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, 10 (1), 118

A tRNA Half Modulates Translation as Stress Response in Trypanosoma Brucei

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A tRNA Half Modulates Translation as Stress Response in Trypanosoma Brucei

Roger Fricker et al. Nat Commun.

Abstract

In the absence of extensive transcription control mechanisms the pathogenic parasite Trypanosoma brucei crucially depends on translation regulation to orchestrate gene expression. However, molecular insight into regulating protein biosynthesis is sparse. Here we analyze the small non-coding RNA (ncRNA) interactome of ribosomes in T. brucei during different growth conditions and life stages. Ribosome-associated ncRNAs have recently been recognized as unprecedented regulators of ribosome functions. Our data show that the tRNAThr 3´half is produced during nutrient deprivation and becomes one of the most abundant tRNA-derived RNA fragments (tdRs). tRNAThr halves associate with ribosomes and polysomes and stimulate translation by facilitating mRNA loading during stress recovery once starvation conditions ceased. Blocking or depleting the endogenous tRNAThr halves mitigates this stimulatory effect both in vivo and in vitro. T. brucei and its close relatives lack the well-described mammalian enzymes for tRNA half processing, thus hinting at a unique tdR biogenesis in these parasites.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Profiling the abundance levels of rancRNA sequencing reads. a The distribution of sequencing reads fractions assigned to different RNA classes among different growth conditions and life stages of T. brucei. Note that the category ‘ncRNA’ includes annotated T. brucei ncRNAs not listed otherwise. b Read length distribution of tRNA-derived sequencing reads observed during different growth conditions. c Sample correlation matrix showing Pearson’s correlation of expression levels of identified tRNA processing products between different growth conditions and life stages. d Ribosome-association of tRNA halves was assessed via northern blot analyses on RNA isolated from the crude ribosomal pellet from exponentially growing cells or from cells starved for two hours by incubating the parasite in PBS. The 5S rRNA (5S) served as loading control. tRNA isoacceptor anticodons and the origin of the tRNA halve (5′ or 3′) are indicated
Fig. 2
Fig. 2
tRNAThr 3′ half accumulates during stress in procyclic and bloodstream T. brucei. a T. brucei procyclic cells were starved for the indicated times by incubation in PBS. Total RNA was extracted and the presence of the tRNAThr 3’ half (37 nucleotides long; see also Supplementary Figure 10) monitored by northern blot analysis. b The presence of the tRNAThr 3′ half was investigated as in a in procyclic cells grown to different cell densities (indicated on top of the panel). c Same as a but cells were allowed to recover in normal media for the indicated periods of time after nutritional stress. In the lower panel the contrast of this part of the blot was adjusted to more clearly see the tRNAThr 3′ half. d T. brucei bloodstream cells were stressed by incubation in PBS and then allowed to recover in normal growth media. The presence of the tRNAThr 3′ half was analyzed as described in a. In all cases the EtBr-stained rRNAs serve as loading controls
Fig. 3
Fig. 3
tRNAAla 5′ half is present under normal growth but disappears upon stress in procyclic and bloodstream T. brucei. a T. brucei procyclic cells were starved for two hours by incubation in PBS. Subsequently, the cells were allowed to recover in normal media for the indicated periods of time. Total RNA was extracted and the presence of the tRNAAla 5′ half (length: 34 nucleotides) monitored by northern blot analysis. b T. brucei bloodstream cells were stressed by incubation in PBS for 30 or 60 min and then allowed to recover in normal growth media. The presence of the tRNAAla 5′ half was analyzed as described in a. c The presence of the tRNAAla 5′ half was investigated as in a in procyclic cells grown to different cell densities (indicated on top of the panel). In all cases the EtBr-stained rRNAs serve as loading controls
Fig. 4
Fig. 4
