The human nucleolar protein FTSJ3 associates with NIP7 and functions in pre-rRNA processing

NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A' to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells.     

NOP3 is an essential yeast protein which is required for pre-rRNA processing

The four nucleolar proteins NOP1, SSB1, GAR1, and NSR1 of Saccharomyces cerevisiae share a repetitive domain composed of repeat units rich in glycine and arginine (GAR domain). We have cloned and sequenced a fifth member of this family, NOP3, and shown it to be essential for cell viability. The NOP3 open reading frame encodes a 415 amino acid protein with a predicted molecular mass of 45 kD, containing a GAR domain and an RNA recognition motif. NOP3-specific antibodies recognize a 60-kD protein by SDS-PAGE and decorate the nucleolus and the surrounding nucleoplasm. A conditional lethal mutation, GAL::nop3, was constructed; growth of the mutant strain in glucose medium represses NOP3 expression. In cells depleted of NOP3, production of cytoplasmic ribosomes is impaired. Northern analysis and pulse-chase labeling indicate that pre-rRNA processing is inhibited at the late steps, in which 27SB pre-rRNA is cleaved to 25S rRNA and 20S pre-rRNA to 18S rRNA.     

Ribosomal protein L35 is required for 27SB pre-rRNA processing in Saccharomyces cerevisiae

Ribosome synthesis involves the concomitance of pre-rRNA processing and ribosomal protein assembly. In eukaryotes, this is a complex process that requires the participation of specific sequences and structures within the pre-rRNAs, at least 200 trans-acting factors and the ribosomal proteins. There is little information on the function of individual 60S ribosomal proteins in ribosome synthesis. Herein, we have analysed the contribution of ribosomal protein L35 in ribosome biogenesis. In vivo depletion of L35 results in a deficit in 60S ribosomal subunits and the appearance of half-mer polysomes. Pulse-chase, northern hybridization and primer extension analyses show that processing of the 27SB to 7S pre-rRNAs is strongly delayed upon L35 depletion. Most likely as a consequence of this, release of pre-60S ribosomal particles from the nucleolus to the nucleoplasm is also blocked. Deletion of RPL35A leads to similar although less pronounced phenotypes. Moreover, we show that L35 assembles in the nucleolus and binds to early pre-60S ribosomal particles. Finally, flow cytometry analysis indicated that L35-depleted cells mildly delay the G1 phase of the cell cycle. We conclude that L35 assembly is a prerequisite for the efficient cleavage of the internal transcribed spacer 2 at site C₂.     

The evolutionarily conserved protein Las1 is required for pre-rRNA processing at both ends of ITS2

Ribosome synthesis entails the formation of mature rRNAs from long precursor molecules, following a complex pre-rRNA processing pathway. Why the generation of mature rRNA ends is so complicated is unclear. Nor is it understood how pre-rRNA processing is coordinated at distant sites on pre-rRNA molecules. Here we characterized, in budding yeast and human cells, the evolutionarily conserved protein Las1. We found that, in both species, Las1 is required to process ITS2, which separates the 5.8S and 25S/28S rRNAs. In yeast, Las1 is required for pre-rRNA processing at both ends of ITS2. It is required for Rrp6-dependent formation of the 5.8S rRNA 3' end and for Rat1-dependent formation of the 25S rRNA 5' end. We further show that the Rat1-Rai1 5'-3' exoribonuclease (exoRNase) complex functionally connects processing at both ends of the 5.8S rRNA. We suggest that pre-rRNA processing is coordinated at both ends of 5.8S rRNA and both ends of ITS2, which are brought together by pre-rRNA folding, by an RNA processing complex. Consistently, we note the conspicuous presence of ~7- or 8-nucleotide extensions on both ends of 5.8S rRNA precursors and at the 5' end of pre-25S RNAs suggestive of a protected spacer fragment of similar length.     

The small nucleolar RNP protein NOP1 (fibrillarin) is required for pre-rRNA processing in yeast.

