Chromogranin A, an "on/off" switch controlling dense-core secretory granule biogenesis

How pre-ribosomes temporally and spatially mature during intranuclear biogenesis is not known. Here, we report three nucleolar proteins, Noc1p to Noc3p, that are required for ribosome maturation and transport. They can be isolated in two distinct complexes: Noc1p/Noc2p associates with 90S and 66S pre-ribosomes and is enriched in the nucleolus, and Noc2p/Noc3p associates with 66S pre-ribosomes and is mainly nucleoplasmic. Mutation of each Noc protein impairs intranuclear transport of 60S subunits at different stages and inhibits pre-rRNA processing. Overexpression of a conserved domain common to Noc1p and Noc3p is dominant-negative for cell growth, with a defect in nuclear 60S subunit transport, but no inhibition of pre-rRNA processing. We propose that the dynamic interaction of Noc proteins is crucial for intranuclear movement of ribosomal precursor particles, and, thereby represent a prerequisite for proper maturation.     

90S pre-ribosomes include the 35S pre-rRNA,the U3 snoRNP,and 40S subunit processing factors but predominantly lack 60S synthesis factors

We report the characterization of early pre-ribosomal particles. Twelve TAP-tagged components each showed nucleolar localization, sedimented at approximately 90S on sucrose gradients, and coprecipitated both the 35S pre-rRNA and the U3 snoRNA. Thirty-five non-ribosomal proteins were coprecipitated, including proteins associated with U3 (Nop56p, Nop58p, Sof1p, Rrp9, Dhr1p, Imp3p, Imp4p, and Mpp10p) and other factors required for 18S rRNA synthesis (Nop14p, Bms1p, and Krr1p). Mutations in components of the 90S pre-ribosomes impaired 40S subunit assembly and export. Strikingly, few components of recently characterized pre-60S ribosomes were identified in the 90S pre-ribosomes. We conclude that the 40S synthesis machinery predominately associates with the 35S pre-rRNA factors, whereas factors required for 60S subunit synthesis largely bind later, showing an unexpected dichotomy in binding.     

Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes

Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.     

Functional and physical interactions of Faf1p, a Saccharomyces cerevisiae nucleolar protein

We report the discovery and characterisation of a novel nucleolar protein of Saccharomyces cerevisiae. We identified this protein encoded by ORF YIL019w, designated in SGD base as Faf1p, in a two hybrid interaction screen using the known nucleolar protein Krr1 as bait. The presented data indicate that depletion of the Faf1 protein has an impact on the 40S ribosomal subunit biogenesis resulting from a decrease in the production of 18S rRNA. The primary defect is apparently due to inefficient processing of 35S rRNA at the A(0), A(1), and A(2) cleavage sites.     

Mrd1p is required for processing of pre-rRNA and for maintenance of steady-state levels of 40 S ribosomal subunits in yeast

Ribosome biogenesis is a conserved process in eukaryotes that requires a large number of small nucleolar RNAs and trans-acting proteins. The Saccharomyces cerevisiae MRD1 (multiple RNA-binding domain) gene encodes a novel protein that contains five consensus RNA-binding domains. Mrd1p is essential for viability. Mrd1p partially co-localizes with the nucleolar protein Nop1p. Depletion of Mrd1p leads to a selective reduction of 18 S rRNA and 40 S ribosomal subunits. Mrd1p associates with the 35 S precursor rRNA (pre-rRNA) and U3 small nucleolar RNAs and is necessary for the initial processing at the A(0)-A(2) cleavage sites in pre-rRNA. The presence of five RNA-binding domains in Mrd1p suggests that Mrd1p may function to correctly fold pre-rRNA, a requisite for proper cleavage. Sequence comparisons suggest that Mrd1p homologues exist in all eukaryotes.     

