U20, a novel small nucleolar RNA, is encoded in an intron of the nucleolin gene in mammals.

We have found that intron 11 of the nucleolin gene in humans and rodents encodes a previously unidentified small nucleolar RNA, termed U20. The single-copy U20 sequence is located on the same DNA strand as the nucleolin mRNA. U20 RNA, which does not possess a trimethyl cap, appears to result from intronic RNA processing and not from transcription of an independent gene. In mammals, U20 RNA is an 80-nucleotide-long, metabolically stable species, present at about 7 x 10(3) molecules per exponentially growing HeLa cell. It has a nucleolar localization, as indicated by fluorescence microscopy following in situ hybridization with digoxigenin-labeled oligonucleotides. U20 RNA contains the box C and box D sequence motifs, hallmarks of most small nucleolar RNAs reported to date, and is immunoprecipitated by antifibrillarin antibodies. It also exhibits a 5'-3' terminal stem bracketing the box C-box D motifs like U14, U15, U16, or Y RNA. A U20 homolog of similar size has been detected in all vertebrate classes by Northern (RNA) hybridization with mammalian oligonucleotide probes. U20 RNA contains an extended region (21 nucleotides) of perfect complementarity with a phylogenetically conserved sequence in 18S rRNA. This complementarity is strongly preserved among distant vertebrates, suggesting that U20 RNA may be involved in the formation of the small ribosomal subunit like nucleolin, the product of its host gene.

Rok1p is a putative RNA helicase required for rRNA processing.

The synthesis of ribosomes involves many small nucleolar ribonucleoprotein particles (snoRNPs) as transacting factors. Yeast strains lacking the snoRNA, snR10, are viable but are impaired in growth and delayed in the early pre-rRNA cleavages at sites A0, A1, and A2, which lead to the synthesis of 18S rRNA. The same cleavages are inhibited by genetic depletion of the essential snoRNP protein Gar1p. Screens for mutations showing synthetic lethality with deletion of the SNR10 gene or with a temperature-sensitive gar1 allele both identified the ROK1 gene, encoding a putative, ATP-dependent RNA helicase of the DEAD-box family. The ROK1 gene is essential for viability, and depletion of Rok1p inhibits pre-rRNA processing at sites A0, A1, and A2, thereby blocking 18S rRNA synthesis. Indirect immunofluorescence by using a ProtA-Rok1p construct shows the protein to be predominantly nucleolar. These results suggest that Rok1p is required for the function of the snoRNP complex carrying out the early pre-rRNA cleavage reactions.

Mitosis-specific phosphorylation of nucleolin by p34cdc2 protein kinase.

Nucleolin is a ubiquitous multifunctional protein involved in preribosome assembly and associated with both nucleolar chromatin in interphase and nucleolar organizer regions on metaphasic chromosomes in mitosis. Extensive nucleolin phosphorylation by a casein kinase (CKII) occurs on serine in growing cells. Here we report that while CKII phosphorylation is achieved in interphase, threonine phosphorylation occurs during mitosis. We provide evidence that this type of in vivo phosphorylation involves a mammalian homolog of the cell cycle control Cdc2 kinase. In vitro M-phase H1 kinase from starfish oocytes phosphorylated threonines in a TPXK motif present nine times in the amino-terminal part of the protein. The same sites which matched the p34cdc2 consensus phosphorylation sequence were used in vivo during mitosis. We propose that successive Cdc2 and CKII phosphorylation could modulate nucleolin function in controlling cell cycle-dependent nucleolar function and organization. Our results, along with previous studies, suggest that while serine phosphorylation is related to nucleolin function in the control of rDNA transcription, threonine phosphorylation is linked to mitotic reorganization of nucleolar chromatin.

Molecular cloning of Xenopus fibrillarin, a conserved U3 small nuclear ribonucleoprotein recognized by antisera from humans with autoimmune disease.

Autoantibodies against U3 small nuclear ribonucleoprotein are associated with scleroderma autoimmune disease. They were shown to react with fibrillarin, a 34- to 36-kilodalton protein that has been detected in all eukaryotes tested from humans to yeasts. We isolated a 1.6-kilobase cDNA encoding fibrillarin from a Xenopus laevis cDNA library. The protein contains a 79-residue-long Gly-Arg-rich domain in its N-terminal region and a putative RNA-binding domain with ribonucleoprotein consensus sequence in its central portion. This is the first report of cloning of fibrillarin, and the deduced protein sequence is in agreement with the involvement of the protein in a ribonucleoprotein particle.

