTP14, encoding components of the SSU processome, as spontaneous suppressors of a bud23Δ mutant. The suppressors improved growth and subunit balance and restored cleavage at site A2. In a directed screen of 50 ribosomal trans-acting factors, we identified strong positive and negative genetic interactions with components of the SSU processome and strong negative interactions with components of RNase MRP. RNase MRP is responsible for cleavage at site A3 in pre-rRNA, an alternative cleavage site for separating the precursor rRNAs. The strong negative genetic interaction between RNase MRP mutants and bud23Δ is likely due to the combined defects in cleavage at A2 and A3. Our results suggest that Bud23 plays a role at the time of A2 cleavage, earlier than previously thought. The genetic interaction with the SSU processome suggests that Bud23 could be involved in triggering disassembly of the SSU processome, or of particular subcomplexes of the processome.     

Assembly of Saccharomyces cerevisiae 60S ribosomal subunits: role of factors required for 27S pre-rRNA processing

The precise functions of most of the ～200 assembly factors and 79 ribosomal proteins required to construct yeast ribosomes in vivo remain largely unexplored. To better understand the roles of these proteins and the mechanisms driving ribosome biogenesis, we examined in detail one step in 60S ribosomal subunit assembly-processing of 27SA(3) pre-rRNA. Six of seven assembly factors required for this step (A(3) factors) are mutually interdependent for association with preribosomes. These A(3) factors are required to recruit Rrp17, one of three exonucleases required for this processing step. In the absence of A(3) factors, four ribosomal proteins adjacent to each other, rpL17, rpL26, rpL35, and rpL37, fail to assemble, and preribosomes are turned over by Rat1. We conclude that formation of a neighbourhood in preribosomes containing the A(3) factors establishes and maintains stability of functional preribosomes containing 27S pre-rRNAs. In the absence of these assembly factors, at least one exonuclease can switch from processing to turnover of pre-rRNA.     

Nol9 is a novel polynucleotide 5'-kinase involved in ribosomal RNA processing

In a cell, an enormous amount of energy is channelled into the biogenesis of ribosomal RNAs (rRNAs). In a multistep process involving a large variety of ribosomal and non-ribosomal proteins, mature rRNAs are generated from a long polycistronic precursor. Here, we show that the non-ribosomal protein Nol9 is a polynucleotide 5'-kinase that sediments primarily with the pre-60S ribosomal particles in HeLa nuclear extracts. Depletion of Nol9 leads to a severe impairment of ribosome biogenesis. In particular, the polynucleotide kinase activity of Nol9 is required for efficient generation of the 5.8S and 28S rRNAs from the 32S precursor. Upon Nol9 knockdown, we also observe a specific maturation defect at the 5' end of the predominant 5.8S short-form rRNA (5.8S(S)), possibly due to the Nol9 requirement for 5'>3' exonucleolytic trimming. In contrast, the endonuclease-dependent generation of the 5'-extended, minor 5.8S long-form rRNA (5.8S(L)) is largely unaffected. This is the first report of a nucleolar polynucleotide kinase with a role in rRNA processing.     

The roles of moonlight ribosomal proteins in the development of human cancers

“Moonlighting protein” is a term used to define a single protein with multiple functions and different activities that are not derived from gene fusions, multiple RNA splicing, or the proteolytic activity of promiscuous enzymes. Different proteinous constituents of ribosomes have been shown to have important moonlighting extra-ribosomal functions. In this review, we introduce the impact of key moonlight ribosomal proteins and dependent signal transduction in the initiation and progression of various cancers. As a future perspective, the potential role of these moonlight ribosomal proteins in the diagnosis, prognosis, and development of novel strategies to improve the efficacy of therapies for human cancers has been suggested.     

Control of ribosomal RNA synthesis by hematopoietic transcription factors

1. Download : Download high-res image (178KB)
2. Download : Download full-size image

     

Henipaviruses and lyssaviruses target nucleolar treacle protein and regulate ribosomal RNA synthesis

The nucleolus is a common target of viruses and viral proteins, but for many viruses the functional outcomes and significance of this targeting remains unresolved. Recently, the first intranucleolar function of a protein of a cytoplasmically-replicating negative-sense RNA virus (NSV) was identified, with the finding that the matrix (M) protein of Hendra virus (HeV) (genus Henipavirus, family Paramyxoviridae) interacts with Treacle protein within nucleolar subcompartments and mimics a cellular mechanism of the nucleolar DNA-damage response (DDR) to suppress ribosomal RNA (rRNA) synthesis. Whether other viruses utilise this mechanism has not been examined. We report that sub-nucleolar Treacle targeting and modulation is conserved between M proteins of multiple Henipaviruses, including Nipah virus and other potentially zoonotic viruses. Furthermore, this function is also evident for P3 protein of rabies virus, the prototype virus of a different RNA virus family (Rhabdoviridae), with Treacle depletion in cells also found to impact virus production. These data indicate that unrelated proteins of viruses from different families have independently developed nucleolar/Treacle targeting function, but that modulation of Treacle has distinct effects on infection. Thus, subversion of Treacle may be an important process in infection by diverse NSVs, and so could provide novel targets for antiviral approaches with broad specificity.     

