Search

Trypanosoma brucei live in mammals as bloodstream forms and in the Tsetse midgut as procyclic forms. Differentiation from one form to the other proceeds via a growth-arrested stumpy form with low messenger RNA (mRNA) content and translation. The parasites have six eIF4Es and five eIF4Gs. EIF4E1 pairs with the mRNA-binding protein 4EIP but not with any EIF4G. EIF4E1 and 4EIP each inhibit expression when tethered to a reporter mRNA, but while tethered EIF4E1 suppresses only when 4EIP is present, suppression by tethered 4EIP does not require the interaction with EIF4E1. In growing bloodstream forms, 4EIP is preferentially associated with unstable mRNAs. Bloodstream- or procyclic-form trypanosomes lacking 4EIP have only a marginal growth disadvantage. Bloodstream forms without 4EIP are, however, defective in translation suppression during stumpy-form differentiation and cannot subsequently convert to growing procyclic forms. Intriguingly, the differentiation defect can be complemented by a truncated 4EIP that does not interact with EIF4E1. In contrast, bloodstream forms lacking EIF4E1 have a growth defect, stumpy formation seems normal, but they appear unable to grow as procyclic forms. We suggest that 4EIP and EIF4E1 fine-tune mRNA levels in growing cells, and that 4EIP contributes to translation suppression during differentiation to the stumpy form.

SummaryThe ribosome is an RNA-protein complex essential for translation in all domains of life. The structural and catalytic core of the ribosome is its ribosomal RNA (rRNA). While mutations in ribosomal protein (RP) genes are known drivers of oncogenesis, oncogenic rRNA variants have remained elusive. We discovered a cancer-specific single nucleotide variation in 18S rRNA at nucleotide 1248.U in up to 45.9% of colorectal carcinoma (CRC) patients and present across >22 cancer types. This is the site of a unique hyper-modified base, 1-methyl-3-α-amino-α-carboxyl-propyl pseudouridine (m1acp3Ψ), a >1 billion years conserved RNA modification at the ribosome’s peptidyl decoding-site. A sub-set of CRC tumors we term ‘hypo-m1acp3Ψ’, show sub-stoichiometric m1acp3Ψ-modification unlike normal control tissues. A m1acp3Ψ knockout model and hypo-m1acp3Ψ patient tumors share a translational signature, characterized by highly abundant ribosomal proteins. Thus, m1acp3Ψ-deficient rRNA forms an uncharacterized class of ‘onco-ribosome’ which may serve as a chemotherapeutic target for treating cancer patients.

Summary/AbstractTrypanosoma brucei live in mammals as bloodstream forms and in the Tsetse midgut as procyclic forms. Differentiation from one form to the other proceeds via a growth-arrested stumpy form with low mRNA content and translation. The parasites have six eIF4Es and five eIF4Gs. EIF4E1 pairs with the mRNA-binding protein 4EIP but not with any EIF4G. EIF4E1 and 4EIP each inhibit expression when tethered to a reporter mRNA. The 4E-binding motif in 4EIP is required for the interaction with EIF4E1 both in vivo and in a 2-hybrid assay, but not for the suppressive activity of 4EIP when tethered. However, the suppressive activity of EIF4E1 when tethered requires 4EIP. Correspondingly, in growing bloodstream forms, 4EIP is preferentially associated with unstable mRNAs. Trypanosomes lacking 4EIP have a marginal growth disadvantage as cultured bloodstream or procyclic forms. Bloodstream forms without 4EIP cannot make differentiation-competent stumpy forms, but the defect can be complemented by a truncated 4EIP that does not interact with EIF4E1. Bloodstream forms lacking EIF4E1 have a growth defect but can differentiate. We suggest that 4EIP and EIF4E1 fine-tune mRNA levels in growing cells, and that 4EIP is required for mRNA suppression during differentiation to the stumpy form.

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., Author Summary When trypanosomes are inside tsetse flies, they have to cope with temperature variations from below 20°C up to 37°C, due to diurnal variations and periodic intake of warm blood. In the laboratory, procyclic forms (the form that multiplies in the midgut), are routinely cultured at 27°C. When procyclic forms are heated to temperatures of 37°C and above, they decrease protein production, and at 41°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.

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.a 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 27C and after 1 hour at 39°C, and the mRNA content of 41°C heat shocks granules. We found that 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. Seventy mRNAs moved towards the polysomal fraction after the 39°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 formsAuthor summaryWhen trypanosomes are inside tsetse flies, they have to cope with temperature variations from below 20°C up to nearly 40°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°C in the laboratory. When they are heated to temperatures of 37°C and above, they shut down protein production, and at 41°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.

