WBSCR22/Merm1 is required for late nuclear pre-ribosomal RNA processing and mediates N7-methylation of G1639 in human 18S rRNA

Ribosomal (r)RNAs are extensively modified during ribosome synthesis and their modification is required for the fidelity and efficiency of translation. Besides numerous small nucleolar RNA-guided 2′-O methylations and pseudouridinylations, a number of individual RNA methyltransferases are involved in rRNA modification. WBSCR22/Merm1, which is affected in Williams–Beuren syndrome and has been implicated in tumorigenesis and metastasis formation, was recently shown to be involved in ribosome synthesis, but its molecular functions have remained elusive. Here we show that depletion of WBSCR22 leads to nuclear accumulation of 3′-extended 18SE pre-rRNA intermediates resulting in impaired 18S rRNA maturation. We map the 3′ ends of the 18SE pre-rRNA intermediates accumulating after depletion of WBSCR22 and in control cells using 3′-RACE and deep sequencing. Furthermore, we demonstrate that WBSCR22 is required for N⁷-methylation of G1639 in human 18S rRNA in vivo. Interestingly, the catalytic activity of WBSCR22 is not required for 18S pre-rRNA processing, suggesting that the key role of WBSCR22 in 40S subunit biogenesis is independent of its function as an RNA methyltransferase.     

The METTL5-TRMT112 N6-methyladenosine methyltransferase complex regulates mRNA translation via 18S rRNA methylation

Ribosomal RNAs (rRNAs) have long been known to carry chemical modifications, including 2′O-methylation, pseudouridylation, N⁶-methyladenosine (m⁶A), and N^6,6-dimethyladenosine. While the functions of many of these modifications are unclear, some are highly conserved and occur in regions of the ribosome critical for mRNA decoding. Both 28S rRNA and 18S rRNA carry single m⁶A sites, and while the methyltransferase ZCCHC4 has been identified as the enzyme responsible for the 28S rRNA m⁶A modification, the methyltransferase responsible for the 18S rRNA m⁶A modification has remained unclear. Here, we show that the METTL5-TRMT112 methyltransferase complex installs the m⁶A modification at position 1832 of human 18S rRNA. Our work supports findings that TRMT112 is required for METTL5 stability and reveals that human METTL5 mutations associated with microcephaly and intellectual disability disrupt this interaction. We show that loss of METTL5 in human cancer cell lines and in mice regulates gene expression at the translational level; additionally, Mettl5 knockout mice display reduced body size and evidence of metabolic defects. While recent work has focused heavily on m⁶A modifications in mRNA and their roles in mRNA processing and translation, we demonstrate here that deorphanizing putative methyltransferase enzymes can reveal previously unappreciated regulatory roles for m⁶A in noncoding RNAs.     

The extended loops of ribosomal proteins L4 and L22 are not required for ribosome assembly or L4-mediated autogenous control

Ribosomal proteins L4 and L22 both have a globular domain that sits on the surface of the large ribosomal subunit and an extended loop that penetrates its core. The tips of both loops contribute to the lining of the peptide exit tunnel and have been implicated in a gating mechanism that might regulate the exit of nascent peptides. Also, the extensions of L4 and L22 contact multiple domains of 23S rRNA, suggesting they might facilitate rRNA folding during ribosome assembly. To learn more about the roles of these extensions, we constructed derivatives of both proteins that lack most of their extended loops. Our analysis of ribosomes carrying L4 or L22 deletion proteins did not detect any significant difference in their sedimentation property or polysome distribution. Also, the role of L4 in autogenous control was not affected. We conclude that these extensions are not required for ribosome assembly or for L4-mediated autogenous control of the S10 operon.     

