ATP-independent Control of Vac8 Palmitoylation by a SNARE Subcomplex on Yeast Vacuoles

Yeast vacuole fusion requires palmitoylated Vac8. We previously showed that Vac8 acylation occurs early in the fusion reaction, is blocked by antibodies against Sec18 (yeast N-ethylmaleimide-sensitive fusion protein (NSF)), and is mediated by the R-SNARE Ykt6. Here we analyzed the regulation of this reaction on purified vacuoles. We show that Vac8 acylation is restricted to a narrow time window, is independent of ATP hydrolysis by Sec18, and is stimulated by the ion chelator EDTA. Analysis of vacuole protein complexes indicated that Ykt6 is part of a complex distinct from the second R-SNARE, Nyv1. We speculate that during vacuole fusion, Nyv1 is the classical R-SNARE, whereas the Ykt6-containing complex has a novel function in Vac8 palmitoylation

A ribosome-bound tRNA half stimulates mitochondrial translation during stress recovery in Trypanosoma brucei.

The protozoan parasite Trypanosoma brucei and its disease-causing relatives are among the few organisms that barely regulate the transcription of protein-coding genes. Yet, alterations in its gene expression are essential to survive in different host environments. Recently, tRNA-derived RNAs have been implicated as regulators of many cellular processes within and beyond translation. Previously, we identified the tRNAThr-3'-half (AGU) as a ribosome-associated non-coding RNA able to enhance global translation. Here we report that the tRNAThr-3'-half is generated upon starvation inside the mitochondria. The tRNAThr-3'-half associates with mitochondrial ribosomes and stimulates translation during stress recovery, positively affecting mitochondrial activity and, consequently, cellular energy production capacity. Our results describe an organelle ribosome-associated ncRNA involved in translation regulation to boost the central hub of energy metabolism as an immediate stress recovery response

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

Sensing of environmental cues is crucial for cell survival. To adapt to changes in their surroundings cells need to tightly control the repertoire of genes expressed at any time. Regulation of translation is key, especially in organisms in which transcription is hardly controlled, like Trypanosoma brucei. In this study, we describe the shortening of the bulk of the cellular tRNAs during stress at the expense of the conserved 3' CCA-tail. This tRNA shortening is specific for nutritional stress and renders tRNAs unsuitable substrates for translation. We uncovered the nuclease LCCR4 (Tb927.4.2430), a homologue of the conserved deadenylase Ccr4, as being responsible for tRNA trimming. Once optimal growth conditions are restored tRNAs are rapidly repaired by the trypanosome tRNA nucleotidyltransferase thus rendering the recycled tRNAs amenable for translation. This mechanism represents a fast and efficient way to repress translation during stress, allowing quick reactivation with a low energy input

Independence from kinetoplast DNA maintenance and expression is associated with multi-drug resistance in Trypanosoma brucei in vitro

It is well known that several antitrypanosomatid drugs accumulate in the parasite's mitochondrion, where they often bind to the organellar DNA, the kinetoplast. To what extent this property relates to the mode of action of these compounds has remained largely unquantified. Here we show that single point mutations that remove the dependence of laboratory strains of the sleeping sickness parasite Trypanosoma brucei on a functional kinetoplast result in significant resistance to the diamidine and phenanthridine drug classes

Association of UBP1 to ribonucleoprotein complexes is regulated by interaction with the trypanosome ortholog of the human multifunctional P32 protein

Regulation of gene expression in trypanosomatid parasitic protozoa is mainly achieved posttranscriptionally. RNA-binding proteins (RBPs) associate to 3′ untranslated regions in mRNAs through dedicated domains such as the RNA recognition motif (RRM). Trypanosoma cruziUBP1 (TcUBP1) is an RRM-type RBP involved in stabilization/degradation of mRNAs. TcUBP1 uses its RRM to associate with cytoplasmic mRNA and to mRNA granules under starvation stress. Here, we show that under starvation stress, TcUBP1 is tightly associated with condensed cytoplasmic mRNA granules. Conversely, under high nutrient/low density-growing conditions, TcUBP1 ribonucleoprotein (RNP) complexes are lax and permeable to mRNA degradation and disassembly. After dissociating from mRNA, TcUBP1 can be phosphorylated only in unstressed parasites. We have identified TcP22, the ortholog of mammalian P32/C1QBP, as an interactor of TcUBP1 RRM. Overexpression of TcP22 decreased the number of TcUBP1 granules in starved parasites in vivo. Endogenous TcUBP1 RNP complexes could be dissociated in vitro by addition of recombinant TcP22, a condition stimulating TcUBP1 phosphorylation. Biochemical and in silico analysis revealed that TcP22 interacts with the RNA-binding surface of TcUBP1 RRM. We propose a model for the decondensation of TcUBP1 RNP complexes in T.cruzi through direct interaction with TcP22 and phosphorylation.Fil: Cassola, Alejandro Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Romaniuk, María Albertina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Primrose, Debora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Cervini Bohm, Gabriela Marta . Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: D'Orso, Iván. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); ArgentinaFil: Frasch, Alberto Carlos C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús). Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas "Dr. Raúl Alfonsín" (sede Chascomús); Argentin