The yeast snoRNP protein, NOP1, is structurally and functionally homologous to vertebrate fibrillarin and is essential for viability. A conditionally lethal allele was constructed by placing NOP1 expression under the control of a GAL promoter. Growth on glucose medium results in the depletion of NOP1 over several generations, during which cell growth is progressively impaired. Pulse labelling of proteins shows that NOP1 depleted strains are greatly impaired in the production of cytoplasmic ribosomes, and they have a reduced level of rRNA. Northern hybridization and pulse-chase labelling of pre-rRNA show a progressive impairment of all pre-rRNA processing steps. The pathway leading to 18S rRNA is particularly affected. Methylation of pre-rRNA is concomitantly impaired and unmethylated pre-rRNA accumulates and is not processed over long periods. NOP1 depletion does not prevent the accumulation of seven snoRNAs tested including U3; the levels of two species, U14 and snR190, decline. The snoRNAs synthesized in the absence of NOP1 retain TMG cap structures. Subnuclear fractionation and immunocytochemistry indicate that they continue to be localized in the nucleolus.     

The structure of the ITS2-proximal stem is required for pre-rRNA processing in yeast.

Accurate and efficient processing of pre-rRNA is critical to the accumulation of mature functional ribosomal subunits for maintenance of cell growth. Processing requires numerous factors which act in trans as well as RNA sequence/ structural elements which function in cis. To examine the latter, we have used directed mutagenesis and expression of mutated pre-rRNAs in yeast. Specifically, we tested requirements for formation of an ITS2-proximal stem on processing, a structure formed by an interaction between sequences corresponding to the 3' end of 5.8S rRNA and the 5' end of 25S. Pre-rRNA processing is inhibited in templates encoding mutations that prevent the formation of the ITS2-proximal stem. Compensatory, double mutations, which alter the sequence of this region but restore the structure of the stem, also restore processing, although at lower efficiency. This reduction in efficiency is reflected in decreased levels of mature 5.8S and 25S rRNA and increased levels of 35S pre-rRNA and certain processing intermediates. This phenotype is reminiscent of the biochemical depletion of U8 snoRNA in vertebrates for which the ITS2-proximal stem has been proposed as a potential site for interaction with U8 RNP. Thus, formation of the ITS2-proximal stem may be a requirement common to yeast and vertebrate pre-rRNA processing.     

The budding yeast homolog of the human EBNA1-binding protein 2 (Ebp2p) is an essential nucleolar protein required for pre-rRNA processing

The human EBP2 protein was found by two-hybrid analysis to interact with the Epstein-Barr virus nuclear antigen 1 (EBNA1). Homologs of human EBP2 can be found in Caenorhabditis elegans, Schizosaccharomyces pombe, and in Saccharomyces cerevisiae, and they all share a conserved 200-300-amino acid block of residues at their C termini. To understand the cellular function of EBP2, we have begun to study the protein in S. cerevisiae. The yeast Ebp2 protein contains N-terminal, nucleolar-associated KKE motifs, and deletion analysis reveals that the C-terminal conserved region is required for the activity of the protein. The EBP2 gene codes for an essential protein that localizes to the nucleolus. Temperature-sensitive ebp2-1 mutants become depleted of ribosomes and cease to divide after several generations at the restrictive temperature of 36 degrees C. This decline in ribosome levels is accompanied by a diminution in the levels of the 35 S-derived recombinant RNAs (rRNAs) (in particular the 25 S and 5.8 S rRNAs). Pulse-chase, Northern, and primer extension analysis of the rRNA biosynthetic pathway indicates that ebp2-1 mutants are defective in processing the 27 SA precursor into the 27 SB pre-rRNA.     

The nucleolar protein SAHY1 is involved in pre-rRNA processing and normal plant growth