ESF1 is required for 18S rRNA synthesis in Saccharomyces cerevisiae

We report that Esf1p (Ydr365cp), an essential, evolutionarily conserved nucleolar protein, is required for the biogenesis of 18S rRNA in Saccharomyces cerevisiae. Depletion of Esf1p resulted in delayed processing of 35S precursor and a striking loss of 18S rRNA. Esf1p physically associated with ribosomal proteins and proteins involved in 18S rRNA biogenesis. Consistent with its role in 18S rRNA biogenesis, Esf1p also physically associated with U3 and U14 snoRNAs, but did not appear to be a core component of the SSU processome. These data indicate that Esf1p plays a direct role in early pre-rRNA processing.     

Bms1p, a G-domain-containing protein, associates with Rcl1p and is required for 18S rRNA biogenesis in yeast

Maturation of 18S rRNA and biogenesis of the 40S ribosomes in yeast requires a large number of trans-acting factors, including the U3 small nucleolar ribonucleoprotein (U3 snoRNP), and the recently characterized cyclase-like protein Rcl1p. U3 snoRNP is a key particle orchestrating early 35S rRNA cleavage events. A unique property of Rcl1p is that it specifically associates with U3 snoRNP, but this association appears to occur only at the level of nascent ribosomes and not with the U3 monoparticle. Here we report the characterization of Bms1p, a protein that associates with Rcl1p in multiple structures, including a specific complex sedimenting at around 10S. Like Rcl1p, Bms1p is an essential, evolutionarily conserved, nucleolar protein, and its depletion interferes with processing of the 35S pre-rRNA at sites A0, A1, and A2, and the formation of 40S subunits. The N-terminal domain of Bms1p has structural features found in regulatory GTPases and we demonstrate that mutations of amino acids implicated in GTP/GDP binding affect Bms1p activity in vivo. The results indicate that Bms1p may act as a molecular switch during maturation of the 40S ribosomal subunit in the nucleolus.     

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.     

Evidence that in higher plants the 25S and 18S rRNA genes are not interspersed with genes for 5S rRNA

DNA samples from various higher plants (Phaseolus aureus, Glycine max, Matthiola incana, Brassica pekinensis, Cucumis melo) were centrifuged in actinomycin-caesium chloride gradients and the genes coding for the ribosomal RNAs were detected by hybridisation with tritium labelled 5S and 25S+18S rRNA, respectively. With DNA of low molecular weight (< 5×10⁶ daltons) the 5S and 25S+18S rRNA genes are often fractionated together. A good separation of the genes for 25S+18S rRNA from the 5S rRNA genes occurred only with high molecular weight DNA (> 10×10⁶ daltons) indicating that at least most of the 5S rRNA genes are not linked to, or interspersed with, the genes coding for 25S and 18S rRNA. This result is in agreement with the situation in animal cells and in contrast to that reported for bacteria, lower eukaryotes and chloroplasts.     

The 60S associated ribosome biogenesis factor LSG1‐2 is required for 40S maturation in Arabidopsis thaliana

Ribosome biogenesis involves a large ensemble of trans‐acting factors, which catalyse rRNA processing, ribosomal protein association and ribosomal subunit assembly. The circularly permuted GTPase Lsg1 is such a ribosome biogenesis factor, which is involved in maturation of the pre‐60S ribosomal subunit in yeast. We identified two orthologues of Lsg1 in Arabidopsis thaliana. Both proteins differ in their C‐terminus, which is highly charged in atLSG1‐2 but missing in atLSG1‐1. This C‐terminus of atLSG1‐2 contains a functional nuclear localization signal in a part of the protein that also targets atLSG1‐2 to the nucleolus. Furthermore, only atLSG1‐2 is physically associated with ribosomes suggesting its function in ribosome biogenesis. Homozygous T‐DNA insertion lines are viable for both LSG1 orthologues. In plants lacking atLSG1‐2 18S rRNA precursors accumulate and a 20S pre‐rRNA is detected, while the amount of pre‐rRNAs that lead to the 25S and 5.8S rRNA is not changed. Thus, our results suggest that pre‐60S subunit maturation is important for the final steps of pre‐40S maturation in plants. In addition, the lsg1‐2 mutants show severe developmental defects, including triple cotyledons and upward curled leaves, which link ribosome biogenesis to early plant and leaf development.     

