Preferential deadenylation of Hsp70 mRNA plays a key role in regulating Hsp70 expression in Drosophila melanogaster.

Abstract

Following a standard heat shock, -40%o ofHsp7O transcripts in Drosophila melanogaster lack a poly(A) tail. Since heat shock disrupts other aspects of RNA processing, this observation suggested that heat might disrupt polyadenylation as well. We find, however, that as the temperature is increased a larger fraction of Hsp7O RNA is polyadenylated. Poly(A)-deficient Hsp7O RNAs arise not from a failure in polyadenylation but from the rapid and selective removal of poly(A) from previously adenylated transcripts. Poly(A) removal is highly regulated: poly(A) is (i) removed much more rapidly from Hsp7O RNAs than from Hsp23 RNAs, (ii) removed more rapidly after mild heat shocks than after severe heat shocks, and (iii) removed more rapidly after a severe heat shock if cells have first been conditioned by a mild heat treatment. Poly(A) seems to be removed by simple deadenylation rather than by endonucleolytic cleavage 5' of the adenylation site. During recovery from heat shock, deadenylation is rapidly followed by degradation. In cells maintained at high temperatures, however, the two processes are uncoupled and Hsp7O RNAs are deadenylated without being degraded. These deadenylated mRNAs are translated with low efficiency. Deadenylation therefore allows Hsp7O synthesis to be repressed even when degradation of the mRNA is blocked. Poly(A) tail shortening appears to play a key role in regulating Hsp7O expression.The most abundant and tightly regulated heat-inducible protein in Drosophila melanogaster is Hsp7O (47,62). Hsp7O and its relatives make up a highly conserved protein family, with eukaryotic, eubacterial, and archaebacterial members sharing at least 50% amino acid identity (27,30,47). Eukaryotes have several Hsp7O proteins. Some are constitutive and are required for normal growth, whereas others are heat inducible and help organisms cope with the toxic effects of heat. In D. melanogaster the haploid genome encodes five virtually identical heat-inducible Hsp7O proteins (32), which share -70 to 80% amino acid identity with their constitutively expressed relatives (61, 64). Consistent with their independent patterns of regulation, the 3' and 5' sequences of the constitutive genes share little or no homology with those of the heat-inducible genes.The heat- strategies are used. First, the absence of introns in the Hsp7O gene (31) allows its transcripts to circumvent the high-temperature-induced block in mRNA splicing and move rapidly into the cytoplasm (91). Second, high temperatures block the turnover of the normally short-lived Hsp7O mRNA, allowing it to accumulate very rapidly to high concentrations (67). Third, the translation of normal cellular mRNAs is inhibited at high temperatures by mechanisms that act at the levels of both initiation and elongation (8,33,45,80). Hsp7O mRNAs are translated efficiently under these conditions because special sequences in their 5' untranslated leaders allow them to circumvent the block in translation (51; for a review, see reference 46).After return to normal temperatures, hsp synthe...

The lack of a stress response in Hydra oligactis is due to reduced hsp70 mRNA stability

“…Poly(A)-tail lengths of hsp70 mRNAs were assayed as described by Dellavalle et al [14]. Total RNA from heat-shocked polyps was hybridized with an oligonucleotide complementary to the 3′ end of the hsp70 coding sequence.…”

“…The length of the poly(A) tail was determined by RNAse H mapping following previously described methods [14]. 5Ϫ15 µg total RNA from polyps heat shocked for 40 min at maximal temperature were annealed to 1 µg primers konoli (5′-GTGTTCGAACTCGTCTTTATC-3′) and konhsp (5′-CTGATCTTTTTCGGCAGTCTG-3′), which are complementary to a region in the 3′ end of Hydra hsp70 mRNA.…”

Subunits of the Drosophila CCR4-NOT complex and their roles in mRNA deadenylation

“…During heat shock, mRNA decay is inhibited, and the hsp70 mRNA is stable. Synthesis of this RNA ceases upon return to the normal growth temperature, and at the same time, its decay is initiated by deadenylation (Dellavalle et al 1994;Temme et al 2004;Bönisch et al 2007). Poly(A) tail lengths at various time points of recovery were measured by Northern blotting after shortening of the RNA with RNase H and a DNA oligonucleotide hybridizing close to the 39 end of the coding sequence.…”

The 2008 Genetics Society of America Medal

“…Her group also established that it is the heat-shock proteins themselves that turn these mechanisms off, at every level Lindquist 1986, 1991;Dellavalle et al 1994;Vogel et al 1995). As it became clear that heat-shock proteins help to prevent and repair the damage caused by stress (a story to which Lindquist also made important contributions), the extremely elegant logic of the regulatory circuitry was revealed: as heat-shock proteins (Hsps) restore normal protein homeostasis they reset the damaged regulatory systems that prevent the transcription, translation, splicing, degradation, and deadenylation of normal mRNAs.…”

“…Her work revealed regulation operating at the level of RNA splicing (Yost and Lindquist 1986, 1991, selective RNA and protein transport in and out of the nucleus (Velazquez et al 1980;Wang and Lindquist 1998), selective RNA degradation Lindquist 1988, 1989), and selective deadenylation (Dellavalle et al 1994).…”