The tRNAThr 3′ half associates with ribosomes in vivo and in vitro. a Polysome profiling with sucrose gradients (10–40%) using total cell lysates prepared from exponentially growing T. brucei cells (gray) and from cells starved for 2 h in PBS (black). b RNA was extracted from fractions containing polysomes, 80S monosomes, 60S, or 40S ribosomal subunits and from the light sucrose gradient fractions (free RNA) and used for northern blot analysis to monitor the tRNAThr 3′ half (length: 37 nucleotides). In a and b representative data of three independent experiments are shown. c Fractions from the polysome profiles of unstressed (no stress) or starved (stress) cells were also investigated for the presence of the YFP-tagged DHH1 (73 kDa) by western blot analysis. The location of molecular weight protein markers are indicated (see also Supplementary Figure 16). Western blots have been repeated twice. d Association of the tRNAThr 3′ half to ribosomes was analyzed employing in vitro filter binding assay using gradient-purified 80S ribosomes isolated from starvation stressed or unstressed cells and 5′ [32P]-end labeled synthetic tRNA halves. The mean and standard deviation of four independent binding experiments are shown underneath the scan, whereas binding efficiency employing unstressed 80S ribosomes was set to 1.00
Fig. 5
Fig. 5
The tRNAThr 3′-half stimulates translation in vitro and in vivo. a On the left, the autoradiographs of two representative SDS polyacrylamide gels of in vitro translation assays performed in the absence (mock) or in the presence of in vitro transcribed tRNAThr 3′ half, either containing a 3′-CCA or 3′-GGU end (+) or lacking it (−), are shown. The mean and standard deviations of four to nine independent in vitro translation experiments in the absence (mock) or presence of tRNA halves (either originating from tRNAThr of tRNAAla) are shown on the right graph. Addition of the translation inhibitor puromycin (Pmn) serves as specificity control for the assay (n = 3). b On the left, the autoradiograph of two representative gels of in vivo translation reactions performed in the absence (mock) or in the presence of electroporated tRNAThr 3′ halves, either containing a 3′-CCA or 3′-GGU end (+) or lacking it (−), are shown. Quantification (mean and standard deviation) of three to ten independent metabolic labeling experiments in the absence (mock) or presence of introduced tRNA halves (either originating from tRNAThr of tRNAAla) is shown on the right graph. c Autoradiograph of an in vivo translation assay using electroporated tRNAThr 3′ halves containing different chemical groups at the 5′ end. A representative gel of in total four independent experiments is shown. 5′-PPP: 5′ triphosphate; 5′-P: 5′ monophosphate. 3′-CCA: indicates the presence or absence of a 3′ CCA tail. Significance in a and b according to paired Student’s t-test: **P ≤ 0.01. d Abundance of tubulin mRNA (1755 nucleotides; see Supplementary Figure 17a) associated with ribosomes (P100) during T. brucei in vitro translation reactions in the presence or absence of the tRNAThr 3′ half was monitored by northern blot analysis (n = 2). S100 indicates the respective post-ribosomal supernatants. Reactions were stopped either after 5 or 20 min of incubation. In all figures either Coomassie stained protein gels or EtBr staining of RNA gels (bottom panels) serve as loading controls
Fig. 6
Fig. 6
Depletion of endogenous tRNAThr 3′ halves alleviates the stimulatory effects during translation. a Scheme of the strategy used for affinity purification of the endogenous tRNAThr 3′ half from the pool of small RNAs (size range between 30–40 nucleotides). As a control the same procedure was performed in the absence of a biotinylated antisense oligonucleotide (ASO). b Northern blot analysis of RNA extracted from the samples obtained after affinity purification showing the successful isolation of the tRNAThr 3′ half (see also Supplementary Figure 10) when using the biotinylated ASO (fraction II+) and its partial depletion from the corresponding flow through (compare fractions III, − and +ASO). A representative blot of in total two independent affinity purification experiments is shown. c Depletion of endogenous tRNAThr 3′ halves from the pool of small RNAs results in reduced in vitro translation activities. The effects of RNA fractions obtained after affinity purification were tested on in vitro translation. Quantification (mean and standard deviation) shows the average of three independent experiments. d Chemically modified ASO complementary to the tRNAThr 3′ half were electroporated into T. brucei cells and their effect on in vivo translation was investigated by metabolic labeling during stress recovery. The autoradiograph shows a representative SDS polyacrylamide gel in which metabolic labeling was performed in cells electroporated without an ASO (−) or cells electroporated with one (a) or two (a+b) different ASO targeting endogenous tRNAThr 3′ halves. Coomassie staining of the gel (bottom panel) serve as loading control. Quantification of three independent metabolic labeling experiments is shown on the right. As specificity control metabolic labeling was performed also after electroporation of an analogous ASO without any sequence complementarity to the tRNAThr 3′ half (ctr). Significance according to paired Student’s t-test: **P ≤ 0.01

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