Bms1p, a novel GTP-binding protein, and the related Tsr1p are required for distinct steps of 40S ribosome biogenesis in yeast

Bms1p and Tsr1p define a novel family of proteins required for synthesis of 40S ribosomal subunits in Saccharomyces cerevisiae. Both are essential and localize to the nucleolus. Tsr1p shares two extended regions of similarity with Bms1p, but the two proteins function at different steps in 40S ribosome maturation. Inactivation of Bms1p blocks at an early step, leading to disappearance of 20S and 18S rRNA precursors. Also, slight accumulation of an aberrant 23S product and significant 35S accumulation are observed, indicating that pre-rRNA processing at sites A0, A1, and A2 is inhibited. In contrast, depletion of Tsr1p results in accumulation of 20S rRNA. Because processing of 20S to 18S rRNA occurs in the cytoplasm, this suggests that Tsr1p is required for assembly of a transport- or maturation-competent particle or is specifically required for transport of 43S pre-ribosomal particles, but not 60S ribosome precursors, from the nucleus to the cytosol. Finally, Bms1p is a GTP-binding protein, the first found to function in ribosome assembly or rRNA processing.     

Phosphorylation of ribosomal protein L18 is required for its folding and binding to 5S rRNA.

Ribosomal protein L18 from Bacillus stearothermophilus (bL18) includes a previously unreported phosphoserine residue. The folded conformation of the protein is stabilized by the dianionic form of the phosphate group of that residue. In the absence of Mg2+, the pK(a) of the phosphate group is so high that the protein is not fully folded at pH 7. In the presence of Mg2+, its pK(a) drops significantly, and consequently the native conformation of bL18 becomes stable at pH 7 and the protein is able to bind to 5S rRNA. Dephosphorylated bL18 does not bind to 5S rRNA at neutral pH.     

Schizosaccharomyces pombe Noc3 is essential for ribosome biogenesis and cell division but not DNA replication

The initiation of eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at chromosomal origins of DNA replication. Pre-RC assembly requires the essential DNA replication proteins ORC, Cdc6, and Cdt1 to load the MCM DNA helicase onto chromatin. Saccharomyces cerevisiae Noc3 (ScNoc3), an evolutionarily conserved protein originally implicated in 60S ribosomal subunit trafficking, has been proposed to be an essential regulator of DNA replication that plays a direct role during pre-RC formation in budding yeast. We have cloned Schizosaccharomyces pombe noc3(+) (Spnoc3(+)), the S. pombe homolog of the budding yeast ScNOC3 gene, and functionally characterized the requirement for the SpNoc3 protein during ribosome biogenesis, cell cycle progression, and DNA replication in fission yeast. We showed that fission yeast SpNoc3 is a functional homolog of budding yeast ScNoc3 that is essential for cell viability and ribosome biogenesis. We also showed that SpNoc3 is required for the normal completion of cell division in fission yeast. However, in contrast to the proposal that ScNoc3 plays an essential role during DNA replication in budding yeast, we demonstrated that fission yeast cells do enter and complete S phase in the absence of SpNoc3, suggesting that SpNoc3 is not essential for DNA replication in fission yeast.     

BCCIP is required for nucleolar recruitment of eIF6 and 12S pre-rRNA production during 60S ribosome biogenesis

