Identification of the HIT-45 protein from <i>Trypanosoma brucei</i> as an FHIT protein/dinucleoside triphosphatase: Substrate specificity studies on the recombinant and endogenous proteins

Banerjee, Hiren; Palenchar, Jennifer B.; Łukaszewicz, Maciej; Bojarska, Elzbieta; Stępiński, Janusz; Jemielity, Jacek; Guranowski, Andrzej; Ng, Stephanie; Wah, David A.; Darżynkiewicz, Edward; Bellofatto, Vivian

doi:10.1261/rna.1426609

Cited by 14 publications

(13 citation statements)

References 60 publications

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“…For example, mammalian eIF4E has been implicated in targeting mRNAs to P-bodies (Andrei et al 2005;Ferraiuolo et al 2005). The provocative possibility of cytosolic mRNA capping activities highlights the elusive identity of the kinetoplastid mRNA 5 ′ -decapping enzyme (Clayton and Shapira 2007;Banerjee et al 2009), a lynchpin in the control of translation initiation. Cytosolic 5 ′ decapping of mRNA is one of two major pathways for eukaryotic mRNA degradation (Coller and Parker 2004), however it is not necessarily the end of life for the mRNA (Topisirovic et al 2011).…”

Section: Discussionmentioning

confidence: 99%

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

Freire

Vashisht

Malvezzi

et al. 2014

RNA

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Members of the eIF4E mRNA cap-binding family are involved in translation and the modulation of transcript availability in other systems as part of a three-component complex including eIF4G and eIF4A. The kinetoplastids possess four described eIF4E and five eIF4G homologs. We have identified two new eIF4E family proteins in Trypanosoma brucei, and define distinct complexes associated with the fifth member, TbEIF4E5. The cytosolic TbEIF4E5 protein binds cap 0 in vitro. TbEIF4E5 was found in association with two of the five TbEIF4Gs. TbIF4EG1 bound TbEIF4E5, a 47.5-kDa protein with two RNA-binding domains, and either the regulatory protein 14-3-3 II or a 117.5-kDa protein with guanylyltransferase and methyltransferase domains in a potentially dynamic interaction. The TbEIF4G2/TbEIF4E5 complex was associated with a 17.9-kDa hypothetical protein and both 14-3-3 variants I and II. Knockdown of TbEIF4E5 resulted in the loss of productive cell movement, as evidenced by the inability of the cells to remain in suspension in liquid culture and the loss of social motility on semisolid plating medium, as well as a minor reduction of translation. Cells appeared lethargic, as opposed to compromised in flagellar function per se. The minimal use of transcriptional control in kinetoplastids requires these organisms to implement downstream mechanisms to regulate gene expression, and the TbEIF4E5/TbEIF4G1/117.5-kDa complex in particular may be a key player in that process. We suggest that a pathway involved in cell motility is affected, directly or indirectly, by one of the TbEIF4E5 complexes.

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Section: Discussionmentioning

confidence: 99%

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

Freire

Vashisht

Malvezzi

et al. 2014

RNA

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“…For most mRNAs, removal of the poly(A) tail precedes degradation of the remainder of the transcript (Manful et al 2011). A typical mRNA degradation machinery is present: an exosome (Estévez et al 2001; a scavenger decapping enzyme (Banerjee et al 2009); an Xrn1 homolog, XRNA (Li et al 2006); PAN2/PAN3 (Schwede et al 2009); PARN (Milone et al 2004;Utter et al 2011); a CAF1/NOT complex composed of CAF1, CAF40, NOT1, NOT2, NOT3/5 (Schwede et al 2008); a CNOT11 homolog (Schwede et al 2008;Mauxion et al 2013); and CNOT10 (Färber et al 2013). Only a Dcp2-like decapping enzyme is missing (Schwede et al 2009).…”

Section: Introductionmentioning

confidence: 99%

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

Fadda

Färber

Droll

et al. 2013

RNA

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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.

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“…Lister 427 procyclic cells were cultured in SDM-79 medium containing 10% fetal calf serum (FCS) at 26°C in 5% CO 2 . Procyclic 29-13 cells were cultured in the presence of 15 g/ml G418 and 50 g/ml hygromycin to maintain expression of T7 RNA polymerase and the tetracycline (Tet) repressor (57).…”

Section: Culturing and Transfection Of Parasites T Bruceimentioning

confidence: 99%

“…Procyclic 29-13 cells were cultured in the presence of 15 g/ml G418 and 50 g/ml hygromycin to maintain expression of T7 RNA polymerase and the tetracycline (Tet) repressor (57). Transfected parasites containing the integrated plasmid were selected for by the addition of 2.5 g/ml phleomycin (2). Lister 427 BF cells were grown in HMI-9 medium containing 10% FCS and 10% Serum Plus (SAFC Biosciences) at 37°C in 5% CO 2 (26).…”

