Christine Clayton | Search bioRxiv

bioRxiv2016

The RNA-binding protein RBP10 controls a regulatory cascade that defines bloodstream-form trypanosome identity

Gene expression control in the pathogen Trypanosoma brucei relies almost exclusively on post-transcriptional mechanisms, so RNA binding proteins must assume the burden that is usually borne by transcription factors. T. brucei multiply in the blood of mammals as bloodstream forms, and in the midgut of Tsetse flies as procyclic forms. We show here that a single RNA-binding protein, RBP10, defines the bloodstream-form trypanosome differentiation state. Depletion of RBP10 from bloodstream-form trypanosomes gives cells that can grow only as procyclic forms; conversely, expression of RBP10 in procyclic forms converts them to bloodstream forms. RBP10 binds to procyclic-specific mRNAs containing an UAUUUUUU motif, targeting them for translation repression and destruction. Products of RBP10 target mRNAs include not only the major procyclic surface protein and enzymes of energy metabolism, but also protein kinases and stage-specific RNA-binding proteins: consequently, alterations in RBP10 trigger a regulatory cascade.

Molecular Biology↗

bioRxiv2016

Gene expression changes after heat shock of procyclic-form Trypanosoma brucei suggest that stress has a role in differentiation to mammalian-infective forms.

Trypanosome procyclic forms multiply in the midgut of Tsetse flies, and are routinely cultured at 27{degrees}C. Heat shocks of 37{degrees}C and above result in general inhibition of translation, and severe heat shock (41{degrees}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.a At 27{degrees}C, ZC3H11 mRNA is predominantly present as an untranslated cytosolic messenger ribonucleoprotein particle, but after heat shocks of 37{degrees}C - 41{degrees}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 27C and after 1 hour at 39{degrees}C, and the mRNA content of 41{degrees}C heat shocks granules. We found that that mRNAs that bind to ZC3H11 remained in polysomes at 39{degrees}C and were protected from sequestration in granules at 41{degrees}C. As previously seen for starvation stress granules, the mRNAs that encode ribosomal proteins were excluded from heat-shock granules. Seventy mRNAs moved towards the polysomal fraction after the 39{degrees}C heat shock; surprisingly, many of these 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\n\nAuthor summaryWhen trypanosomes are inside tsetse flies, they have to cope with temperature variations from below 20{degrees}C up to nearly 40{degrees}C, due to diurnal variations and periodic intake of warm blood. The procyclic forms, which usually multiply in the midgut, are routinely cultured at 27{degrees}C in the laboratory. When they are heated to temperatures of 37{degrees}C and above, they shut down protein production, and at 41{degrees}C, mRNAs aggregate into granules. We show here that quite a large number of mRNAs are not included in granules and continue to be used for making proteins. Some of the proteins that continue to be made are needed in order to defend the cells against the effects of heat shock. Interestingly, however, a moderate heat shock stimulates expression of genes needed for the parasites to develop further into forms that can colonise the salivary glands. It thus seems possible that in the field, temperature variations might influence the efficiency with which of trypanosomes in tsetse flies become infective for mammals.

Cell Biology↗