The Trypanosoma brucei RNA-binding protein DRBD18 ensures correct mRNA trans splicing and polyadenylation patterns.

The parasite Trypanosoma brucei grows as bloodstream forms in mammals, and as procyclic forms in tsetse flies. Transcription is polycistronic, all mRNAs are trans spliced, and polyadenylation sites are defined by downstream splicing signals. Expression regulation therefore depends heavily on post-transcriptional mechanisms. The RNA-binding protein DRBD18 was previously implicated in the export of some mRNAs from the nucleus in procyclic forms. It copurifies the outer ring of the nuclear pore, mRNA export factors and exon-junction-complex proteins. We show that for more than 200 mRNAs, DRBD18 depletion caused preferential accumulation of versions with shortened 3'-untranslated regions, arising from use of polyadenylation sites that were either undetectable or rarely seen in nondepleted cells. The shortened mRNAs were often, but not always, more abundant in depleted cells than the corresponding longer versions in normal cells. Their appearance was linked to the appearance of trans-spliced, polyadenylated RNAs containing only downstream 3'-untranslated region-derived sequences. Experiments with one mRNA suggested that nuclear retention alone, through depletion of MEX67, did not affect mRNA length, suggesting a specific effect of DRBD18 on processing. DRBD18-bound mRNAs were enriched in polypyrimidine tract motifs, and DRBD18 was found in both the nucleus and the cytoplasm. We therefore suggest that in the nucleus, DRBD18 might bind to polypyrimidine tracts in 3'-UTRs of mRNA precursors. Such binding might both prevent recognition of mRNA-internal polypyrimidine tracts by splicing factors, and promote export of the processed bound mRNAs to the cytosol.

Roles and interactions of the specialized initiation factors EIF4E2, EIF4E5, and EIF4E6 in Trypanosoma brucei: EIF4E2 maintains the abundances of S-phase mRNAs.

Trypanosoma brucei has six versions of the cap-binding translation initiation factor EIF4E. We investigated the functions of EIF4E2, EIF4E3, EIF4E5, and EIF4E6 in bloodstream forms. We confirmed the protein associations previously found in procyclic forms and detected specific copurification of some RNA-binding proteins. Bloodstream forms lacking EIF4E5 grew normally and differentiated to replication-incompetent procyclic forms. Depletion of EIF4E6 inhibited bloodstream-form trypanosome growth and translation. EIF4E2 copurified only the putative RNA-binding protein SLBP2. Bloodstream forms lacking EIF4E2 multiplied slowly, had a low maximal cell density, and expressed the stumpy-form marker PAD1 but showed no evidence for enhanced stumpy-form signaling. EIF4E2 knock-out cells differentiated readily to replication-competent procyclic forms. EIF4E2 was strongly associated with a subset of mRNAs that are maximally abundant in the S-phase, and these all had decreased abundances in EIF4E2 knock-out cells. Three EIF4E2 target mRNAs are also bound and stabilized by the Pumilio domain protein PUF9. Yeast 2-hybrid results suggested that PUF9 interacts directly with SLBP2, but PUF9 was not detected in EIF4E2 pull-downs. We speculate that the EIF4E2-SLBP2 complex might interact with its target mRNAs, perhaps via PUF9, only early during G1/S, stabilizing the mRNAs in preparation for translation later in S-phase or in early G2.

Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase.

The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this process.

Interactions of the Trypanosoma brucei brucei zinc-finger-domain protein ZC3H28.

