The U14 snoRNA is required for 2'-O-methylation of the pre-18S rRNA in Xenopus oocytes.

We have studied the role of the U14 small nucleolar RNA (snoRNA) in pre-rRNA methylation and processing in Xenopus oocytes. Depletion of U14 in Xenopus oocytes was achieved by co-injecting two nonoverlapping antisense oligonucleotides. Focusing on the earliest precursor, depletion experiments revealed that the U14 snoRNA is essential for 2'-O-ribose methylation at nt 427 of the 18S rRNA. Injection of U14-depleted oocytes with specific U14 mutant snoRNAs indicated that conserved domain B, but not domain A, of U14 is required for the methylation reaction. When the effect of U14 on pre-rRNA processing is assayed, we find only modest effects on 18S rRNA levels, and no effect on the type or accumulation of 18S precursors, suggesting a role for U14 in a step in ribosome biogenesis other than cleavage of the pre-rRNA. Xenopus U14 is, therefore, a Box C/D fibrillarin-associated snoRNA that is required for site-specific 2'-O-ribose methylation of pre-rRNA.     

Secondary methylation of yeast ribosomal precursor RNA.

The timing of methylation of the ribosomal sequences of ribosomal precursor RNA (pre-rRNA) from the yeast Saccharomyces carlsbergensis was investigated by fingerprint analysis of the methylated oligonucleotides derived from the various precursors. From the total of 37 ribose and 6 base-methyl groups found in 26-S rRNA, the two copies of the base-methylated nucleoside m3U as well as the doubly methylated sequence Um-Gm psi are not yet present in 37-S RNA, the predominant common precursor of 26-S and 17-S rRNA. Introduction of these methyl groups into the ribosomal sequences appears to take place at the level of 29-S pre-rRNA, the immediate precursor to 26-S rRNA. From the total of 18 ribose-methylated and 6 base-methylated nucleosides found in 17-S rRNA, the latter group (one copy of m7G, the m62A-m62A- sequence and the hypermodified methylated nucleoside "mX") is completely missing in 37-S pre-rRNA. The methyl group of m7G is introduced into 18-S pre-rRNA, the direct precursor of 17-S rRNA, in the nucleus. The -m62A-m62A- sequence is methylated after transport of the 18-S pre-rRNA to the cytoplasm prior to the final maturation into 17-S rRNA.     

Ribosomal RNA synthesis in liver of adrenalectomized rats after partial hepatectomy

The ribosome formation in four experimental groups: normal, adrenalectomized, partially hepatectomized and adrenalectomized — partially hepatectomized rats was studied. Ribosomal RNA was labelled for different intervals and the transfer of the radioactivity from 45 S pre-rRNA through the nucleolar pre-rRNA and rRNA pools into cytoplasmic 28S and 18S rRNA was followed. The results show that there are at least two ways of positive control of rRNA synthesis, one of them being glucocorticoid-dependent. The acceleration of the pre-rRNA processing through the shortest maturation pathway in regenerating liver is reduced in the absence of the hormone. Glucocorticoids do not influence nucleo-cytoplasmic rRNA transport.     

Alterations in the processing of rat-liver ribosomal RNA caused by cycloheximide inhibition of protein synthesis.

Cycloheximide given in vivo at low doses (2--5 mg/kg body weight) causes within 30 min a complete inhibition of protein synthesis in rat liver. The labelling of nuclear proteint is also strongly inhibited. Under these conditions, the amount of nucleolar 45-S pre-rRNA and its [14C]-orotate labelling remain unaffected for at least 4 h. These results show that initially the rates of synthesis and processing of 45-S pre-rRNA are not appreciably altered. On the other hand, drastic alterations in the 45-S pre-rRNA processing pathways occur at the early stages of cycloheximide action. Formation of 18-S rRNA is abolished and that of 28S rRNA is reduced to about half the level in control rats. This dichotomy in the production of the two ribosomal particles may be correlated with a block in the formation of 41-S and 21-S pre-rRNA. Generation of 36-S and 32-S pre-rRNA is still taking place, but the rate of their processing to nucleolar 28-S rRNA is decreased, thus causing the accumulation of these two pre-rRNA species. In parallel, processing of 45-S pre-rRNA to an aberrant 39-S rRNA species is markedly enhanced. The results obtained show that the channelling of nucleolar pre-rRNA along alternative processing pathways is under stringent control by the continuous supply of critical protein(s).     

Depletion of U3 small nucleolar RNA inhibits cleavage in the 5'' external transcribed spacer of yeast pre-ribosomal RNA and impairs formation of 18S ribosomal RNA.

Multiple processing events are required to convert a single eukaryotic pre-ribosomal RNA (pre-rRNA) into mature 18S (small subunit), 5.8S and 25-28S (large subunit) rRNAs. We have asked whether U3 small nucleolar RNA is required for pre-rRNA processing in vivo by depleting Saccharomyces cerevisiae of U3 by conditional repression of U3 synthesis. The resulting pattern of accumulation and depletion of specific pre-rRNAs indicates that U3 is required for multiple events leading to the maturation of 18S rRNA. These include an initial cleavage within the 5' external transcribed spacer, resembling the U3 dependent initial processing event of mammalian pre-rRNA. Formation of large subunit rRNAs is unaffected by U3 depletion. The similarity between the effects of U3 depletion and depletion of U14 small nucleolar RNA and the nucleolar protein fibrillarin (NOP1) suggests that these could be components of a single highly conserved processing complex.     