Although the nucleolus is involved in ribosome biogenesis, the functions of numerous nucleolus-localized proteins remain unclear. In this study, we genetically isolated Arabidopsis thaliana salt hypersensitive mutant 1 (sahy1), which exhibits slow growth, short roots, pointed leaves, and sterility. SAHY1 encodes an uncharacterized protein that is predominantly expressed in root tips, early developing seeds, and mature pollen grains and is mainly restricted to the nucleolus. Dysfunction of SAHY1 primarily causes the accumulation of 32S, 18S-A₃, and 27SB pre-rRNA intermediates. Coimmunoprecipitation experiments further revealed the interaction of SAHY1 with ribosome proteins and ribosome biogenesis factors. Moreover, sahy1 mutants are less sensitive to protein translation inhibitors and show altered expression of structural constituents of ribosomal genes and ribosome subunit profiles, reflecting the involvement of SAHY1 in ribosome composition and ribosome biogenesis. Analyses of ploidy, S-phase cell cycle progression, and auxin transport and signaling indicated the impairment of mitotic activity, translation of auxin transport carrier proteins, and expression of the auxin-responsive marker DR5::GFP in the root tips or embryos of sahy1 plants. Collectively, these data demonstrate that SAHY1, a nucleolar protein involved in ribosome biogenesis, plays critical roles in normal plant growth in association with auxin transport and signaling.

SALT-HYPERSENSITIVE MUTANT 1, a nucleolar protein involved in ribosome biogenesis, regulates the auxin-mediated development of vegetative and reproductive tissues.     

Nop53p, an essential nucleolar protein that interacts with Nop17p and Nip7p, is required for pre-rRNA processing in Saccharomyces cerevisiae

In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form large and intriguingly organized complexes. A novel nucleolar protein, Nop53p, was isolated by using Nop17p as bait in the yeast two-hybrid system. Nop53p also interacts with a second nucleolar protein, Nip7p. A carbon source-conditional strain with the NOP53 coding sequence under the control of the GAL1 promoter did not grow in glucose-containing medium, showing the phenotype of an essential gene. Under nonpermissive conditions, the conditional mutant strain showed rRNA biosynthesis defects, leading to an accumulation of the 27S and 7S pre-rRNAs and depletion of the mature 25S and 5.8S mature rRNAs. Nop53p did not interact with any of the exosome subunits in the yeast two-hybrid system, but its depletion affects the exosome function. In pull-down assays, protein A-tagged Nop53p coprecipitated the 27S and 7S pre-rRNAs, and His-Nop53p also bound directly 5.8S rRNA in vitro, which is consistent with a role for Nop53p in pre-rRNA processing.     

The helicase Has1p is required for snoRNA release from pre-rRNA

Synthesis of rRNA in eukaryotes involves the action of a large population of snoRNA-protein complexes (snoRNPs), which create modified nucleotides and participate in cleavage of pre-rRNA. The snoRNPs mediate these functions through direct base pairing, in many cases through long complementary sequences. This feature suggests that RNA helicases may be involved in the binding and release of snoRNPs from pre-rRNA. In this study, we determined that the DEAD box helicase Has1p, a nucleolar protein required for the production of 18S rRNA, copurifies with the snR30/U17 processing snoRNP but is also present with other snoRNPs. Blocking Has1p expression causes a substantial increase in snoRNPs associated with 60S-90S preribosomal RNP complexes, including the U3 and U14 processing snoRNPs and several modifying snoRNPs examined. Cosedimentation persisted even after deproteinization. This effect was not observed with depletion of two nonhelicase proteins, Esf1p and Dim2p, that are also required for 18S rRNA production. Point mutations in ATPase and helicase motifs of Has1p block U14 release from pre-rRNA. Surprisingly, depletion of Has1p causes a reduction in the level of free U6 snRNP. The results indicate that the Has1p helicase is required for snoRNA release from pre-rRNA and production of the U6 snRNP.     

Nop2p is required for pre-rRNA processing and 60S ribosome subunit synthesis in yeast.

To investigate the function of the nucleolar protein Nop2p in Saccharomyces cerevisiae, we constructed a strain in which NOP2 is under the control of a repressible promoter. Repression of NOP2 expression lengthens the doubling time of this strain about fivefold and reduces steady-state levels of 60S ribosomal subunits, 80S ribosomes, and polysomes. Levels of 40S subunits increase as the free pool of 60S subunits is reduced. Nop2p depletion impairs processing of the 35S pre-rRNA and inhibits processing of 27S pre-rRNA, which results in lower steady-state levels of 25S rRNA and 5.8S rRNA. Processing of 20S pre-rRNA to 18S rRNA is not significantly affected. Processing at sites A2, A3, B1L, and B1S and the generation of 5' termini of different pre-rRNA intermediates appear to be normal after Nop2p depletion. Sequence comparisons suggest that Nop2p may function as a methyltransferase. 2'-O-ribose methylation of the conserved site UmGm psi UC2922 is known to take place during processing of 27S pre-rRNA. Although Nop2p depletion lengthens the half-life of 27S pre-RNA, methylation of UmGm psi UC2922 in 27S pre-rRNA is low during Nop2p depletion. However, methylation of UmGm psi UC2922 in mature 25S rRNA appears normal. These findings provide evidence for a close interconnection between methylation at this conserved site and the processing step that yields the 25S rRNA.     