Section: Culturing and Transfection Of Parasites T Bruceimentioning

confidence: 99%

“…Transfected parasites containing the integrated plasmid were selected for by the addition of 2.5 g/ml phleomycin (2). Lister 427 BF cells were grown in HMI-9 medium containing 10% FCS and 10% Serum Plus (SAFC Biosciences) at 37°C in 5% CO 2 (26). Single-marker BF cells (a gift from G. Cross) were cultured in the presence of 2.5 g/ml G418 to maintain the T7 RNA polymerase and Tet repressor (57), and transfected parasites were obtained using an Amaxa system and selected for using 2.5 g/ml phleomycin.…”

Section: Culturing and Transfection Of Parasites T Bruceimentioning

confidence: 99%

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Poly(A)-Specific Ribonuclease (PARN-1) Function in Stage-Specific mRNA Turnover in Trypanosoma brucei

Utter¹,

Garcia²,

Milone³

et al. 2011

Eukaryot Cell

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Deadenylation is often the rate-limiting event in regulating the turnover of cellular mRNAs in eukaryotes. Removal of the poly(A) tail initiates mRNA degradation by one of several decay pathways, including deadenylation-dependent decapping, followed by 5 to 3 exonuclease decay or 3 to 5 exosome-mediated decay. In trypanosomatids, mRNA degradation is important in controlling the expression of differentially expressed genes. Genomic annotation studies have revealed several potential deadenylases. Poly(A)-specific RNase (PARN) is a key deadenylase involved in regulating gene expression in mammals, Xenopus oocytes, and higher plants. Trypanosomatids possess three different PARN genes, PARN-1, -2, and -3, each of which is expressed at the mRNA level in two life-cycle stages of the human parasite Trypanosoma brucei. Here we show that T. brucei PARN-1 is an active deadenylase. To determine the role of PARN-1 on mRNA stability in vivo, we overexpressed this protein and analyzed perturbations in mRNA steady-state levels as well as mRNA half-life. Interestingly, a subset of mRNAs was affected, including a family of mRNAs that encode stage-specific coat proteins. These data suggest that PARN-1 functions in stage-specific protein production. Regulation of gene expression in the protozoan parasiteTrypanosoma brucei allows the organism to adapt and survive during its life cycle in two very different environments, the mammalian bloodstream and the tsetse fly. Expression of numerous protein-coding genes is regulated posttranscriptionally, particularly at the level of mRNA stability (4,11,25). For example, differential mRNA stability accounts for the stagespecific expression of procyclins, hexose transporters, and phosphoglycerate kinases (6,20,27,28,53).In the well-studied Saccharomyces and mammalian systems, mRNA decay is a tightly controlled, multistep, and multipathway process. Various cis-acting elements, embedded in specific mRNAs, are recognized by RNA-binding proteins (7,52,55), which stabilize mRNAs or recruit RNases to carry out mRNA degradation and inhibit translation (8,34,39,47). The removal of the mRNA 3Ј poly(A) tail by 3Ј to 5Ј exoribonucleases (deadenylases) is often the rate-limiting step in mRNA degradation in vertebrates and thus a key point in regulation of mRNA turnover (19,40).A number of different deadenylases exist in eukaryotes, including poly(A)-specific RNase (PARN), the CCR4/CAF1/ NOT complex, and the PAN2/PAN3 complex (reviewed in reference 23). The specific role of each of these proteins remains largely unknown, although evidence suggests that each enzyme may recognize a particular set of mRNA substrates (46). PARN functions in the targeted degradation of specific mRNAs in humans, Xenopus, and higher plants (1,3,22,31,32,36,58). To date, no PARN-encoding genes have been characterized in any single-cell eukaryote (56). In humans, PARN initiates decay of mRNAs containing AU-rich elements or nonsense codons. In Xenopus, PARN regulates oocyte maturation, whereas in Arabidopsis, PARN regulates embryogen...

show abstract

Identification of the HIT-45 protein from Trypanosoma brucei as an FHIT protein/dinucleoside triphosphatase: Substrate specificity studies on the recombinant and endogenous proteins

Cited by 14 publications

References 60 publications

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

eIF4F-like complexes formed by cap-binding homolog TbEIF4E5 with TbEIF4G1 or TbEIF4G2 are implicated in post-transcriptional regulation in Trypanosoma brucei

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

Poly(A)-Specific Ribonuclease (PARN-1) Function in Stage-Specific mRNA Turnover in Trypanosoma brucei

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