Has1 regulates consecutive maturation and processing steps for assembly of 60S ribosomal subunits

Ribosome biogenesis requires ∼200 assembly factors in Saccharomyces cerevisiae. The pre-ribosomal RNA (rRNA) processing defects associated with depletion of most of these factors have been characterized. However, how assembly factors drive the construction of ribonucleoprotein neighborhoods and how structural rearrangements are coupled to pre-rRNA processing are not understood. Here, we reveal ATP-independent and ATP-dependent roles of the Has1 DEAD-box RNA helicase in consecutive pre-rRNA processing and maturation steps for construction of 60S ribosomal subunits. Has1 associates with pre-60S ribosomes in an ATP-independent manner. Has1 binding triggers exonucleolytic trimming of 27SA₃ pre-rRNA to generate the 5′ end of 5.8S rRNA and drives incorporation of ribosomal protein L17 with domain I of 5.8S/25S rRNA. ATP-dependent activity of Has1 promotes stable association of additional domain I ribosomal proteins that surround the polypeptide exit tunnel, which are required for downstream processing of 27SB pre-rRNA. Furthermore, in the absence of Has1, aberrant 27S pre-rRNAs are targeted for irreversible turnover. Thus, our data support a model in which Has1 helps to establish domain I architecture to prevent pre-rRNA turnover and couples domain I folding with consecutive pre-rRNA processing steps.     

Ebp2 and Brx1 function cooperatively in 60S ribosomal subunit assembly in Saccharomyces cerevisiae

The yeast protein Ebp2 is required for early steps in production of 60S ribosomal subunits. To search for cofactors with which Ebp2 functions, or substrates on which it acts, we screened for mutants that were synthetically lethal (sl) with the ebp2-14 mutation. Four different mutant alleles of the 60S ribosomal subunit assembly factor Brx1 were found. To investigate defects of the double mutant, we constructed strains conditional for the ebp2-14 brx1- synthetic lethal phenotype. These ebp2-14 brx1 mutants were defective in processing of 27S pre-rRNA and production of 60S subunits, under conditions where each single mutant was not. Ebp2 and Brx1 exhibit a strong two-hybrid interaction, which is eliminated by some combinations of brx1 and ebp2 mutations. In one such mutant, Ebp2 and Brx1 can still associate with pre-ribosomes, but subunit maturation is perturbed. Depletion of either Ebp2 or Brx1 revealed that Brx1 requires Ebp2 for its stable association with pre-ribosomes, but Ebp2 does not depend on the presence of Brx1 to enter pre-ribosomes. These results suggest that assembly of 60S ribosomal subunits requires cooperation of Ebp2 with Brx1, together with other molecules present in pre-ribosomes, potentially including several found in assembly subcomplexes with Brx1 and Ebp2.     

Yeast polypeptide exit tunnel ribosomal proteins L17, L35 and L37 are necessary to recruit late-assembling factors required for 27SB pre-rRNA processing

Ribosome synthesis involves the coordinated folding and processing of pre-rRNAs with assembly of ribosomal proteins. In eukaryotes, these events are facilitated by trans-acting factors that propel ribosome maturation from the nucleolus to the cytoplasm. However, there is a gap in understanding how ribosomal proteins configure pre-ribosomes in vivo to enable processing to occur. Here, we have examined the role of adjacent yeast r-proteins L17, L35 and L37 in folding and processing of pre-rRNAs, and binding of other proteins within assembling ribosomes. These three essential ribosomal proteins, which surround the polypeptide exit tunnel, are required for 60S subunit formation as a consequence of their role in removal of the ITS2 spacer from 27SB pre-rRNA. L17-, L35- and L37-depleted cells exhibit turnover of aberrant pre-60S assembly intermediates. Although the structure of ITS2 does not appear to be grossly affected in their absence, these three ribosomal proteins are necessary for efficient recruitment of factors required for 27SB pre-rRNA processing, namely, Nsa2 and Nog2, which associate with pre-60S ribosomal particles containing 27SB pre-rRNAs. Altogether, these data support that L17, L35 and L37 are specifically required for a recruiting step immediately preceding removal of ITS2.     

Fcf1p and Fcf2p are novel nucleolar Saccharomyces cerevisiae proteins involved in pre-rRNA processing

The uncharacterized Saccharomyces cerevisiae proteins Fcf1 and Fcf2, encoded by the ORFs YDR339c and YLR051c, respectively, were identified in a tandem affinity purification experiment of the known 40S factor Faf1p. Most of the proteins associated with TAP-Faf1p are trans-acting factors involved in pre-rRNA processing and 40S subunit biogenesis, in agreement with the previously observed role of Faf1p in 18S rRNA synthesis. Fcf1p and Fcf2p are both essential and localize to the nucleolus. Depletion of Fcf1p and Fcf2p leads to a decrease in synthesis of the 18S rRNA, resulting in a deficit in 40S ribosomal subunits. Northern analysis indicates inefficient processing of pre-rRNA at the A(0), A(1), and A(2) cleavage sites.     