In Trypanosoma brucei and related Kinetoplastids, regulation of gene expression occurs mostly post-transcriptionally, and RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Trypanosoma brucei ZC3H28 is a 114 KDa cytoplasmic mRNA-binding protein with a single C(x)7C(x)5C(x)sH zinc finger at the C-terminus and numerous proline-, histidine- or glutamine-rich regions. ZC3H28 is essential for normal bloodstream-form trypanosome growth, and when tethered to a reporter mRNA, ZC3H28 increased reporter mRNA and protein levels. Purification of N-terminally tagged ZC3H28 followed by mass spectrometry showed enrichment of ribosomal proteins, various RNA-binding proteins including both poly(A) binding proteins, the translation initiation complex EIF4E4/EIF4G3, and the activator MKT1. Tagged ZC3H28 was preferentially associated with long RNAs that have low complexity sequences in their 3'-untranslated regions; their coding regions also have low ribosome densities. In agreement with the tethering results, after ZC3H28 depletion, the levels of a significant proportion of its bound mRNAs decreased. We suggest that ZC3H28 is implicated in the stabilization of long mRNAs that are poorly translated.

A potential role for a novel ZC3H5 complex in regulating mRNA translation in Trypanosoma brucei.

In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein. RNAi targeting ZC3H5 causes accumulation of precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500, and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5'-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of "halfmer" disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of suboptimal open reading frames.

The RNA-associated proteins MKT1 and MKT1L form alternative PBP1-containing complexes in Trypanosoma brucei.

Control of gene expression in kinetoplastids such as trypanosomes depends heavily on RNA-binding proteins that influence mRNA decay and translation. We previously showed that the trypanosome protein MKT1 forms a multicomponent protein complex: MKT1 interacts with PBP1, which in turn recruits LSM12 and poly(A)-binding protein. MKT1 is recruited to mRNAs by sequence-specific RNA-binding proteins, resulting in stabilization of the bound mRNA. We here show that PBP1, LSM12, and a 117-residue protein, XAC1 (Tb927.7.2780), are present in complexes that contain either MKT1 or an MKT1-like protein, MKT1L (Tb927.10.1490). All five proteins are present predominantly in the complexes, and we found evidence for a minor subset of complexes containing both MKT1 and MKT1L. XAC1-containing complexes reproducibly contained RNA-binding proteins that were previously found associated with MKT1. Moreover, XAC1- or MKT1-containing complexes specifically recruited one of the two poly(A)-binding proteins, PABP2, and one of the six cap-binding translation initiation complexes, EIF4E6-EIF4G5. Yeast two-hybrid assay results indicated that MKT1 directly interacts with EIF4G5. MKT1-PBP1 complexes can therefore interact with mRNAs via their poly(A) tails and caps, as well as through sequence-specific RNA-binding proteins. Correspondingly, MKT1 is associated with many mRNAs, although not with those encoding ribosomal proteins. Meanwhile, MKT1L resembles MKT1 at the C terminus but additionally features an N-terminal extension with low-complexity regions. Although MKT1L depletion inhibited cell proliferation, we found no evidence that it specifically interacts with RNA-binding proteins or mRNA. We speculate that MKT1L may compete with MKT1 for PBP1 binding and thereby modulate the function of MKT1-containing complexes.

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

ZC3H20 and ZC3H21 are related trypanosome proteins with two C(x)8 C(x)5 C(x)3 H zinc finger motifs. ZC3H20 is present at a low level in replicating mammalian-infective bloodstream forms, but becomes more abundant when they undergo growth arrest at high density; ZC3H21 appears only in the procyclic form of the parasite, which infects Tsetse flies. Each protein binds to several hundred mRNAs, with overlapping but not identical specificities. Both increase expression of bound mRNAs, probably through recruitment of the MKT1-PBP1 complex. At least 28 of the bound mRNAs decrease after depletion of ZC3H20, or of ZC3H20 and ZC3H21 together; their products include procyclic-specific proteins of the plasma membrane and energy metabolism. Simultaneous depletion of ZC3H20 and ZC3H21 causes procyclic forms to shrink and stop growing; in addition to decreases in target mRNAs, there are other changes suggestive of loss of developmental regulation. The bloodstream-form-specific protein RBP10 controls ZC3H20 and ZC3H21 expression. Interestingly, some ZC3H20/21 target mRNAs also bind to and are repressed by RBP10, allowing for dynamic regulation as RBP10 decreases and ZC3H20 and ZC3H21 increase during differentiation.