Mammalian cells do not have a stringent response

A key attribute of the stringent response of bacteria is the rapid inhibition of ribosomal RNA synthesis mediated by unusual nucleotides in response to uncharged tRNA. The question as to whether mammalian cells show a stringent response analogous to that of bacteria was critically tested by the effective rapid amino acid starvation of both normal and transformed cells. Rapid starvation giving a high proportion of uncharged tRNA for leucine was produced within 7 minutes of expression of a nonleaky ts leucyl tRNA synthetase mutation in transformed CHO cells (tsH1) and in its normal growth control revertant (L-73). To control for the effect of temperature alone, tsrevertants of tsH1 and L-73 were included in the study, and to control for effects due simply to the inhibition of protein synthesis, the translational elongation inhibitor cycloheximide was used. In addition, rapid starvation for histidine was effected by incubation of both the CHO cell lines and of freshly explanted normal Chinese hamster embryo fibroblasts in histidine-free medium containing high concentrations of histidinol. The rate of preribosomal RNA synthesis and the extent of its maturation to mature rRNA was measured using (³H-methyl) methionine as a donor of methyl groups during synthesis and methylation of pre-rRNA. There was no effect on pre-rRNA synthesis of the rapid generation of uncharged tRNA for 45 minutes for any of the cell types tested. A nonspecific inhibition of maturation of 18S rRNA and late (3 hour) inhibition of pre-rRNA synthesis was observed, but could be mimicked by the inhibition of protein synthesis to comparable levels with cycloheximide. Less severe amino acid starvation resulting in a more physiological inhibition of protein synthesis to 30% also had no specific effect on pre-rRNA synthesis and maturation. Intracellular nucleotide pools were also examined for the appearance of unusual nucleotides such as guanosine tetraphosphate or pentaphosphate and for changes in the levels of normal nucleotides after severe amino acid starvation. No such changes could be detected. We conclude that although mammalian cells may have some biochemical reactions which respond to uncharged tRNA, they do not possess a macromolecular control system analogous to the stringent response of bacteria.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

Mutational analysis of an essential binding site for the U3 snoRNA in the 5' external transcribed spacer of yeast pre-rRNA.

The small nucleolar RNA U3 is essential for viability in yeast. We have previously shown that U3 can be cross-linked in vivo to the pre-rRNA in the 5' external transcribed spacer (ETS), at +470. This ETS region contains 10 nucleotides of perfect complementarity to U3. In a genetic background where the mutated rDNA is the only transcribed rDNA repeat, the deletion of the 10 nt complementary to U3 is lethal. Cells lacking the U3 complementary sequence in pre-rRNA fail to accumulate 18S rRNA: pre-rRNA processing is inhibited at sites A0 in the 5' ETS, A1 at the 5' end of 18S rRNA and A2 in ITS1. We show here that effects on processing at site A0 are specific for U3 and its associated proteins and are not seen on depletion of other snoRNP components. The deletion of the sequence complementary to U3 in the ETS therefore mimics all the known effects of the depletion of U3 in trans. This indicates that we have identified an essential U3 binding site on pre-rRNA, required in cis for the maturation of 18S rRNA.     

5'ETS rRNA processing facilitated by four small RNAs: U14, E3, U17, and U3.

The nucleolus, the compartment in which the large ribosomal RNA precursor (pre-rRNA) is synthesized, processed through a series of nucleolytic cleavages and modifications into the mature 18S, 5.8S, and 28S rRNAs, and assembled with proteins to form ribosomal subunits, also contains many small nucleolar RNAs (snoRNAs). We present evidence that the first processing event in mouse rRNA maturation, cleavage within the 5' external transcribed spacer, is facilitated by at least four snoRNAs: U14, U17(E1), and E3, as well as U3. These snoRNAs do not augment this processing by directing 2'-O-methylation of the pre-rRNA. A macromolecular complex in which this 5'ETS processing occurs may then function in the processing of 18S rRNA.     

Cell division and nucleolar ribonucleic acid synthesis in synchronous cultures

Summary Nucleolar RNA synthesis is inhibited and cell division delayed in synchronous cultures of mouse fibroblasts (strain L-929) treated with actinomycin D (0.04 μg per ml). The gradual loss of actinomycin D from the cells during a 2-hr period following incubation is accompanied by an increase in the rate of nucleolar RNA synthesis to the control level. Following this the rate of protein synthesis is decreased by 25% for approximately 9 hr. The length of time that nucleolar RNA and protein synthesis are inhibited accounts for the delay in mitosis 1 1/2 cell cycles later. These data support the contention that certain proteins produced during one interphase are prerequisite for division in a subsequent cycle.