Ribosome biogenesis is a fundamental process required for cell proliferation. Although evolutionally conserved, the mammalian ribosome assembly system is more complex than in yeasts. BCCIP was originally identified as a BRCA2 and p21 interacting protein. A partial loss of BCCIP function was sufficient to trigger genomic instability and tumorigenesis. However, a complete deletion of BCCIP arrested cell growth and was lethal in mice. Here, we report that a fraction of mammalian BCCIP localizes in the nucleolus and regulates 60S ribosome biogenesis. Both abrogation of BCCIP nucleolar localization and impaired BCCIP–eIF6 interaction can compromise eIF6 recruitment to the nucleolus and 60S ribosome biogenesis. BCCIP is vital for a pre-rRNA processing step that produces 12S pre-rRNA, a precursor to the 5.8S rRNA. However, a heterozygous Bccip loss was insufficient to impair 60S biogenesis in mouse embryo fibroblasts, but a profound reduction of BCCIP was required to abrogate its function in 60S biogenesis. These results suggest that BCCIP is a critical factor for mammalian pre-rRNA processing and 60S generation and offer an explanation as to why a subtle dysfunction of BCCIP can be tumorigenic but a complete depletion of BCCIP is lethal.     

The UBA domains of NUB1L are required for binding but not for accelerated degradation of the ubiquitin-like modifier FAT10

Proteins selected for degradation are labeled with multiple molecules of ubiquitin and are subsequently cleaved by the 26 S proteasome. A family of proteins containing at least one ubiquitin-associated (UBA) domain and one ubiquitin-like (UBL) domain have been shown to act as soluble ubiquitin receptors of the 26 S proteasome and introduce a new level of specificity into the degradation system. They bind ubiquitylated proteins via their UBA domains and the 26 S proteasome via their UBL domain and facilitate the contact between substrate and protease. NEDD8 ultimate buster-1 long (NUB1L) belongs to this class of proteins and contains one UBL and three UBA domains. We recently reported that NUB1L interacts with the ubiquitin-like modifier FAT10 and accelerates its degradation and that of its conjugates. Here we show that a deletion mutant of NUB1L lacking the UBL domain is still able to bind FAT10 but not the proteasome and no longer accelerates FAT10 degradation. A version of NUB1L lacking all three UBA domains, on the other hand, looses the ability to bind FAT10 but is still able to interact with the proteasome and accelerates the degradation of FAT10. The degradation of a FAT10 mutant containing only the C-terminal UBL domain is also still accelerated by NUB1L, even though the two proteins do not interact. In addition, we show that FAT10 and either one of its UBL domains alone can interact directly with the 26 S proteasome. We propose that NUB1L not only acts as a linker between the 26 S proteasome and ubiquitin-like proteins, but also as a facilitator of proteasomal degradation.     

Yeast Krr1p physically and functionally interacts with a novel essential Kri1p, and both proteins are required for 40S ribosome biogenesis in the nucleolus

Using a two-hybrid screening with TOM1, a putative ubiquitin-ligase gene of Saccharomyces cerevisiae, we isolated KRR1, a homologue of human HRB2 (for human immunodeficiency virus type 1 Rev-binding protein 2). To characterize the gene function, we constructed temperature-sensitive krr1 mutants and isolated two multicopy suppressors. One suppressor is RPS14A, encoding a 40S ribosomal protein. The C-terminal-truncated rpS14p, which was reported to have diminished binding activity to 18S rRNA, failed to suppress the krr1 mutant. The other suppressor is a novel gene, KRI1 (for KRR1 interacting protein; YNL308c). KRI1 is essential for viability, and Kri1p is localized to the nucleolus. We constructed a galactose-dependent kri1 strain by placing KRI1 under control of the GAL1 promoter, so that expression of KRI1 was shut off when transferring the culture to glucose medium. Polysome and 40S ribosome fractions were severely decreased in the krr1 mutant and Kri1p-depleted cells. Pulse-chase analysis of newly synthesized rRNAs demonstrated that 18S rRNA is not produced in either mutant. However, wild-type levels of 25S rRNA are made in either mutant. Northern analysis revealed that the steady-state levels of 18S rRNA and 20S pre-rRNAs were reduced in both mutants. Precursors for 18S rRNA were detected but probably very unstable in both mutants. A myc-tagged Kri1p coimmunoprecipitated with a hemagglutinin-tagged Krr1p. Furthermore, the krr1 mutant protein was defective in its interaction with Kri1p. These data lead us to conclude that Krr1p physically and functionally interacts with Kri1p to form a complex which is required for 40S ribosome biogenesis in the nucleolus.     

