Inhibition of protein synthesis stabilizes histone mRNA.

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167

We have examined the posttranscriptional regulation of hsp70 gene expression in two human cell lines, HeLa and 293 cells, which constitutively express high levels of HSP70. HSP70 mRNA translates with high efficiency in both control and heat-shocked cells. Therefore, heat shock is not required for the efficient translation of HSP70 mRNA. Rather, the main effect of heat shock on translation is to suppress the translatability of non-heat shock mRNAs. Heat shock, however, has a marked effect on the stability of HSP70 mRNA; in non-heatshocked cells the half-life of HSP70 mRNA is approximately 50 min, and its stability increases at least 10-fold upon heat shock. Moreover, HSP70 mRNA is more stable in cells treated with protein synthesis inhibitors, suggesting that a heat shock-sensitive labile protein regulates its turnover. An additional effect on posttranscriptional regulation of hsp7O expression can be found in adenovirus-infected cells, in which HSP70 mRNA levels decline precipititously late during infection although hsp7O transcription continues unabated.Expression of the major heat shock protein, HSP70, is under complex regulatory controls exerted at both the transcriptional and translational levels (18,43,55). Transcription of the hsp70 gene is transiently induced by environmental stress such as exposure to heat shock or heavy metals; in human cells the hsp70 gene is also expressed during normal conditions of cell growth at the Gl/S boundary of the cell cycle (49) and in response to serum stimulation (71).The effects of heat shock on posttranscriptional events include the preferential translation of heat shock mRNAs (32,41,42,62). During heat shock, HSP70 mRNA appears to be translated efficiently whereas most preexisting cellular mRNAs (hereafter referred to as "control mRNAs") are poorly translated. The selective translation of heat shock mRNAs is in part due to features within the 5' noncoding region of the mRNA (15,34,46). Although it has been suggested that heat shock mRNAs may require heat shock conditions for their efficient translation (20), there are at least two lines of evidence to suggest that these mRNAs can translate efficiently under non-stress conditions. First, heat shock mRNAs isolated from a variety of organisms translate in extracts prepared from non-heat-shocked wheat germ, Drosophila, rabbit reticulocyte, and other animal cells (for example, see references 5, 32, 37, 39, 47, 56, and 62). Second, heat shock mRNAs are transcribed and translated under a variety of normal conditions of cell growth such as the Gl/S boundary of the cell cycle (49) and during development and differentiation (9, 10, 59; S. S. Banerji, K. L. Laing, and R. I. Morimoto, Genes Devel., in press).Other posttranscriptional events induced by heat shock include a block in pre-mRNA processing (72) and effects on mRNA stability (24). During heat shock, HSP70 mRNA appears to be stable relative to its short half-life in cells recovering from heat shock (22, 23). For example, HSP70 mRNA persists in heat-shocked chicken cells wel...

Section: Discussionmentioning

confidence: 99%

Posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability.

Theodorakis¹,

Morimoto²

1987

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167

“…Changes in the rate of transcription of specific genes or alterations in transcript stability during inhibition of protein synthesis are frequently considered evidence for labile negative or positive regulatory proteins (13,19,22,23,28,35,39,45). In the present case, a short-lived positive regulatory VOL.…”

Section: Discussionmentioning

confidence: 99%

RNA turnover in Trypanosoma brucei.