Seven novel methylation guide small nucleolar RNAs are processed from a common polycistronic transcript by Rat1p and RNase III in yeast.

Through a computer search of the genome of the yeast Saccharomyces cerevisiae, the coding sequences of seven different box C/D antisense small nucleolar RNAs (snoRNAs) with the structural hallmarks of guides for rRNA ribose methylation have been detected clustered over a 1.4-kb tract in an inter-open reading frame region of chromosome XIII. The corresponding snoRNAs have been positively identified in yeast cells. Disruption of the nonessential snoRNA gene cluster specifically suppressed the seven cognate rRNA ribose methylations but did not result in any growth delay under the conditions of yeast culture tested. The seven snoRNAs are processed from a common polycistronic transcript synthesized from an independent promoter, similar to some plant snoRNAs but in marked contrast with their vertebrate functional homologues processed from pre-mRNA introns containing a single snoRNA. Processing of the polycistronic precursor requires nucleases also involved in rRNA processing, i.e., Rnt1p and Rat1p. After disruption of the RNT1 gene, the yeast ortholog of bacterial RNase III, production of the seven mature snoRNAs was abolished, while the polycistronic snoRNA precursor accumulated. In cells lacking functional Rat1p, an exonuclease involved in the processing of both pre-rRNA and intron-encoded snoRNAs, several processing intermediates of the polycistronic precursor accumulated. This allowed for the mapping in the precursor of the presumptive Rnt1p endonucleolytic cuts which provide entry sites for subsequent exonucleolytic trimming of the pre-snoRNAs. In line with known properties of double-stranded RNA-specific RNase III, pairs of Rnt1p cuts map next to each other on opposite strands of long double-helical stems in the secondary structure predicted for the polycistronic snoRNA precursor.

The role of the Schizosaccharomyces pombe gar2 protein in nucleolar structure and function depends on the concerted action of its highly charged N terminus and its RNA-binding domains.

Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe. We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure. Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain. These drastic functional defects correlate with striking nucleolar hypertrophy. Point mutations in the conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA-protein interactions are sufficient to induce severe nucleolar modifications but only in the presence of the N-terminal domain of the protein. Gar2 and its mutants also distribute differently in glycerol gradients: gar2 lacking its RBDs is found either free or assembled into significantly larger complexes than the wild-type protein. We propose that gar2 helps the assembly on rRNA of factors necessary for 40S subunit synthesis by providing a physical link between them. These factors may be recruited by the N-terminal domain of gar2 and may not be released if interaction of gar2 with rRNA is impaired.

Accumulation of H/ACA snoRNPs depends on the integrity of the conserved central domain of the RNA-binding protein Nhp2p.

Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. How box H/ACA snoRNPs are assembled remains unknown. Here we show that yeast Nhp2p, a core component of these particles, directly binds RNA. In vitro, Nhp2p interacts with high affinity with RNAs containing irregular stem-loop structures but shows weak affinity for poly(A), poly(C) or for double-stranded RNAs. The central region of Nhp2p is believed to function as an RNA-binding domain, since it is related to motifs found in various RNA-binding proteins. Removal of two amino acids that shortens a putative beta-strand element within Nhp2p central domain impairs the ability of the protein to interact with H/ACA snoRNAs in cell extracts. In vivo, this deletion prevents cell viability and leads to a strong defect in the accumulation of H/ACA snoRNAs and Gar1p. These data suggest that proper direct binding of Nhp2p to H/ACA snoRNAs is required for the assembly of H/ACA snoRNPs and hence for the stability of some of their components. In addition, we show that converting a highly conserved glycine residue (G(59)) within Nhp2p central domain to glutamate significantly reduces cell growth at 30 and 37 degrees C. Remarkably, this modification affects the steady-state levels of H/ACA snoRNAs and the strength of Nhp2p association with these RNAs to varying degrees, depending on the nature of the H/ACA snoRNA. Finally, we show that the modified Nhp2p protein whose interaction with H/ACA snoRNAs is impaired cannot accumulate in the nucleolus, suggesting that only the assembled H/ACA snoRNP particles can be efficiently retained in the nucleolus.

Stable expression in yeast of the mature form of human telomerase RNA depends on its association with the box H/ACA small nucleolar RNP proteins Cbf5p, Nhp2p and Nop10p.