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.     

The complete amino acid sequences of the 5 S rRNA binding proteins L5 and L18 from the moderate thermophile Bacillus stearothermophilus ribosome

The complete amino acid sequences of the 5 S rRNA binding proteins L5 and L18 isolated from ribosomes of the moderate thermophile Bacillus stearothermophilus are presented. This has been achieved by the sequence analysis of peptides derived by enzymatic digestions with trypsin, chymotrypsin, pepsin, and Staphylococcus aureus protease, as well as by chemical cleavage with cyanogen bromide. The proteins L5 and L18 consist of 179 and 120 amino acid residues, and have Mr values of 20,163 and 13,473, respectively. A comparison of the sequences with their counterparts from the Escherichia coli ribosome reveals 59% identical residues for L5, and 53% for L18. For both proteins, the distribution of conserved regions is not random along the protein chains: some regions are highly conserved while others are not. The regions which are conserved during evolution may be important for the interaction with the 5 S rRNA molecule.     

Nob1p is required for cleavage of the 3' end of 18S rRNA

We report the characterization of a novel factor, Nob1p (Yor056c), which is essential for the synthesis of 40S ribosome subunits. Genetic depletion of Nob1p strongly inhibits the processing of the 20S pre-rRNA to the mature 18S rRNA, leading to the accumulation of high levels of the 20S pre-rRNA together with novel degradation intermediates. 20S processing occurs within a pre-40S particle after its export from the nucleus to the cytoplasm. Consistent with a direct role in this cleavage, Nob1p was shown to be associated with the pre-40S particle and to be present in both the nucleus and the cytoplasm. This suggests that Nob1p accompanies the pre-40S ribosomes during nuclear export. Pre-40S export is not, however, inhibited by depletion of Nob1p.     

CetSINEs and AREs are not SINEs but are parts of cetartiodactyl L1

Short interspersed repetitive elements (SINEs) are widely distributed among the genomes of eukaryotes. We proposed previously that a SINE should be defined by the presence of a region homologous to a tRNA or to 7SL RNA, together with A-box and B-box promoter sequences, in order to distinguish SINEs from other short repetitive sequences, such as short segments of LINEs (long interspersed repetitive elements; Okada et al. Gene 205, 229–243, 1997). Numerous SINE sequences have been deposited to date in DNA databases. In some cases, however, designation of a particular sequence is problematic when the short repetitive sequence has been defined as a SINE without reference to the presence or absence of promoter elements specific for RNA polymerase III. We demonstrate here that four different sequences, namely, ARE1p, ARE2p, CetSINE1, and CetSINE2, each of which has been reported as a SINE, are, in fact, only partial sequences of members of a new subfamily of L1. We also demonstrate that members of this subfamily are distributed specifically among the genomes of cetartiodactyls. Received: 3 May 2000 / Accepted: 22 August 2000     

Accessibility of 18S rRNA in human 40S subunits and 80S ribosomes at physiological magnesium ion concentrations--implications for the study of ribosome dynamics

Protein biosynthesis requires numerous conformational rearrangements within the ribosome. The structural core of the ribosome is composed of RNA and is therefore dependent on counterions such as magnesium ions for function. Many steps of translation can be compromised or inhibited if the concentration of Mg(2+) is too low or too high. Conditions previously used to probe the conformation of the mammalian ribosome in vitro used high Mg(2+) concentrations that we find completely inhibit translation in vitro. We have therefore probed the conformation of the small ribosomal subunit in low concentrations of Mg(2+) that support translation in vitro and compared it with the conformation of the 40S subunit at high Mg(2+) concentrations. In low Mg(2+) concentrations, we find significantly more changes in chemical probe accessibility in the 40S subunit due to subunit association or binding of the hepatitis C internal ribosomal entry site (HCV IRES) than had been observed before. These results suggest that the ribosome is more dynamic in its functional state than previously appreciated.