Genetic Reconstruction of Protozoan rRNA Decoding Sites Provides a Rationale for Paromomycin Activity against Leishmania and Trypanosoma

Aminoglycoside antibiotics target the ribosomal decoding A-site and are active against a broad spectrum of bacteria. These compounds bind to a highly conserved stem-loop-stem structure in helix 44 of bacterial 16S rRNA. One particular aminoglycoside, paromomycin, also shows potent antiprotozoal activity and is used for the treatment of parasitic infections, e.g. by Leishmania spp. The precise drug target is, however, unclear; in particular whether aminoglycoside antibiotics target the cytosolic and/or the mitochondrial protozoan ribosome. To establish an experimental model for the study of protozoan decoding-site function, we constructed bacterial chimeric ribosomes where the central part of bacterial 16S rRNA helix 44 has been replaced by the corresponding Leishmania and Trypanosoma rRNA sequences. Relating the results from in-vitro ribosomal assays to that of in-vivo aminoglycoside activity against Trypanosoma brucei, as assessed in cell cultures and in a mouse model of infection, we conclude that aminoglycosides affect cytosolic translation while the mitochondrial ribosome of trypanosomes is not a target for aminoglycoside antibiotics

A role for Caf1 in mRNA deadenylation and decay in trypanosomes and human cells

The eukaryotic Ccr4/Caf1/Not complex is involved in deadenylation of mRNAs. The Caf1 and Ccr4 subunits both potentially have deadenylating enzyme activity. We investigate here the roles of Ccr4 and Caf1 in deadenylation in two organisms that separated early in eukaryotic evolution: humans and trypanosomes. In Trypanosoma brucei, we found a complex containing CAF1, NOT1, NOT2 and NOT5, DHH1 and a possible homologue of Caf130; no homologue of Ccr4 was found. Trypanosome CAF1 has deadenylation activity, and is essential for cell survival. Depletion of trypanosome CAF1 delayed deadenylation and degradation of constitutively expressed mRNAs. Human cells have two isozymes of Caf1: simultaneous depletion of both inhibited degradation of an unstable reporter mRNA. In both species, depletion of Caf1 homologues inhibited deadenylation of bulk RNA and resulted in an increase in average poly(A) tail length

Rapid decay of unstable Leishmania mRNAs bearing a conserved retroposon signature 3′-UTR motif is initiated by a site-specific endonucleolytic cleavage without prior deadenylation

We have previously shown that the Leishmania genome possess two widespread families of extinct retroposons termed Short Interspersed DEgenerated Retroposons (SIDER1/2) that play a role in post-transcriptional regulation. Moreover, we have demonstrated that SIDER2 retroposons promote mRNA degradation. Here we provide new insights into the mechanism by which unstable Leishmania mRNAs harboring a SIDER2 retroposon in their 3′-untranslated region are degraded. We show that, unlike most eukaryotic transcripts, SIDER2-bearing mRNAs do not undergo poly(A) tail shortening prior to rapid turnover, but instead, they are targeted for degradation by a site-specific endonucleolytic cleavage. The main cleavage site was mapped in two randomly selected SIDER2-containing mRNAs in vivo between an AU dinucleotide at the 5′-end of the second 79-nt signature (signature II), which represents the most conserved sequence amongst SIDER2 retroposons. Deletion of signature II abolished endonucleolytic cleavage and deadenylation-independent decay and increased mRNA stability. Interestingly, we show that overexpression of SIDER2 anti-sense RNA can increase sense transcript abundance and stability, and that complementarity to the cleavage region is required for protecting SIDER2-containing transcripts from degradation. These results establish a new paradigm for how unstable mRNAs are degraded in Leishmania and could serve as the basis for a better understanding of mRNA decay pathways in general

The role of deadenylation in the degradation of unstable mRNAs in trypanosomes

Removal of the poly(A) tail is the first step in the degradation of many eukaryotic mRNAs. In metazoans and yeast, the Ccr4/Caf1/Not complex has the predominant deadenylase activity, while the Pan2/Pan3 complex may trim poly(A) tails to the correct size, or initiate deadenylation. In trypanosomes, turnover of several constitutively-expressed or long-lived mRNAs is not affected by depletion of the 5′–3′ exoribonuclease XRNA, but is almost completely inhibited by depletion of the deadenylase CAF1. In contrast, two highly unstable mRNAs, encoding EP procyclin and a phosphoglycerate kinase, PGKB, accumulate when XRNA levels are reduced. We here show that degradation of EP mRNA was partially inhibited after CAF1 depletion. RNAi-targeting trypanosome PAN2 had a mild effect on global deadenylation, and on degradation of a few mRNAs including EP. By amplifying and sequencing degradation intermediates, we demonstrated that a reduction in XRNA had no effect on degradation of a stable mRNA encoding a ribosomal protein, but caused accumulation of EP mRNA fragments that had lost substantial portions of the 5′ and 3′ ends. The results support a model in which trypanosome mRNAs can be degraded by at least two different, partially independent, cytoplasmic degradation pathways attacking both ends of the mRNA