A small ribosomal subunit (SSU) processome component, the human U3 protein 14A (hUTP14A) binds p53 and promotes p53 degradation

Ribosome biogenesis is required for normal cell function, and aberrant ribosome biogenesis can lead to p53 activation. However, how p53 is activated by defects of ribosome biogenesis remains to be determined. Here, we identified human UTP14a as an SSU processome component by showing that hUTP14a is nucleolar, associated with U3 snoRNA and involved in 18 S rRNA processing. Interestingly, ectopic expression of hUTP14a resulted in a decrease and knockdown of hUTP14a led to an increase of p53 protein levels. We showed that hUTP14a physically interacts with p53 and functionally promotes p53 turn-over, and that hUTP14a promotion of p53 destabilization is sensitive to a proteasome inhibitor but independent of ubiquitination. Significantly, knockdown of hUTP14a led to cell cycle arrest and apoptosis. Our data identified a novel pathway for p53 activation through a defect in rRNA processing and suggest that a ribosome biogenesis factor itself could act as a sensor for nucleolar stress to regulate p53.     

The role of RbfA in 16S rRNA processing and cell growth at low temperature in Escherichia coli

RbfA, a 30S ribosome-binding factor, is a multicopy suppressor of a cold-sensitive C23U mutation of the 16S rRNA and is required for efficient processing of the 16S rRNA. At 37 degrees C, DeltarbfA cells show accumulation of ribosomal subunits and 16S rRNA precursor with a significantly reduced polysome profile in comparison with wild-type cells. RbfA is also a cold-shock protein essential for Escherichia coli cells to adapt to low temperature. In this study, we examined its association with the ribosome and its role in 16S rRNA processing and ribosome profiles at low temperature. In wild-type cells, following cold shock at 15 degrees C, the amount of free RbfA remained largely stable, while that of its 30S subunit-associated form became several times greater than that at 37 degrees C and a larger fraction of total 30S subunits was detected to be RbfA-containing. In DeltarbfA cells, the pre-16S rRNA amount increased after cold shock with a concomitant reduction of the mature 16S rRNA amount and the formation of polysomes was further reduced. A closer examination revealed that 30S ribosomal subunits of DeltarbfA cells at low temperature contained primarily pre-16S rRNA and little mature 16S rRNA. Our results indicate that the cold sensitivity of DeltarbfA cells is directly related to their lack of translation initiation-capable 30S subunits containing mature 16S rRNA at low temperature. Importantly, when the C-terminal 25 residue sequence was deleted, the resulting RbfADelta25 lost the abilities to stably associate with the 30S subunit and to suppress the dominant-negative, cold-sensitive phenotype of the C23U mutation in 16S rRNA but was able to suppress the 16S rRNA processing defect and the cold-sensitive phenotype of the DeltarbfA cells, suggesting that RbfA may interact with the 30S ribosome at more than one site or function in more than one fashion in assisting the 16S rRNA maturation at low temperature.     

The role of human ribosomal proteins in the maturation of rRNA and ribosome production

Production of ribosomes is a fundamental process that occurs in all dividing cells. It is a complex process consisting of the coordinated synthesis and assembly of four ribosomal RNAs (rRNA) with about 80 ribosomal proteins (r-proteins) involving more than 150 nonribosomal proteins and other factors. Diamond Blackfan anemia (DBA) is an inherited red cell aplasia caused by mutations in one of several r-proteins. How defects in r-proteins, essential for proliferation in all cells, lead to a human disease with a specific defect in red cell development is unknown. Here, we investigated the role of r-proteins in ribosome biogenesis in order to find out whether those mutated in DBA have any similarities. We depleted HeLa cells using siRNA for several individual r-proteins of the small (RPS6, RPS7, RPS15, RPS16, RPS17, RPS19, RPS24, RPS25, RPS28) or large subunit (RPL5, RPL7, RPL11, RPL14, RPL26, RPL35a) and studied the effect on rRNA processing and ribosome production. Depleting r-proteins in one of the subunits caused, with a few exceptions, a decrease in all r-proteins of the same subunit and a decrease in the corresponding subunit, fully assembled ribosomes, and polysomes. R-protein depletion, with a few exceptions, led to the accumulation of specific rRNA precursors, highlighting their individual roles in rRNA processing. Depletion of r-proteins mutated in DBA always compromised ribosome biogenesis while affecting either subunit and disturbing rRNA processing at different levels, indicating that the rate of ribosome production rather than a specific step in ribosome biogenesis is critical in patients with DBA.