Heat shock causes a decrease in polysomes and the appearance of stress granules in trypanosomes independently of eIF2α phosphorylation at Thr169

Abstract

In trypanosomes there is an almost total reliance on posttranscriptional mechanisms to alter gene expression; here, heat shock was used to investigate the response to an environmental signal. Heat shock rapidly and reversibly induced a decrease in polysome abundance, and the consequent changes in mRNA metabolism were studied. Both heat shock and polysome dissociation were necessary for (1) a reduction in mRNA levels that was more rapid than normal turnover, (2) an increased number of P-body-like granules that contained DHH1, SCD6 and XRNA, (3) the formation of stress granules that remained largely separate from the P-body-like granules and localise to the periphery of the cell and, (4) an increase in the size of a novel focus located at the posterior pole of the cell that contain XRNA, but neither DHH1 nor SCD6. The response differed from mammalian cells in that neither the decrease in polysomes nor stress-granule formation required phosphorylation of eIF2α at the position homologous to that of serine 51 in mammalian eIF2α and in the occurrence of a novel XRNA-focus.Supplementary material available online at http://jcs.biologists.org/cgi/content/full/121/18/3002/DC1 Key words: Heat shock, Trypanosoma brucei, Stress granules, eIF2 alpha, P-bodies SummaryHeat shock causes a decrease in polysomes and the appearance of stress granules in trypanosomes independently of eIF2α phosphorylation at Thr169 Journal of Cell Science 3003 Heat shock stress granules and P-bodies in trypanosomes from a single promoter (Johnson et al., 1987; Kooter et al., 1987;Martinez-Calvillo et al., 2004;Martinez-Calvillo et al., 2003), and monocistronic mRNAs result from trans-splicing of a short, capped leader to the 5Ј end and linked 3Ј cleavage and polyadenylation of the upstream mRNA (Campbell et al., 1984;LeBowitz et al., 1993;Liang et al., 2003;Matthews et al., 1994;Schürch et al., 1994;Ullu et al., 1993). Consequently, the regulation of gene expression in trypanosomes is predominantly post-transcriptional (Clayton and Shapira, 2007). There is evidence for the presence of P-bodies: DHH1, XRNA and one Pumilio-family protein are located in cytoplasmic granules in normally growing cells (Caro et al., 2006;Cassola et al., 2007;Dallagiovanna et al., 2007;Dallagiovanna et al., 2005;Holetz et al., 2007), although any role in the regulation of gene expression has yet to be determined. In contrast to mRNA synthesis, the mechanisms of translation initiation and elongation appear to be typical for a eukaryote; all the factors identified in metazoa and yeast are present in the trypanosome genome (Ivens et al., 2005), although a functional analysis has only been performed on a small number (Dhalia et al., 2006;Dhalia et al., 2005) and very little is known about regulation of translation (Clayton and Shapira, 2007).Little is known about how the overall rate of gene expression is regulated, for example when trypanosomes stop growth and enter stationary phase. The only such phenomenon that has been investigated in any detail is the response to heat sh...

Metazoan stress granule assembly is mediated by P-eIF2α-dependent and -independent mechanisms

“…A recent study in S. cerevisiae indicated that yeast heat shock SGs are not regulated by eIF2a phosphorylation, as deletion of the sole yeast eIF2a kinase, Gcn2p, did not inhibit SG formation (Groušl et al 2009). Similarly, heatinduced SGs in trypanosomes are also eIF2a-independent (Kramer et al 2008), as mutant trypanosomes with an eIF2a Thr169Ala mutation still formed SGs in response to heat shock. Thr169 is thought to be functionally analogous to Ser51 in other eukaryotes, although it has not yet been directly demonstrated that phosphorylation of trypanosome eIF2a at Thr169 is a physiologically relevant mode of translational control in response to stress in this organism.…”

“…Until recently, bona fide SGs fitting all the above criteria were mainly reported in mammalian cells. However, several new studies have demonstrated the formation of SGs or SG-like bodies in Caenorhabditis elegans (Jud et al 2008), Trypanosoma brucei (Kramer et al 2008), Schizosaccharomyces pombe (Dunand-Sauthier et al 2002), and Saccharomyces cerevisiae (Buchan et al 2008;Groušl et al 2009), although SGs have yet to be documented in some other common model organisms such as Xenopus laevis, Danio rerio, and Drosophila melanogaster. The apparent evolutionary conservation of SGs as a stress survival mechanism underscores their importance throughout eukaryota.…”

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

“…The four published kinetoplastid EIF4E protein family members are either cytosolic or present in both the nucleus and cytosol (Kramer et al 2008;Freire et al 2011). The family member involved in the lion's share of mRNA translation should be critical for cell survival.…”

Hyperosmotic Stress Induces Aquaporin-dependent Cell Shrinkage, Polyphosphate Synthesis, Amino Acid Accumulation, and Global Gene Expression Changes in Trypanosoma cruzi

“…High osmolarity has also been shown to elicit global genomic expression responses in yeast (7). Another form of environmental stress produced by an increase in temperature (heat stress) has been reported to reduce the steady-state levels of most mRNAs in Trypanosoma brucei procyclic stages, although a number of mRNAs were increased (14).…”