The F-box protein Fbw7 is required for cerebellar development

The F-box protein Fbw7 (also known as Fbxw7, hCdc4 and Sel-10) functions as a substrate recognition component of a SCF-type E3 ubiquitin ligase. SCF(Fbw7) facilitates polyubiquitination and subsequent degradation of various proteins such as Notch, cyclin E, c-Myc and c-Jun. Fbw7 is highly expressed in the nervous system and controls neural stem cell differentiation and apoptosis via Notch and c-Jun during embryonic development (Hoeck et al., 2010). Fbw7 deletion in the neural lineage is perinatal lethal and thus prohibits studying the role of Fbw7 in the adult nervous system. fbw7 mRNA is highly expressed in the postnatal brain and to gain insights into the function of Fbw7 in postnatal neurogenesis we analysed Fbw7 function in the cerebellum. We generated conditional Fbw7-knockout mice (fbw7^?Cb) by inactivating Fbw7 specifically in the cerebellar anlage. This resulted in decreased cerebellar size, reduced Purkinje cell number and defects in axonal arborisation. Moreover, Fbw7-deficient cerebella showed supranumeral fissures and aberrant progenitor cell migration. Protein levels of the Fbw7 substrates Notch1 and N-terminally phosphorylated c-Jun were upregulated in fbw7^?Cb mice. Concomitant deletion of c-Jun, and also the junAA knock-in mutation which specifically abrogates c-Jun N-terminal phosphorylation, rescued Purkinje cell numbers and arborisation in the fbw7^?Cb background. Taken together these data demonstrate that Fbw7 is essential during cerebellar development, and identify N-terminally phosphorylated c-Jun as an important substrate of SCF(Fbw7) during neurogenesis.     

The C-terminus of MIP-T3 protein is required for ubiquitin-proteasome-mediated degradation in human cells

The intraflagellar transport (IFT) complex is essential for the formation and functional maintenance of eukaryotic cilia which play a vital role in development and tissue homeostasis. However, the biochemical characteristics and precise functions of IFT proteins remain unknown. Here, we report that MIP-T3, a human microtubule-interacting protein recently identified as a novel conserved component of the IFT complex, is an easily degradable protein in human cell lines. Protein degradation is mediated by the ubiquitin-proteasome system, and the C-terminus is required for ubiquitination and proteasome-mediated degradation of MIP-T3 protein. This study provides the first evidence for regulation of IFT protein stability.     

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.     

Neural Wiskott-Aldrich syndrome protein is required for accurate chromosome congression and segregation

The accurate distribution and segregation of replicated chromosomes through mitosis is crucial for cellular viability and development of organisms. Kinetochores are responsible for the proper congression and segregation of chromosomes. Here, we show that neural Wiskott-Aldrich syndrome protein (N-WASP) localizes to and forms a complex with kinetochores in mitotic cells. Depletion of NWASP by RNA interference causes chromosome misalignment, prolonged mitosis, and abnormal chromosomal segregation, which is associated with decreased proliferation of N-WASP-deficient cells. N-WASP-deficient cells display defects in the kinetochores recruitment of inner and outer kinetochore components, CENP-A, CENP-E, and Mad2. Live-cell imaging analysis of GFP-α-tubulin revealed that depletion of N-WASP impairs microtubule attachment to chromosomes in mitotic cells. All these results indicate that N-WASP plays a role in efficient assembly of kinetochores and attachment of microtubules to chromosomes, which is essential for accurate chromosome congression and segregation.