Heat shock alters nuclear ribonucleoprotein assembly in Drosophila cells.

Abstract

Heterogeneous nuclear RNA is normally complexed with a specific set of proteins, forming ribonucleoprotein particles termed hnRNP. These particles are likely to be involved in mRNA processing. We have found that the structure of hnRNP is profoundly altered during the heat shock response in Drosophila cultured cells. Although hnRNA continues to be synthesized at a near-normal rate during heat shock, its assembly into hnRNP is incomplete, as evidenced by a greatly decreased protein content of the particles in Cs2SO4 density gradients. RNA-protein cross-linking conducted in vivo (Mayrand and Pederson, Proc. Natl. Acad. Sci. U.S. A. 78:2208-2212, 1981) also reveals that hnRNA made during heat shock is complexed with greatly reduced amounts of protein. The block of hnRNP assembly occurs immediately upon heat shock, even before the onset of heat shock protein synthesis. Additional experiments reveal that hnRNP assembled normally at 25°C subsequently disassembles during heat shock. The capacity for normal hnRNP assembly is gradually restored after heat-shocked cells are returned to 25°C. Heat-shocked mammalian cells also show a similar block in hnRNP assembly. We suggest that incomplete assembly of hnRNP during heat shock leads to abortive processing of most mRNA precursors and favors the processing or export (or both) of others whose pathway of nuclear maturation is less dependent on, or even independent of, normal hnRNP particle structure. This hypothesis is compatible with a large number of previous observations. Exposure of Drosophila embryos or cultured cells to elevated temperature elicits major changes in gene transcription and protein synthesis (2). Transcription of a small set of genes is activated by heat shock (4, 11), and the resulting heat shock mRNAs are rapidly exported to the cytoplasm where they are preferentially translated (19, 29, 47). Meanwhile, the appearance of new non-heat shock mRNAs in the cytoplasm ceases (46). It is not known whether this latter effect of heat shock reflects decreased rates of transcription, a reduced efficiency of mRNA processing, or both. We have previously reported that the rate of actin mRNA transcription rapidly decreases to a low level during heat shock in Drosophila cultured cells, suggesting that this particular mRNA may be regulated chiefly at the transcriptional level (11). However, there are other observations which indicate that transcription of heterogeneous nuclear RNA (hnRNA) continues during heat shock. First, although the rates of heat shock gene transcription are very high, these mRNAs nevertheless are found to comprise a surprisingly small fraction (2%) of the nonribosomal nuclear t Paper no. 22 in a series entitled "Ribonucleoprotein Organization of Eukaryotic RNA."RNA that is synthesized during heat shock (11). Second, the size distribution of nonribosomal nuclear RNA made during heat shock is heterogeneous and is similar to that of nuclear RNA from non-heat-shocked Drosophila cells (23)(24)(25)(26). In the present investigation we examined hnRNA transcr...

Reassembly and protection of small nuclear ribonucleoprotein particles by heat shock proteins in yeast cells

“…Protection of tri-snRNP levels in pretreated heat-shocked cells+ Cell extracts from wild-type (JN55) and mutant strains, as indicated above the lanes, were electrophoresed on nondenaturing polyacrylamide gels and transferred to nylon membranes+ The membranes were probed with a U6 specific DNA probe+ The positions of the U4/U6+U5 tri-snRNP, the U4/U6 and U6 snRNPs are indicated+ Lanes 1, 4, 7, 10: extracts from untreated cells (25 8C)+ Lanes 2, 5, 8, 11: extracts from cells pretreated at 37 8C for 45 min and subsequently heat shocked at 42 8C for 1 h (TT)+ Lanes 3, 6, 9, 12: extracts from cells heat shocked at 42 8C without a prior pretreatment at 37 8C+ B: Cell extracts from strain SL304A (HSP104 ) were electrophoresed for 7 h (see Materials and Methods) to illustrate the upward shift in the U4/U6+U5 and the U4/U6 snRNPs+ Lane 1: untreated cells (25 8C)+ Lane 2: cells treated at 37 8C for 45 min+ Lane 3: cells treated at 37 8C for 45 min and then heat shocked at 42 8C for 1 h+ Lane 4: cells heat shocked at 42 8C for 1 h+ in heat-shocked cells (Fig+ 2A, lane 3, see arrowheads)+ These species are also observed in untreated cells but in much lower amounts, which suggests that they are precursors of the U4/U6 and/or the tri-snRNP+ Thus, heat shock treatments may block the assembly of snRNPs or alternatively, may disassemble snRNPs into bona fide precursor species+ Pulse chase experiments will be required to differentiate between these two possibilities+ In general, the levels of snRNAs remain relatively constant following a heat shock: we observed no more than a twofold reduction in U4 and U6 snRNAs, whereas U5, U1, and U2 levels were less affected (data not shown)+ snRNPS do not appear to be the only RNPs affected by heat shock+ A number of studies have also reported alterations in hnRNP particles in heat-shocked cells (Mayrand & Pederson, 1983;Lutz et al+, 1988;Mähl et al+, 1989;de Graaf et al+, 1992;Buchenau et al+, 1997)+ Many of these alterations are associated with mRNA splicing inactivation (Gattoni et al+, 1996;Mahé et al+, 1997)+ Thus, the inactivation of mRNA splicing in heat-shocked cells appears to be multi-faceted, but specifically involves alterations in RNP complexes+…”

“…The process of mRNA splicing is extremely sensitive in vivo thermal inactivation in many different organisms (Bond & Schlesinger, 1986;Yost & Lindquist, 1986;Bond, 1988;Vogel et al+, 1995)+ Treatment of cells at sublethal heat shock temperatures for an hour or more results in a complete but reversible inactivation of mRNA splicing+ In heat-shocked HeLa cells, alterations in snRNPs have been correlated with the inactivation of mRNA splicing (Bond, 1988;Shukla et al+, 1990;Utans et al+, 1992)+ Alterations of hnRNP particles have also been observed in heat-shocked HeLa and Drosophila cells, suggesting that RNP particles, in general, are particularly sensitive to heat shock (Mayrand & Pederson, 1983;Gattoni et al+, 1996;Mahé et al+, 1997)+ Despite the numerous effects of heat on RNPs, studies have shown that in vitro splicing activity in extracts prepared from heat-shocked cells can be restored by the addition of a protein fraction consisting minimally of five proteins (Utans et al+, 1992)+ This fraction is capable of reassembling the U4/U6 and the U5 snRNPs into the U4/U6+U5 tri-snRNP, suggesting that the primary splicing defect in heat-shocked HeLa cells may be the lack of a functional tri-snRNP+…”

Hsp70 accelerates the recovery of nucleolar morphology after heat shock.

“…(Welch and Feramisco, 1984), and is consistent with cell fractionation and autoradiographic studies on Drosophila salivary gland and tissue-culture cells (Arrigo et al, 1980;Velazquez et al, 1980) and tomato cells (Neumann et al, 1984). Interestingly, at least one other heat shock protein (a mammalian hspl 1O) is found in nucleoli, both at normal and high temperatures (Subjeck et al, 1983 (Mayrand and Pederson, 1983) and have been reported to bind hsp70 (Kloetzel and Bautz, 1983).…”

In vitro effect of the Escherichia coli heat shock regulatory protein on expression of heat shock genes

“…In E. coli, it has been established that this increased synthesis reflects enhanced transcription of heat shock genes (12,25,26,(28)(29)(30)(31). Transcriptional control is a general feature of the heat shock response in many organisms, and in addition, translational control has been described for Drosophila (20) and Xenopus oocytes (2).…”