Telomerase is a ribonucleoprotein (RNP) particle required for the replication of telomeres. The RNA component, termed hTR, of human telomerase contains a domain structurally and functionally related to box H/ACA small nucleolar RNAs (snoRNAs). Furthermore, hTR is known to be associated with two core components of H/ACA snoRNPs, hGar1p and Dyskerin (the human counterpart of yeast Cbf5p). To assess the functional importance of the association of hTR with H/ACA snoRNP core proteins, we have attempted to express hTR in a genetically tractable system, Saccharomyces cerevisiae. Both mature non-polyadenylated and polyadenylated forms of hTR accumulate in yeast. The former is associated with all yeast H/ACA snoRNP core proteins, unlike TLC1 RNA, the endogenous RNA component of yeast telomerase. We show that the presence of the H/ACA snoRNP proteins Cbf5p, Nhp2p and Nop10p, but not Gar1p, is required for the accumulation of mature non-polyadenylated hTR in yeast, while accumulation of TLC1 RNA is not affected by the absence of any of these proteins. Our results demonstrate that yeast telomerase is unrelated to H/ACA snoRNPs. In addition, they show that the accumulation in yeast of the mature RNA component of human telomerase depends on its association with three of the four core H/ACA snoRNP proteins. It is likely that this is the case in human cells as well.

The post-transcriptional steps of eukaryotic ribosome biogenesis.

One of the most important tasks of any cell is to synthesize ribosomes. In eukaryotes, this process occurs sequentially in the nucleolus, the nucleoplasm and the cytoplasm. It involves the transcription and processing of pre-ribosomal RNAs, their proper folding and assembly with ribosomal proteins and the transport of the resulting pre-ribosomal particles to the cytoplasm where final maturation events occur. In addition to the protein and RNA constituents of the mature cytoplasmic ribosomes, this intricate process requires the intervention of numerous protein and small RNA trans-acting factors. These transiently interact with pre-ribosomal particles at various stages of their maturation. Most of the constituents of pre-ribosomal particles have probably now been identified and research in the field is starting to unravel the timing of their intervention and their precise mode of action. Moreover, quality control mechanisms are being discovered that monitor ribosome synthesis and degrade the RNA components of defective pre-ribosomal particles.

The SpGAR1 gene of Schizosaccharomyces pombe encodes the functional homologue of the snoRNP protein GAR1 of Saccharomyces cerevisiae.

GAR1 is a nucleolar protein which is associated with small nucleolar RNAs (snoRNAs) and which is required for pre-ribosomal RNA processing. In Saccharomyces cerevisiae, the GAR1 gene is essential for cell viability. We have cloned and sequenced the GAR1 gene from the distantly related yeast Schizosaccharomyces pombe. The SpGAR1 gene, which contains two small introns, codes for a 194 amino-acid protein of 20 kDa. A protein sequence comparison indicates that SpGAR1 is 65% identical to ScGAR1. Anti-ScGAR1 antibodies recognize SpGAR1, emphasizing the structural conservation of the protein. Immunostaining of S.pombe cells with these antibodies reveals that SpGAR1 is localized in the nucleolus, as is the case in S.cerevisiae. Moreover, SpGAR1 can substitute for GAR1 in S.cerevisiae, indicating that the two proteins are functionally equivalent. These results suggest a parallel evolutionary conservation of proteins and RNAs with which GAR1 interacts in mediating its pre-rRNA processing and viability functions. After fibrillarin, GAR1 is the second protein of the snoRNPs shown to have been conserved throughout evolution.

The family of box ACA small nucleolar RNAs is defined by an evolutionarily conserved secondary structure and ubiquitous sequence elements essential for RNA accumulation.

Eukaryotic cells contain a large number of small nucleolar RNAs (snoRNAs). A major family of snoRNAs features a consensus ACA motif positioned 3 nucleotides from the 3' end of the RNA. In this study we have characterized nine novel human ACA snoRNAs (U64-U72). Structural probing of U64 RNA followed by systematic computer modeling of all known box ACA snoRNAs revealed that this class of snoRNAs is defined by a phylogenetically conserved secondary structure. The ACA snoRNAs fold into two hairpin structures connected by a single-stranded hinge region and followed by a short 3' tail. The hinge region carries an evolutionarily conserved sequence motif, called box H (consensus, AnAnnA). The H box, probably in concert with the flanking helix structures and the ACA box characterized previously, plays an essential role in the accumulation of human U64 intronic snoRNA. The correct processing of a yeast ACA snoRNA, snR36, in mammalian cells demonstrated that the cis- and trans-acting elements required for processing and accumulation of ACA snoRNAs are evolutionarily conserved. The notion that ACA snoRNAs share a common secondary structure and conserved box elements that likely function as binding sites for common proteins (e.g., GAR1) suggests that these RNAs possess closely related nucleolar functions.

