Changes in RNA metabolism and accumulation of presumptive messenger RNA during transition from the growing to the quiescent state of cultured mouse fibroblasts

Ps, Rudland; Weil, Susan C.; Hunter, A. R.

doi:10.1016/0022-2836(75)90150-3

Cited by 108 publications

(40 citation statements)

References 38 publications

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“…2). Moreover, bop1 mRNA levels begin to rise in mid to late G 1 , coincident with the timing of rRNA synthesis (24,65,70). As expected of a protein that plays a role in ribosome maturation, Bop1 appears to be highly conserved throughout evolution.…”

Section: Discussionmentioning

confidence: 91%

Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

Strezoska

Pestov

Lau

2000

Molecular and Cellular Biology

166

182

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We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G 1 in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1⌬, which lacks 231 amino acids in the N-terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1⌬ leads to cell growth arrest in the G 1 phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1⌬ expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1⌬ in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.Biogenesis of the eukaryotic ribosomes occurs in the nucleolus, a complex nuclear organelle that forms around the nucleolar organizer regions located in heterochromatic chromosomal sites containing multiple rRNA genes (69, 81). The organization of the nucleolus and the assembly of ribosomes are coupled to transcription of rDNA by RNA polymerase I, which synthesizes a large primary precursor transcript. This precursor transcript is then processed into the mature 18S, 5.8S, and 28S/25S rRNAs. These rRNAs are assembled into preribosomes with some 80 ribosomal proteins that are transported into the nucleus, and with the 5S rRNA, transcribed by RNA polymerase III outside of the nucleolus (28). A large number of small nucleolar RNAs (snoRNAs) and nonribosomal proteins are also recruited to the nucleolus to participate in the modification, processing, and assembly of the rRNAs and proteins into ribonucleoprotein (RNP) particles. These preribosomal RNP (pre-rRNP) particles mature into nearly complete ribosomal subunits prior to their export out of the nucleus.Upon synthesis, the primary precursor rRNA transcript is modified by ribose methylation and pseudouridine conversion and processed through a series of nucleolytic cleavages into the matured rRNAs (21) (see Fig. 6). In vertebrates, the arrangement of the 47S primary precursor transcript begins with a 5Ј external transcribed spacer (5Ј-ETS), followed by the 18S rRNA, internal transcribed spacer 1 (ITS1), 5.8S rRNA, internal transcribed spacer 2 (ITS2), 28S rRNA, and t...

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Section: Discussionmentioning

confidence: 91%

Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

Strezoska

Pestov

Lau

2000

Molecular and Cellular Biology

166

182

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“…We do not know if the stability of the inherited embryonic mRNA sequences also changes with larval growth rate. However, previous investigations have determined that polyadenylated RNA is equally stable in resting and growing tissue culture cells (1, 32,37) and that the half-life of specific mRNA species does not change as a function of Escherichia coli growth rate (11,19). In any case our major concern is the relative stability of mRNAs in this system.…”

Section: Drosophila Embryonic Mrnas Decay With a Continuum Of Half-livesmentioning

confidence: 95%

Differential stability of Drosophila embryonic mRNAs during subsequent larval development.

Winkles¹,

Grainger²

1985

The Journal of Cell Biology

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The relative stabilities of specific embryonic mRNAs that persist in Drosophila melanogaster larvae were determined using an approach that combined RNA density labeling with cell-free translation. Unlike the other methods commonly used to measure the decay of individual mRNAs, the density labeling approach does not depend on the use of transcriptional inhibitors or on the measurement of precursor pool specific activities. Using this approach, we have determined that different embryonic mRNA species persist for varying periods during subsequent development, with half-lives ranging from ~2 to ~30 h. The embryonic histone mRNAs are relatively unstable; they are no longer detectable by 9 h of larval development. By 41 h of larval development, 90% of the nonhistone mRNAs assayed have decayed considerably; computerized scanning densitometry of translation products indicates that these transcripts are not decaying as members of discrete half-life classes. The persisting mRNAs that remain are very long-lived; their in vitro translation products can still be detected after 91 h of larval development. We have tentatively identified the mRNAs that encode actin, tropomyosin, and tubulin as members of this stable mRNA population. Although embryonic mRNAs do fall into these three broad classes of stability, they appear to decay with a continuum of half-lives. Because the range of half-lives is so great, mRNA stability is probably an important factor controlling mRNA abundance during Drosophila development.The cytoplasmic abundance of a particular mRNA can be controlled at both the transcriptional and posttranscriptional levels. One of the most poorly understood posttranscriptional controls is that which defines the stability of specific mRNA sequences. The importance of turnover rate in controlling mRNA abundance, and thus gene expression, is best illustrated by the observation that many of the mRNAs that encode the most highly abundant cell type-specific polypeptides are preferentially stabilized over other mRNAs present within the same cytoplasm (6,21,25,29,44).The stability of specific eucaryotic mRNAs has most often been determined by assaying for the presence of particular mRNAs after treating cells with the transcriptional inhibitor actinomycin D (4,13,25,34,43,46). However, this kind of analysis is subject to several possible artifacts. In addition to a secondary effect on cellular protein synthesis (12,15,39,40) it has also been shown that actinomycin D treatment can both decrease the stability of total mRNA populations (41) and increase the half-life of specific mRNAs (9, 42). Also, if 1808 the actinomycin D "chase" does not begin immediately or is not completely effective, then the measured half-life of a mRNA species will appear longer than it actually is.The best experimental system to use for studying the differential stability of individual mRNAs would be one in which no transcriptional inhibition is necessary. For example, by measurement of the rate of radiolabel accumulation into specific mRNAs by usi...

