tRNA 3′ shortening by LCCR4 as a response to stress in Trypanosoma brucei

Figures

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  Figure 1.. tRNAs are trimmed upon stress and repaired once normal growth conditions are restored. (A) Northern blot analyses of tRNAs on total RNA extracted from T. brucei. RNA was isolated from procyclic T. brucei cells either during the exponential growth phase (Exponential) or after heat shock, nutritional deprivation (Nutr. stress), oxidative stress, during the stationary phase or from exponentially growing bloodstream form (Exponential BSF). Full length tRNAs and 3′ trimmed tRNAs are indicated with filled and open arrowheads, respectively. (B) Total RNA extracted from cells growing exponentially (expo), subjected to nutritional stress (stress), or allowed to recover in normal media for 15 or 30 min after stress, was analyzed by northern blot for tRNAVal and tRNAArg (NB, upper panel). Full length tRNAs and 3′ trimmed tRNAs are indicated with filled and open arrowheads, respectively. Ethidium bromide staining (EtBr, lower panel) shows the migration of the bulk of the tRNAs. (C) Decay of total endogenous RNA was investigated by a 2-day pulse using α32P-UTP followed by washes and chase with cold UTP. Total RNA extracted from cells growing exponentially (expo), stressed for 2h in PBS…

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  Figure 2.. The CCA-adding enzyme repairs trimmed tRNAs. (A) The levels of an in situ HA-tagged CCA-adding enzyme (CAE-HA) were investigated by western blot on cells growing exponentially or upon nutritional stress. EF1A serves as loading control. (B) The tagged CAE was purified from parasites growing exponentially (expo) or subjected to nutritional stress (stress) and its activity was tested using tRNAs purified from stressed cells and α32P-CTP after 5, 15 and 30 min of incubation (upper panel). A mock purification was done using cells not expressing the tagged CAE. A representative gel is shown. The levels of the CAE used were analyzed by western blot (lower panel). (C) Signals from experiments in B were quantified and normalized to the intensity of the product generated by the CAE purified from exponentially growing cells and incubated for 5 min. Depicted is the mean of 3 independent experiments ± SD. (D, E) Cells allowing inducible overexpression (D) or depletion (E) of the CAE were stressed and then allowed to recover in normal media. Uninduced cells (−) exposed to the same conditions serve as control. RNA was extracted at the indicated time points and shortening of tRNAVal was analyzed…

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  Figure 3.. The nuclease trimming the tRNAs is constitutively expressed in cells and can be inhibited by ribonucleoside-vanadyl complex and Mg2+ chelators. (A) The tRNA trimming activity was tested in vitro by incubating total cell lysates from cells grown exponentially (expo) or nutritionally stressed (stress) with 5′ 32P-labeled tRNAVal. After the indicated times total RNA was precipitated, separated on 8% polyacrylamide/urea gels, dried and the radioactivity detected by exposure to phosphoimager screens. Incubation of the substrate under the same conditions in the absence of cell lysate (−) serves as control. (B, C) Cell lysates were treated with specific RNase inhibitors (Ribolock, Protector or RVC; B) or different concentrations of divalent cations chelators (C) and the cellular tRNA trimming activity tested as before. (D) The effect of aminoacylation on tRNA trimming was tested using RNA extracted from cells expressing a tagged tRNAGlu. After incubation of total cell lysates with total RNA either aminoacylated (untreated) or subjected to alkaline deacylation RNA was extracted and tRNA shortening investigated by northern blot against tRNAVal. Full length tRNAVal and fully 3′ trimmed…

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  Figure 4.. LCCR4 is involved in stress-triggered tRNA shortening. (A) Cells induced for LCCR4 RNAi or kept uninduced were grown exponentially or under nutritional stress and tRNA shortening analyzed by northern blot detecting tRNAVal. tRNAs containing a complete CCA-tail are marked with a full arrowhead while incomplete and totally shortened species are marked with an asterisk or open arrowheads, respectively. (B) As in (A) but cells were allowed to recover in complete media for the indicated times after nutritional stress. (C) Cells induced for LCCR4-HA overexpression or left uninduced and growing exponentially or under nutritional stress were allowed to recover in normal media for the indicated times. The upper panel shows a northern blot where tRNAVal was investigated while the lower panel corresponds to the same gel stained with ethidium bromide (EtBr). The region corresponding to the bulk cellular tRNAs is indicated. The percentage of full length tRNAVal present is indicated (%FL).

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  Figure 5.. LCCR4 interacts with tRNAs. (A) Inducibly expressed LCCR4-HA was immunoprecipitated and samples obtained from each purification step were analyzed by western blot using antiHA antibodies. (B) LCCR4-HA was immunoprecipitated and its interaction with tRNAVal was investigated by RT-PCR. M6 rRNA serves as specificity control. in: input, FT: flow-through; B: bead-bound.

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  Figure 6.. LCCR4 can cleave tRNAs in vitro. (A) LCCR4-HA expression was induced and the protein was purified from cells growing exponentially (expo) or subjected to nutritional stress (stress) and its activity tested on beads using total RNA as substrate. After incubation for the indicated times the RNA was extracted and analyzed by northern blot for tRNAVal and tRNAHis. Samples for the mock control were obtained from uninduced cells. Full length tRNAs are marked with a full arrowhead while shortened species are marked with open arrowheads. (B) Western blot analysis of samples used in (A). (C) Cells overexpressing the mutant LCCR4 N454A were subjected to stress and then allowed to recover in fresh media for the indicated times. RNA was extracted at each time point and tRNA shortening analyzed by northern blot against tRNAVal. (D) Wild type and N454A mutant LCCR4-HA were purified and their activity tested in vitro as in (A). (E) Western blot analysis of samples used in (D). in: input, FT: flow-through; B: bead-bound.

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  Figure 7.. Repairing of shortened tRNAs is crucial for translation during stress recovery. (A) In vitro aminoacylation was tested on S100 cell fractions from cells grown exponentially or stressed for 2 h in PBS, using as substrate either the endogenous tRNAs (endog. tRNAs) or after supplementation with total yeast tRNA (yeast tRNA). Dark and light grey bars correspond to signals from exponential or stressed samples, respectively. The percentage of aminoacylation was calculated by normalization to the signal for S100 from exponentially growing cells. Depicted is the mean of three experiments ± SD. (B) Cells induced for LCCR4-HA overexpression or not were stressed and translation efficiency investigated during stress recovery by metabolic labeling with 35S-Methionine. After the indicated times cells were pelleted, proteins separated by SDS-PAGE and incorporated radioactivity detected by exposing to phosphorimager screens. Unstressed cells labeled for 30min were also tested (two most right lanes). The upper panel shows a representative autoradiogram of the metabolic labeling experiment while the lower panel is the corresponding gel stained with coomassie. (C) Quantification of 35S-Methionine…