Cloning of cDNA encoding a 100 kDa nucleolar protein (nucleoline) of Chinese hamster ovary cells.

Nucleoline (100 kDa) is the major nucleolar protein in exponentially growing cells that behaves like a nucleolar organizer protein and plays a key role in rDNA transcription and prerRNA processing. We reported the isolation of 5 cDNA clones by probing a cDNA library, constructed in the expression vector lambda gt11, with a polyclonal serum raised against nucleoline. A new immunoassay, using hybrid proteins (beta gal-cDNA encoded protein) was developed to establish that the isolated cDNAs encoded parts of nucleoline. A further confirmation resulted from the sequence comparison between the cDNA encoded peptide and a 42 aa peptide isolated from rat nucleoline (1). The 5 cDNAs overlapped extensively and covered more than 90% of a full length cDNA. By probing a Northern blot with the 100 kDa cDNA, a 2650 nucleotide polyA+ RNA was detected that contained just enough information to code for nucleoline.

Interrelations between the maturation of a 100 kDa nucleolar protein and pre rRNA synthesis in CHO cells.

The synthesis of preribosomal RNA is inhibited "in vivo" and "in vitro" by the protease inhibitor leupeptin. "In vivo" leupeptin decreases by 74% the incorporation of labeled uridine into 45S pre rRNA while the synthesis of other RNA species is only slightly decreased. "In vitro", the elongation of already initiated pre rRNA chains that is achieved by incubation of isolated nucleoli is blocked by leupeptin. On the other hand, "in vitro" leupeptin has no direct effect on RNA polymerase I, tested in a nonspecific transcriptional system with Calf thymus DNA as template and in run off experiments with a cloned DNA containing the initiation site of the rDNA gene. A 100 kDa nucleolar protein which has been shown to be endoproteolytic cleaved "in vivo" (1) acts as an inhibitor of rDNA transcription in presence of leupeptin but produces little effect on the nonspecific transcription. In absence of the drug, the 100 kDa protein is processed in specific peptides which appeared to be similar to the "in vivo" maturation products. The possible role of the 100 kDa maturation process in the regulation of rDNA transcription is discussed.

Histones from Chinese hamster ovary cells. Multiple modified forms: a quantitative approach.

A quantitative analysis of histone variants and of their modified forms in exponentially growing Chinese hamster ovary cells is described. A two-step analytical procedure has been developed. Total nuclear proteins were first fractionated on a sodium dodecyl sulfate/polyacrylamide slab gel electrophoresis. After electroelution from the corresponding bands cut out of the gel, each individual histone was analysed on an acetic acid/urea/Triton X-100 slab gel. All the variants and most of the acetylated and phosphorylated molecules were thus resolved. In absence of butyrate, we have detected four, three and two different levels of acetylation respectively for H4, H3, H2A and H2B. Scanning of the stained gel has allowed the determination of the relative amount of each forms. H3 and H4 present a higher turnover of the acetate groups than H2A and H2B. For the four histones, acetate label appeared preferentially in the multi-acetylated forms then gradually in the less acetylated forms. The data suggest that the deacetylation-acetylation process of a given histone molecule involves all the acetate groups of the molecule (three for a triacetylated histone, one for a monoacetylated histone).

Developmental expression of fibrillarin and U3 snRNA in Xenopus laevis.

Fibrillarin is one of the protein components that together with U3 snRNA constitute the U3 snRNP, a small nuclear ribonucleoprotein particle involved in ribosomal RNA processing in eucaryotic cells. Using an antifibrillarin antiserum for protein detection and a fibrillarin cDNA and a synthetic oligonucleotide complementary to U3 snRNA as hybridization probes, the expression of these two components has been studied during Xenopus development. Fibrillarin mRNA is accumulated early in oogenesis, like many other messengers, and translated during oocyte growth. Fibrillarin protein is thus progressively accumulated throughout oogenesis to be assembled with U3 snRNA and used for ribosome production in the amplified nucleoli. After fertilization, the amount of U3 snRNA decreases while the maternally accumulated fibrillarin mRNA is maintained and utilized to produce more protein. After the mid-blastula transition, stored fibrillarin is assembled with newly synthesized U3 snRNA and becomes localized in the prenucleolar bodies and reforming nucleoli.