Insights into the functions and RNA binding of Trypanosoma brucei ZC3H22, RBP9 and DRBD7.

Trypanosoma brucei is unusually reliant on mRNA-binding proteins to control mRNA fate, because its protein-coding genes lack individual promoters. We here focus on three trypanosome RNA-binding proteins. ZC3H22 is specific to Tsetse fly forms, RBP9 is preferentially expressed in bloodstream forms; and DRBD7 is constitutively expressed. Depletion of RBP9 or DRBD7 did not affect bloodstream-form trypanosome growth. ZC3H22 depletion from procyclic forms caused cell clumping, decreased expression of genes required for cell growth and proliferation, and increased expression of some epimastigote markers. Apart from decreases in mRNAs encoding enzymes of glucose metabolism, levels of most ZC3H22-bound transcripts were unaffected by ZC3H22 depletion. We compared ZC3H22, RBP9 and DRBD7 RNA binding with that of 16 other RNA-binding proteins. ZC3H22, PUF3 and ERBP1 show a preference for ribosomal protein mRNAs. RBP9 preferentially binds mRNAs that are more abundant in bloodstream forms than in procyclic forms. RBP9, ZC3H5, ZC3H30 and DRBD7 prefer mRNAs with long coding regions; UBP1-associated mRNAs have long 3'-untranslated regions; and RRM1 prefers mRNAs with long 3'or 5'-untranslated regions. We suggest that proteins that prefer long mRNAs may have relatively short or degenerate binding sites, and that preferences for A or U increase binding in untranslated regions.

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing.

Kinetoplastid parasites-trypanosomes and leishmanias-infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition.

The suppressive cap-binding complex factor 4EIP is required for normal differentiation.

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.

Expression of the RNA-binding protein RBP10 promotes the bloodstream-form differentiation state in Trypanosoma brucei.

In nearly all eukaryotes, cellular differentiation is governed by changes in transcription, and stabilized by chromatin and DNA modification. Gene expression control in the pathogen Trypanosoma brucei, in contrast, 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, promotes 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: this suggests that alterations in RBP10 trigger a regulatory cascade.

Regulating a Post-Transcriptional Regulator: Protein Phosphorylation, Degradation and Translational Blockage in Control of the Trypanosome Stress-Response RNA-Binding Protein ZC3H11.

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.

Integrative analysis of the Trypanosoma brucei gene expression cascade predicts differential regulation of mRNA processing and unusual control of ribosomal protein expression.

BACKGROUND: Trypanosoma brucei is a unicellular parasite which multiplies in mammals (bloodstream form) and Tsetse flies (procyclic form). Trypanosome RNA polymerase II transcription is polycistronic, individual mRNAs being excised by trans splicing and polyadenylation. We previously made detailed measurements of mRNA half-lives in bloodstream and procyclic forms, and developed a mathematical model of gene expression for bloodstream forms. At the whole transcriptome level, many bloodstream-form mRNAs were less abundant than was predicted by the model. RESULTS: We refined the published mathematical model and extended it to the procyclic form. We used the model, together with known mRNA half-lives, to predict the abundances of individual mRNAs, assuming rapid, unregulated mRNA processing; then we compared the results with measured mRNA abundances. Remarkably, the abundances of most mRNAs in procyclic forms are predicted quite well by the model, being largely explained by variations in mRNA decay rates and length. In bloodstream forms substantially more mRNAs are less abundant than predicted. We list mRNAs that are likely to show particularly slow or inefficient processing, either in both forms or with developmental regulation. We also measured ribosome occupancies of all mRNAs in trypanosomes grown in the same conditions as were used to measure mRNA turnover. In procyclic forms there was a weak positive correlation between ribosome density and mRNA half-life, suggesting cross-talk between translation and mRNA decay; ribosome density was related to the proportion of the mRNA on polysomes, indicating control of translation initiation. Ribosomal protein mRNAs in procyclics appeared to be exceptionally rapidly processed but poorly translated. CONCLUSIONS: Levels of mRNAs in procyclic form trypanosomes are determined mainly by length and mRNA decay, with some control of precursor processing. In bloodstream forms variations in nuclear events play a larger role in transcriptome regulation, suggesting aquisition of new control mechanisms during adaptation to mammalian parasitism.