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“…Furthermore, it has been shown that mRNAs from quiescent and growing cells are similar (43). However, quiescent cultures contain a great deal of nontranslated mRNA (4,44) and the activity of many enzymes is changing during GI (45). Thus, phosphorylation of S6 could play a role in the regulation of expression of mRNA during this phase of the cell cycle.…”

Section: Discussionmentioning

confidence: 99%

Multiple phosphorylation of ribosomal protein S6 during transition of quiescent 3T3 cells into early G1, and cellular compartmentalization of the phosphate donor.

Thomas¹,

Siegmann²,

Gordon³

1979

Proc. Natl. Acad. Sci. U.S.A.

137

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At 5 min after quiescent cells are induced to enter G1 there is a large increase in the amount of 32p incorporated into 40S ribosomal protein S6. Here we show that changes in the specific activities of 32Pi and compared to quiescent cultures do not account for this large increase. Instead, we demonstrate by decreased electrophoretic mobility on two-dimensional polyacrylamide gels that this increase is due to a quantitative increase in the total amount of phosphate incorporated into S6. Furthermore, pulse-chase experiments show that the phosphate that is incorporated into S6 is metabolically stable during at least the first 60 min of induction and that the incorporation of 32P into S6 responds immediately to the replacement of 32Pi by Pi in the medium, in contrast to ["y-32PJATP which changes very slowly. Thus, the S6 phosphate donor must be a compartment separate from that of the total cellular ATP.Regulation of cellular proliferation in the whole animal and in tissue culture is controlled at some point in the cell cycle after mitosis and before cells have progressed far into G1 (1, 2). Elucidation of the molecular mechanisms of this phase of the cell cycle therefore is crucial to our-understanding of the biochemical processes that control cellular proliferation. One of the earliest events affected when animal cells are induced to reenter the G1 phase of the cell cycle is a dramatic shift of monosomes to polysomes accompanied by a large increase in protein synthesis (3-5). Because protein phosphorylation/ dephosphorylation reactions may be common mediators of cellular regulatory signals (6-8) and because ribosomal proteins are known to be phosphorylated both in vitro and in vivo (for reviews, see refs. 9 and 10), we have explored the possibility that phosphorylation of specific ribosomal proteins may be involved in the transition of quiescent cultures into the G, phase of the cell cycle.It has been reported (11) that at 5 min after induction of secondary chicken embryo fibroblasts with serum, insulin, or insulin-like growth factor, there is a large increase in the amount of 32P incorporated into 40S ribosomal protein S6. In the study described here, using 3T3 fibroblasts we found that the increase in incorporation of 32p was due to a quantitative increase in the phosphorylation of S6. This was carried out by comparison of the specific activities of 32p in Pi and ATP y-phosphate pools and by examination of the change in S6 mobility on two-dimensional polyacrylamide gels. We have also examined the metabolic stability of the phosphate in S6 during the stimulation. In so doing, we have discovered evidence for the presence of a compartment for the phosphate donor pool, distinct from the total cellular ATP pool. Finally, the implications of S6 phosphorylation in relation to other known cellular events taking place during early G1 phase of the growth cycle are discussed.MATERIALS AND METHODS Cell Cultures. Swiss mouse 3T3 fibroblasts were seeded at 5 X 105 cells per 80-mm tissue culture plate in 10 ml of Dulbe...

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Changes in RNA metabolism and accumulation of presumptive messenger RNA during transition from the growing to the quiescent state of cultured mouse fibroblasts

Cited by 108 publications

References 38 publications

Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

Differential stability of Drosophila embryonic mRNAs during subsequent larval development.

Multiple phosphorylation of ribosomal protein S6 during transition of quiescent 3T3 cells into early G1, and cellular compartmentalization of the phosphate donor.

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