Synthesis and behaviour of small RNA species of CHO cells submitted to a heat chick.

Incubation of Chinese hamster ovary (CHO) cells for 1 hour at 43 degrees C results in an inhibition of high molecular weight RNA synthesis while most of the ow molecular weight RNAs are still synthesized. In cells returned to 37 degrees C, the transcription of high molecular weight RNA is reinitiated after 7 h recovery. The synthesis of snRNA A, C, D which are transcribed by RNA polymerase B (II) is inhibited in cells incubated at 43 degrees C while the synthesis of 4S, 5S, L and K components is not affected. During the recovery period the synthesis of low molecular weight RNA is increased variously according to the components relative to control cells : x 1.5 for 5S RNA to x 8 for snK. After 9 h recovery at 37 degrees C, snA and SnD are again synthesized but newly synthesized snC does not appear in the nucleus while a putative preC component accumulates in the cytoplasm. On the other hand, the distribution of all the pre-existing low molecular weight RNAs is not affected by the heat shock.

Detection and localization of a class of proteins immunologically related to a 100-kDa nucleolar protein.

A high-molecular-mass nucleolar protein (100-kDa protein) is associated with nascent pre-rRNA and pre-ribosomes in Chinese hamster ovary cells. We have prepared antiserum against the 100-kDa protein and we have used it to study the intracellular localization and the possible processing of this protein. Serologically related proteins were detected in the nucleolus and in ribosomes. Proteins of various subcellular fractions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, reacted with serum and the protein-immunoglobulin complexes were revealed by 125I-labeled protein A. In the nucleolus, four proteins with molecular masses of 100 kDa, 95 kDa, 76 kDa and 70 kDa were thus visualized. In the ribosomes, two proteins (of 100 kDa and 76 kDa) gave a strong reaction while six others (of 70 kDa, 60 kDa, 50 kDa, 30 kDa, 21 kDa, 18 kDa) reacted slightly. By immunological precipitation of total cell extracts, we have shown that the 100-kDa protein antiserum cross-reacts with five proteins with molecular masses of 120 kDa, 100 kDa, 95 kDa, 70 kDa and 60 kDa. Specific degradation of the 100-kDa protein into similar peptides with molecular masses of 95 kDa, 76 kDa, 70 kDa, 60 kDa and 50 kDa can be achieved by incubation of isolated nucleoli or of purified 100-kDa protein in vitro. Cleavage of the protein is due to a thiol endoprotease which is tightly bound to the 100-kDa protein. Possible relations between the maturation of this preribosomal protein into ribosomal proteins and the processing of preribosomal RNA into mature ribosomal RNA are discussed.

Rrp8p is a yeast nucleolar protein functionally linked to Gar1p and involved in pre-rRNA cleavage at site A2.

Chemical modifications and processing of the 18S, 5.8S, and 25S ribosomal RNAs from the 35S pre-ribosomal RNA depend on an important set of small nucleolar ribonucleoprotein particles (snoRNPs). Genetic depletion of yeast Gar1p, an essential common component of H/ACA snoRNPs, leads to inhibition of uridine isomerizations to pseudo-uridines on the 35S pre-rRNA and of the early pre-rRNA cleavages at sites A1 and A2, resulting in a loss of mature 18S rRNA synthesis. To identify Gar1p functional partners, we screened for mutations that are synthetically lethal with a gar1 mutant allele encoding a Gar1p mutant protein lacking its two glycine/arginine-rich (GAR) domains. We identified a previously uncharacterized Saccharomyces cerevisiae open reading frame, YDR083W (now designated RRP8), that encodes a highly conserved protein containing motifs found in methyltransferases. Rrp8p localizes to the nucleolus. A yeast strain lacking this protein is viable at 30 degrees C but displays strong growth impairment at lower temperatures. In this strain, cleavage of the pre-rRNA at site A2 is strongly affected whereas cleavages at sites A0 and A1 are only slightly inhibited or delayed.