A genome-wide tethering screen reveals novel potential post-transcriptional regulators in Trypanosoma brucei.

In trypanosomatids, gene expression is regulated mainly by post-transcriptional mechanisms, which affect mRNA processing, translation and degradation. Currently, our understanding of factors that regulate either mRNA stability or translation is rather limited. We know that often, the regulators are proteins that bind to the 3'-untranslated region; they presumably interact with ribonucleases and translation factors. However, very few such proteins have been characterized in any detail. Here we describe a genome-wide screen to find proteins implicated in post-transcriptional regulation in Trypanosoma brucei. We made a library of random genomic fragments in a plasmid that was designed for expression of proteins fused to an RNA-binding domain, the lambda-N peptide. This was transfected into cells expressing mRNAs encoding a positive or negative selectable marker, and bearing the "boxB" lambda-N recognition element in the 3'-untranslated region. The screen identified about 300 proteins that could be implicated in post-transcriptional mRNA regulation. These included known regulators, degradative enzymes and translation factors, many canonical RNA-binding proteins, and proteins that act via multi-protein complexes. However there were also nearly 150 potential regulators with no previously annotated function, or functions unrelated to mRNA metabolism. Almost 50 novel regulators were shown to bind RNA using a targeted proteome array. The screen also provided fine structure mapping of the hit candidates' functional domains. Our findings not only confirm the key role that RNA-binding proteins play in the regulation of gene expression in trypanosomatids, but also suggest new roles for previously uncharacterized proteins.

Trypanosome MKT1 and the RNA-binding protein ZC3H11: interactions and potential roles in post-transcriptional regulatory networks.

The trypanosome zinc finger protein ZC3H11 binds to AU-rich elements in mRNAs. It is essential for survival of the mammalian-infective bloodstream form, where it stabilizes several mRNAs including some encoding chaperones, and is also required for stabilization of chaperone mRNAs during the heat-shock response in the vector-infective procyclic form. When ZC3H11 was artificially 'tethered' to a reporter mRNA in bloodstream forms it increased reporter expression. We here show that ZC3H11 interacts with trypanosome MKT1 and PBP1, and that domains required for both interactions are necessary for function in the bloodstream-form tethering assay. PBP1 interacts with MKT1, LSM12 and poly(A) binding protein, and localizes to granules during parasite starvation. All of these proteins are essential for bloodstream-form trypanosome survival and increase gene expression in the tethering assay. MKT1 is cytosolic and polysome associated. Using a yeast two-hybrid screen and tandem affinity purification we found that trypanosome MKT1 interacts with multiple RNA-binding proteins and other potential RNA regulators, placing it at the centre of a post-transcriptional regulatory network. A consensus interaction sequence, H(E/D/N/Q)PY, was identified. Recruitment of MKT1-containing regulatory complexes to mRNAs via sequence-specific mRNA-binding proteins could thus control several different post-transcriptional regulons.

Transcriptomes of newly-isolated Trypanosoma brucei rhodesiense reveal hundreds of mRNAs that are co-regulated with stumpy-form markers.

BACKGROUND: During natural Trypanosoma brucei infections, the parasites differentiate spontaneously into a non-dividing "stumpy" form when a certain level of parasitaemia is attained. This form is metabolically adapted for rapid further differentiation into procyclic forms upon uptake by Tsetse flies. RESULTS: We describe here four central Ugandan isolates of Trypanosoma brucei rhodesiense that have undergone only three rodent passages since isolation from human patients. As expected, SNP analysis shows that these isolates are more closely related to each other than to the commonly used strains Lister 427, Antat1.1, and TREU927. TREU927 generally has smaller copy numbers of repeated genes than the other strains, while Lister 427 trypanosomes with a 30-year history of in vitro culture and cloning have more histone genes than the other isolates. The recently isolated trypanosomes were grown in rats, and their transcriptomes characterised. In comparison with cultured procyclic and bloodstream forms, there were increases in mRNAs encoding the stumpy-form markers ESAG9 and PIP39, with coordinated alterations in the levels of over 600 additional mRNAs. Numerous mRNAs encoding proteins of no known function were either increased or decreased. The products of the mRNAs that were increased in parallel with PIP39 included not only enzymes of procyclic-form metabolism, but also components of the translational and RNA control machineries. Many of the mRNAs that were decreased in cells with elevated PIP39 reflected reduced cell division. CONCLUSIONS: These transcriptomes suggest new avenues for research into the regulation of trypanosome differentiation.

