Translation Regulation and RNA Granule Formation after Heat Shock of Procyclic Form Trypanosoma brucei: Many Heat-Induced mRNAs Are also Increased during Differentiation to Mammalian-Infective Forms

Abstract

African trypanosome procyclic forms multiply in the midgut of tsetse flies, and are routinely cultured at 27°C. Heat shocks of 37°C and above result in general inhibition of translation, and severe heat shock (41°C) results in sequestration of mRNA in granules. The mRNAs that are bound by the zinc-finger protein ZC3H11, including those encoding refolding chaperones, escape heat-induced translation inhibition. At 27°C, ZC3H11 mRNA is predominantly present as an untranslated cytosolic messenger ribonucleoprotein particle, but after heat shocks of 37°C—41°C, the ZC3H11 mRNA moves into the polysomal fraction. To investigate the scope and specificities of heat-shock translational regulation and granule formation, we analysed the distributions of mRNAs on polysomes at 27°C and after 1 hour at 39°C, and the mRNA content of 41°C heat shock granules. We found that mRNAs that bind to ZC3H11 remained in polysomes at 39°C and were protected from sequestration in granules at 41°C. As previously seen for starvation stress granules, the mRNAs that encode ribosomal proteins were excluded from heat-shock granules. 70 mRNAs moved towards the polysomal fraction after the 39°C heat shock, and 260 increased in relative abundance. Surprisingly, many of these mRNAs are also increased when trypanosomes migrate to the tsetse salivary glands. It therefore seems possible that in the wild, temperature changes due to diurnal variations and periodic intake of warm blood might influence the efficiency with which procyclic forms develop into mammalian-infective forms.

The zinc finger proteins ZC3H20 and ZC3H21 stabilise mRNAs encoding membrane proteins and mitochondrial proteins in insect‐form Trypanosoma brucei

“…We suggest that they are likely to be secondary effects. One possibility is a response to stress, although there was no correlation with the effects of heat shock (Minia, Merce, Terrao, & Clayton, 2016) or starvation (Fritz et al, 2015). Altered metabolite levels could also be responsible (Bringaud et al, 2006;Fernandez-Moya, Carrington, & Estevez, 2014;Qiu et al, 2018;Vassella et al, 2000Vassella et al, , 2004; and it is conceivable that one or more of the directly regulated surface proteins acts as a receptor for a signal that maintains procyclic identity.…”

“…Before RNA sequencing, ribosomal RNA was depleted from the flow-through samples by incubation with specific oligonucleotides and RNaseH (Minia et al, 2016). RNASeq analysis was performed on eluted (bound) and flow-through (unbound) samples, and analysed as described previously (Droll et al, 2013;Leiss, Merce, Muchunga, & Clayton, 2016;Mugo & Clayton, 2017;Mulindwa et al, 2018).…”

Expression of normal levels of the Pumilio domain protein PUF3 is required for optimal growth of Trypanosoma brucei

“…The unbound RNA fraction had been subjected to rRNA depletion using RNase H and oligonucleotides complementary to rRNA [5]. To check the effect of this procedure on the transcriptome we sequenced the input RNA from the experiment, either with or without rRNA depletion.…”

“…RNA concentrations were determined using a Nanodrop spectrophotometer and the RNA stored at -80°C or used immediately. rRNAs were depleted from whole RNA extracts before RNAseq using a cocktail of DNA oligonucleotides targeting rRNAs with RNaseH [5]. For blotting, RNA was electrophoresed on a formaldehyde agarose gel, blotted on a nylon membrane by downward capillary for at least 4 hrs followed by UV cross-linking of RNA to the membrane using Stratagene® UV cross-linker.…”

The endoplasmic reticulum-associated mRNA-binding proteins ERBP1 and ERBP2 interact in bloodstream-formTrypanosoma brucei

“…64 mRNAs were enriched at least 2-fold in both pull-downs relative to all controls. Strikingly, nearly half of them (29) encode ribosomal proteins. Two glycosomal membrane protein mRNAs, PMP4 and PEX11, were also enriched, but those encoding other PEX proteins -including other glycosomal membrane proteins -were not, so the significance of this is uncertain.…”