Yeast Rrp14p is a nucleolar protein involved in both ribosome biogenesis and cell polarity

We previously cloned RRP14/YKL082c, whose product exhibits two-hybrid interaction with Ebp2p, a regulatory factor of assembly of 60S ribosomal subunits. Depletion of Rrp14p results in shortage of 60S ribosomal subunits and retardation of processing from 27S pre-rRNA to 25S rRNA. Furthermore, 35S pre-rRNA synthesis appears to decline in Rrp14p-depleted cells. Rrp14p interacts with regulatory factors of 60S subunit assembly and also with Utp11p and Faf1p, which are regulatory factors required for assembly of 40S ribosomal subunits. We propose that Rrp14p is involved in ribosome synthesis from the beginning of 35S pre-rRNA synthesis to assembly of the 60S ribosomal subunit. Disruption of RRP14 causes an extremely slow growth rate of the cell, a severe defect in ribosome synthesis, and a depolarized localization of cortical actin patches throughout the cell cycle. These results suggest that Rrp14p has dual functions in ribosome synthesis and polarized cell growth.     

The KRR1 gene encodes a protein required for 18S rRNA synthesis and 40S ribosomal subunit assembly in Saccharomyces cerevisiae

The newly discovered Saccharomyces cerevisiae gene KRR1 (YCL059c) encodes a protein essential for cell viability. Krr1p contains a motif of clustered basic amino acids highly conserved in the evolutionarly distant species from yeast to human. We demonstrate that Krr1p is localized in the nucleolus. The KRR1 gene is highly expressed in dividing cells and its expression ceases almost completely when cells enter the stationary phase. In vivo depletion of Krr1p leads to drastic reduction of 40S ribosomal subunits due to defective 18S rRNA synthesis. We propose that Krr1p is required for proper processing of pre-rRNA and the assembly of preribosomal 40S subunits.     

Coevolution of ribosomal RNA expansion segment 7L and assembly factor Noc2p specializes the ribosome biogenesis pathway between Saccharomyces cerevisiae and Candida albicans

Ribosomes of different species share an evolutionarily conserved core, exhibiting flexible shells formed partially by the addition of species-specific ribosomal RNAs (rRNAs) with largely unexplored functions. In this study, we showed that by swapping the Saccharomyces cerevisiae 25S rRNA genes with non-S. cerevisiae homologs, species-specific rRNA variations caused moderate to severe pre-rRNA processing defects. Specifically, rRNA substitution by the Candida albicans caused severe growth defects and deficient pre-rRNA processing. We observed that such defects could be attributed primarily to variations in expansion segment 7L (ES7^L) and could be restored by an assembly factor Noc2p mutant (Noc2p-K384R). We showed that swapping ES7^L attenuated the incorporation of Noc2p and other proteins (Erb1p, Rrp1p, Rpl6p and Rpl7p) into pre-ribosomes, and this effect could be compensated for by Noc2p-K384R. Furthermore, replacement of Noc2p with ortholog from C. albicans could also enhance the incorporation of Noc2p and the above proteins into pre-ribosomes and consequently restore normal growth. Taken together, our findings help to elucidate the roles played by the species-specific rRNA variations in ribosomal biogenesis and further provide evidence that coevolution of rRNA expansion segments and cognate assembly factors specialized the ribosome biogenesis pathway, providing further insights into the function and evolution of ribosome.     

Ribosomal proteins L7 and L8 function in concert with six A3 assembly factors to propagate assembly of domains I and II of 25S rRNA in yeast 60S ribosomal subunits

Ribosome biogenesis is a complex multistep process that involves alternating steps of folding and processing of pre-rRNAs in concert with assembly of ribosomal proteins. Recently, there has been increased interest in the roles of ribosomal proteins in eukaryotic ribosome biogenesis in vivo, focusing primarily on their function in pre-rRNA processing. However, much less is known about participation of ribosomal proteins in the formation and rearrangement of preribosomal particles as they mature to functional subunits. We have studied ribosomal proteins L7 and L8, which are required for the same early steps in pre-rRNA processing during assembly of 60S subunits but are located in different domains within ribosomes. Depletion of either leads to defects in processing of 27SA(3) to 27SB pre-rRNA and turnover of pre-rRNAs destined for large ribosomal subunits. A specific subset of proteins is diminished from these residual assembly intermediates: six assembly factors required for processing of 27SA(3) pre-rRNA and four ribosomal proteins bound to domain I of 25S and 5.8S rRNAs surrounding the polypeptide exit tunnel. In addition, specific sets of ribosomal proteins are affected in each mutant: In the absence of L7, proteins bound to domain II, L6, L14, L20, and L33 are greatly diminished, while proteins L13, L15, and L36 that bind to domain I are affected in the absence of L8. Thus, L7 and L8 might establish RNP structures within assembling ribosomes necessary for the stable association and function of the A(3) assembly factors and for proper assembly of the neighborhoods containing domains I and II.     