Transcriptome-wide analysis of trypanosome mRNA decay reveals complex degradation kinetics and suggests a role for co-transcriptional degradation in determining mRNA levels.

African trypanosomes are an excellent system for quantitative modelling of post-transcriptional mRNA control. Transcription is constitutive and polycistronic; individual mRNAs are excised by trans splicing and polyadenylation. We here measure mRNA decay kinetics in two life cycle stages, bloodstream and procyclic forms, by transcription inhibition and RNASeq. Messenger RNAs with short half-lives tend to show initial fast degradation, followed by a slower phase; they are often stabilized by depletion of the 5'-3' exoribonuclease XRNA. Many longer-lived mRNAs show initial slow degradation followed by rapid destruction: we suggest that the slow phase reflects gradual deadenylation. Developmentally regulated mRNAs often show regulated decay, and switch their decay pattern. Rates of mRNA decay are good predictors of steady state levels for short mRNAs, but mRNAs longer than 3 kb show unexpectedly low abundances. Modelling shows that variations in splicing and polyadenylation rates can contribute to steady-state mRNA levels, but this is completely dependent on competition between processing and co-transcriptional mRNA precursor destruction.

The roles of 3'-exoribonucleases and the exosome in trypanosome mRNA degradation.

The degradation of eukaryotic mRNAs can be initiated by deadenylation, decapping, or endonuclease cleavage. This is followed by 5'-3' degradation by homologs of Xrn1, and/or 3'-5' degradation by the exosome. We previously reported that, in African trypanosome Trypanosoma brucei, most mRNAs are deadenylated prior to degradation, and that depletion of the major 5'-3' exoribonuclease XRNA preferentially stabilizes unstable mRNAs. We now show that depletion of either CAF1 or CNOT10, two components of the principal deadenylation complex, strongly inhibits degradation of most mRNAs. RNAi targeting another deadenylase, PAN2, or RRP45, a core component of the exosome, preferentially stabilized mRNAs with intermediate half-lives. RRP45 depletion resulted in a 5' bias of mRNA sequences, suggesting action by a distributive 3'-5' exoribonuclease. Results suggested that the exosome is involved in the processing of trypanosome snoRNAs. There was no correlation between effects on half-lives and on mRNA abundance.

Post-transcriptional regulation of the trypanosome heat shock response by a zinc finger protein.

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.

Depletion of the Trypanosome Pumilio domain protein PUF2 or of some other essential proteins causes transcriptome changes related to coding region length.

Pumilio domain RNA-binding proteins are known mainly as posttranscriptional repressors of gene expression that reduce mRNA translation and stability. Trypanosoma brucei has 11 PUF proteins. We show here that PUF2 is in the cytosol, with roughly the same number of molecules per cell as there are mRNAs. Although PUF2 exhibits a low level of in vivo RNA binding, it is not associated with polysomes. PUF2 also decreased reporter mRNA levels in a tethering assay, consistent with a repressive role. Depletion of PUF2 inhibited growth of bloodstream-form trypanosomes, causing selective loss of mRNAs with long open reading frames and increases in mRNAs with shorter open reading frames. Reexamination of published RNASeq data revealed the same trend in cells depleted of some other proteins. We speculate that these length effects could be caused by inhibition of the elongation phase of transcription or by an influence of translation status or polysomal conformation on mRNA decay.