Ehlers¹,

Czichos²,

Overath³

1987

Regulation of variant surface glycoprotein (VSG) mRNA turnover in Trypanosoma brucei was studied in bloodstream forms, in procyclic cells, and during in vitro transformation of bloodstream forms to procycic cells by approach-to-equilibrium labeling and pulse-chase experiments. Upon initiation of transformation at 27°C in the presence of citrate-cis-aconitate, the half-life of VSG mRNA was reduced from 4.5 h in bloodstream forms to 1.2 h in transforming cells. Concomitantly, an approximately 25-fold decrease in the rate of transcription was observed, resulting in a 100-fold reduction in the steady-state level of de novo-synthesized VSG mRNA. This low level of expression was maintained for at least 7 h, finally decreasing to an undetectable level after 24 h. Transcription of the VSG gene in established procyclic cells was undetectable. For comparison, the turnover of polyadenylated and nonpolyadenylated RNA, ,-tubulin mRNA, and mini-exon-derived RNA (medRNA) was studied. For medRNA, no significant changes in the rate of transcription or stability were observed during differentiation. In contrast, while the rate of transcription of ,B-tubulin mRNA in in vitro-cultured bloodstream forms, transforming cells, and established procyclic cells was similar, the half life was four to five times longer in procyclic cells (tj/2, 7 h) than in cultured bloodstream forms (t1/2, 1.4 h) or transforming cells (t012, 1.7 h). Inhibition of protein synthesis in bloodstream forms at 37°C caused a dramatic 20-fold decrease in the rate of VSG mRNA synthesis and a 6-fold decrease in half-life to 45 min, while f8-tubulin mRNA was stabilized 2-to 3-fold and medRNA stability remained unaffected. It is postulated that triggering transformation or inhibiting protein synthesis induces changes in the abundance of the same regulatory molecules which effect the shutoff of VSG gene transcription in addition to shortening the half-life of VSG mRNA.The regulation of mRNA synthesis in the parasitic protozoan Trypanosoma brucei represents an interesting problem. First, throughout its complex life cycle in the mammalian host and the tsetse fly, this unicellular flagellate experiences dramatic changes in environment, to which it adapts by regulating the expression of numerous genes (42); second, it has the unique feature of discontinuous mRNA synthesis (4). In an as yet undefined mechanism, a 140-nucleotide (nt) transcript, designated mini-exon-derived RNA (medRNA), donates a 35-nt sequence located at its 5' end (mini-exon) to the 5' end of all mRNAs coding for proteins (8,9,12,21,26,44 (17,36,40). The mini-exon has been explicitly demonstrated at the 5' end of the VSG and tubulin mRNAs (3,17,36,41). medRNA is transcribed from mini-exon genes present in about 200 copies per nucleus. Most of these genes are clustered in the genome in tandemly linked 1.35-kilobase (kb) repeats (11,27 bloodstream forms to procyclic cells. In a second, related part, mRNA synthesis and decay in the absence of protein synthesis were investigated. MATERIALS AND METHODSTrypanos...

“…If protein synthesis, then, is also inhibited in such a culture, the histone mRNA level rises, in some cases reaching levels above that found in the uninhibited control (9,22,27,31). If these larger changes in histone mRNA levels are reflected in a larger recovery of histone protein synthesis, the relationship between these two would be further substantiated.…”

mentioning

confidence: 91%

Mechanism for differential sensitivity of the chromosome and growth cycles of mammalian cells to the rate of protein synthesis.

Wu¹,

Bonner²

1985

It has been docuiliented widely that when the generation times of eucaryotic cells are lengthened by slowing the rate of protein synthesis, the duration of the chromosotne cycle (S, G2, and M phases) remains relatively invariant. Paradoxically, when the growth of exponentially growing cultures of CHO cells is partially inhibited with inhibitors of protein synthesis, the immediate effect is a proportionate reduction in the rate of total protein, histone protein, and DNA synthesis. However, on -further investigation it was found that over the next 2 h the rates of histone protein and DNA synthesis recover, in some cases completely to the uninhibited rate, while the synthesis rates of other proteins do. not recover. We called this process chromosome cycle compensation. The amount of compensation seen in CHO cell cultures can account quantitatively for the relative invariance in the length of the chromosome cycle (S, G2, and M phases) reported for these cells. The mechanism for this compensation involves a specific increase in the levels of histone mRNAs. An invariant chromosome cycle coupled with a lengthening growth cycle must result in a disproportionate lengthening of the GI phase. Thus, these results suggest that chromosome cycle invariance may be due more to specific cellular compensation mechanisms rather than to the more usual interpretation involving a rate-limiting step for cell cycle progression in the Gl phase.When inhibitors of protein synthesis are added to cultures of exponentially growing cells, their doubling time lengthens approximately in proportion to the decreased rate of protein synthesis. With moderate inhibition of protein synthesis, almost all of the lengthening occurs in the G1 interval while the S, G2, and M intervals, named collectively by Mitchison (17) as the DNA division or chromosome cycle, are relatively unaffected (14,15,19,20,21). Rossow et al. (21) studied a Swiss 3T3 cell line which grew with a doubling time of about 16 h, 4 h in the G1 phase and 12 h in the chromosome cycle (S, G2, and M phases). When the rate of protein synthesis in this culture was inhibited to about 35% of the control rate by the addition of cycloheximide, the doubling time increased to about 63 h with a greatly increased G1 phase of about 47 h but only a slightly increased chromosome cycle of 16 h. Liskay et al. (14) have studied the Chinese hamster cell line V79-8 which had no measurable G1 interval; the chromosome cycle of 9.2 h accounted for the doubling time. When the rate of protein synthesis in these cultures was inhibited to 60% that of the control, their doubling times increased to 14.2 h with a G1 phase of 4.2 h but only a slightly increased chromosome cycle of 10.2 h. A relative invariance for the chromosome cycle has also been reported for yeasts (11) and other fungi (16). These results are commonly interpreted in terms of a step just before the S phase, usually in the G1 phase, which is rate limiting for cell cycle progression and which is particularly sensitive to the rate of protein synthesis ...