The life cycle of the mammalian pathogen Trypanosoma brucei involves commuting between two markedly different environments: the homeothermic mammalian host and the poikilothermic invertebrate vector. The ability to resist temperature and other stresses is essential for trypanosome survival. Trypanosome gene expression is mainly post-transcriptional, but must nevertheless be adjusted in response to environmental cues, including host-specific physical and chemical stresses. We investigate here the control of ZC3H11, a CCCH zinc finger protein which stabilizes stress response mRNAs. ZC3H11 protein levels increase at least 10-fold when trypanosomes are stressed by heat shock, proteasome inhibitors, ethanol, arsenite, and low doses of puromycin, but not by various other stresses. We found that increases in protein stability and translation efficiency both contribute to ZC3H11 accumulation. ZC3H11 is an in vitro substrate for casein kinase 1 isoform 2 (CK1.2), and results from CK1.2 depletion and other experiments suggest that phosphorylation of ZC3H11 can promote its instability in vivo. Results from sucrose density centrifugation indicate that under normal culture conditions translation initiation on the ZC3H11 mRNA is repressed, but after suitable stresses the ZC3H11 mRNA moves to heavy polysomes. The ZC3H11 3'-UTR is sufficient for translation suppression and a region of 71 nucleotides is required for the regulation. Since the control works in both bloodstream forms, where ZC3H11 translation is repressed at 37°C, and in procyclic forms, where ZC3H11 translation is activated at 37°C, we predict that this regulatory RNA sequence is targeted by repressive trans acting factor that is released upon stress., Author Summary Like other organisms, the mammalian pathogen Trypanosoma brucei is able to sense environmental changes and to change its gene expression accordingly. In contrast with other organisms, however, trypanosomes and related kinetoplastids effect these changes almost exclusively by controlling the translation of mRNAs into protein, and by adjusting the rate at which the mRNAs are degraded. ZC3H11 is an RNA binding protein, which stabilizes mRNAs that encode chaperones. Chaperones are needed to refold proteins after stress. Under normal growth conditions ZC3H11 protein is very unstable, and in addition, not much of the protein is made. Although ZC3H11 mRNA is present under normal, unstressed conditions, most of it is not translated. However, when the cells were stressed by elevated temperature, arsenite, ethanol, puromycin or proteasome inhibitors the amount of ZC3H11 rose almost 10-fold. This was caused by a combination of increased protein stability and enhanced translation of the mRNA. We found that a 71 nucleotide segment of the 3'-untranslated region of the ZC3H11 mRNA was responsible for the regulated translational blockage. We also obtained evidence that casein kinase 1 isoform 2 might phosphorylate ZC3H11, and that phosphorylation can promote ZC3H11 protein degradation. Overall, our results show that the increase in the ZC3H11 level after stress occurs because of changes in protein synthesis, phosphorylation, and stability.

In most organisms, the heat-shock response involves increased heat-shock gene transcription. In Kinetoplastid protists, however, virtually all control of gene expression is post-transcriptional. Correspondingly, Trypanosoma brucei heat-shock protein 70 (HSP70) synthesis after heat shock depends on regulation of HSP70 mRNA turnover. We here show that the T. brucei CCCH zinc finger protein ZC3H11 is a post-transcriptional regulator of trypanosome chaperone mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of insect-form trypanosomes from heat shock. ZC3H11 binds to mRNAs encoding heat-shock protein homologues, with clear specificity for the subset of trypanosome chaperones that is required for protein refolding. In procyclic forms, ZC3H11 was required for stabilisation of target chaperone-encoding mRNAs after heat shock, and the HSP70 mRNA was also decreased upon ZC3H11 depletion in bloodstream forms. Many mRNAs bound to ZC3H11 have a consensus AUU repeat motif in the 3′-untranslated region. ZC3H11 bound preferentially to AUU repeats in vitro, and ZC3H11 regulation of HSP70 mRNA in bloodstream forms depended on its AUU repeat region. Tethering of ZC3H11 to a reporter mRNA increased reporter expression, showing that it is capable of actively stabilizing an mRNA. These results show that expression of trypanosome heat-shock genes is controlled by a specific RNA-protein interaction. They also show that heat-shock-induced chaperone expression in procyclic trypanosome enhances parasite survival at elevated temperatures., Author Summary When organisms are placed at a temperature that is higher than normal, their proteins start to unfold. The organisms protect themselves by increasing the synthesis of “heat-shock” proteins which can re-fold other proteins when the temperature returns to normal. In trypanosomes, the degradation of mRNAs that encode heat-shock proteins is slowed down at elevated temperatures. Trypanosoma brucei multiplies as “bloodstream forms” in the blood of mammals, at temperatures between 37–39°C; and as “procyclic forms” in Tsetse flies, which are usually at 20–37°C but can survive at 41°C. In this paper we show that in Trypanosoma brucei, a protein called ZC3H11 can bind to many heat-shock-protein mRNAs. ZC3H11 is essential in bloodstream-form trypanosomes and for recovery of procyclic-form trypanosomes after heat shock. ZC3H11 binds to an AUU repeat motif which is found in parts of the target mRNAs that do not encode protein. Several heat-shock-protein RNAs were decreased when we decreased the amount of ZC3H11 in bloodstream-form trypanosomes. These and other results show that expression of the specific subset of trypanosome heat-shock proteins is controlled by the interaction of ZC3H11 with the relevant mRNAs. They also show that the heat-shock response could enhance survival of trypanosomes in over-heated Tsetse flies.