The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast

We have conducted a genetic screen in order to identify ribosomal proteins of Saccharomyces cerevisiae involved in nuclear export of the small subunit precursors. This has led us to distinguish Rps15p as a protein dispensable for maturation of the pre-40S particles, but whose assembly into the pre-ribosomes is a prerequisite to their nuclear exit. Upon depletion of Rps15p, 20S pre-rRNA is released from the nucleolus and retained in the nucleus, without alteration of the pre-rRNA early cleavages. In contrast, Rps18p, which contacts Rps15p in the small subunit, is required upstream for pre-rRNA processing at site A2. Most pre-40S specific factors are correctly associated with the intermediate particles accumulating in the nucleus upon Rps15p depletion, except the late-binding proteins Tsr1p and Rio2p. Here we show that these two proteins are dispensable for nuclear exit; instead, they participate in 20S pre-rRNA processing in the cytoplasm. We conclude that, during the final maturation steps in the nucleus, incorporation of the ribosomal protein Rps15p is specifically required to render the pre-40S particles competent for translocation to the cytoplasm.     

Structural and functional analysis of the Rpf2-Rrs1 complex in ribosome biogenesis

Proteins Rpf2 and Rrs1 are required for 60S ribosomal subunit maturation. These proteins are necessary for the recruitment of three ribosomal components (5S ribosomal RNA [rRNA], RpL5 and RpL11) to the 90S ribosome precursor and subsequent 27SB pre-rRNA processing. Here we present the crystal structure of the Aspergillus nidulans (An) Rpf2-Rrs1 core complex. The core complex contains the tightly interlocked N-terminal domains of Rpf2 and Rrs1. The Rpf2 N-terminal domain includes a Brix domain characterized by similar N- and C-terminal architecture. The long α-helix of Rrs1 joins the C-terminal half of the Brix domain as if it were part of a single molecule. The conserved proline-rich linker connecting the N- and C-terminal domains of Rrs1 wrap around the side of Rpf2 and anchor the C-terminal domain of Rrs1 to a specific site on Rpf2. In addition, gel shift analysis revealed that the Rpf2-Rrs1 complex binds directly to 5S rRNA. Further analysis of Rpf2-Rrs1 mutants demonstrated that Saccharomyces cerevisiae Rpf2 R236 (corresponds to R238 of AnRpf2) plays a significant role in this binding. Based on these studies and previous reports, we have proposed a model for ribosomal component recruitment to the 90S ribosome precursor.     

NSR1 is required for pre-rRNA processing and for the proper maintenance of steady-state levels of ribosomal subunits.

NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.     

Elucidation of the assembly events required for the recruitment of Utp20, Imp4 and Bms1 onto nascent pre-ribosomes

The 90S pre-ribosome, also known as the small subunit (SSU) processome, is a large multisubunit particle required for the production of the 18S rRNA from a pre-rRNA precursor. Recently, it has been shown that the formation of this particle entails the initial association of the tUTP subunit with the nascent pre-RNA and, subsequently, the binding of Rrp5/UTP-C and U3 snoRNP/UTP-B subunits in two independent assembly branches. However, the mode of assembly of other 90S pre-ribosome components remains obscure as yet. In this study, we have investigated the assembly of three proteins (Utp20, Imp4 and Bms1) previously regarded as potential nucleating factors of the 90S particle. Here, we demonstrate that the loading of those three proteins onto the pre-rRNA takes place independently of Rrp5/UTP-C and, instead, occurs downstream of the tUTP and U3/UTP-B subcomplexes. We also demonstrate that Bms1 and Utp20 are required for the recruitment of a subset of proteins to nascent pre-ribosomes. Finally, we show that proteins associated through secondary steps condition the stability of the two assembly branches in partially assembled pre-ribosomes. These results provide new information about the functional relationships among 90S particle components and the events that are required for their stepwise incorporation onto the primary pre-rRNA.