An RNA-binding protein complex regulates the purine-dependent expression of a nucleobase transporter in trypanosomes

Rico-Jiménez, Míriam; Ceballos-Pérez, Gloria; Gómez-Liñán, Claudia; Estévez, Antonio M.

doi:10.1093/nar/gkab181

Cited by 14 publications

(12 citation statements)

References 52 publications

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“…TAP-purified material from two independent purifications was subjected to LC-MS/MS, and proteins were identified as described in the Materials and Methods. As controls, TAPs were carried out in parallel using protein extracts obtained from cell lines expressing the TAP tag alone ( Supplementary Table S16 ) or the ribonucleoprotein complex PuREBP1/2 ( 47 ) using either PuREBP1–TAP or TAP–PuREBP2 as baits ( Supplementary Table S17 ). We also compared the identified RBP33-associated proteins with a list of putative contaminants commonly observed in TAP purifications ( 64 ).…”

Section: Resultsmentioning

confidence: 99%

“…For enhanced yellow fluorescent protein (eYFP) expression, the eYPF open reading frame (ORF) from p2675 ( 46 ) was PCR-amplified and cloned in reverse orientation in the plasmid pGR12 ( 47 ) to yield pGR417, where the eYFP ORF is flanked by the EP1 5'-untranslated region (5'-UTR) and the actin 3'-UTR. The rDNA spacer targeting sequence in pGR417 was replaced with DNA fragments corresponding to genes Tb427_110143800 or Tb427_100047800 to yield pGR431 and pGR433, respectively.…”

Section: Methodsmentioning

confidence: 99%

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The RNA-binding protein RBP33 dampens non-productive transcription in trypanosomes

Gómez-Liñán

Gómez-Díaz

Ceballos-Pérez

et al. 2022

Nucleic Acids Research

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In-depth analysis of the transcriptomes of several model organisms has revealed that genomes are pervasively transcribed, giving rise to an abundance of non-canonical and mainly antisense RNA polymerase II-derived transcripts that are produced from almost any genomic context. Pervasive RNAs are degraded by surveillance mechanisms, but the repertoire of proteins that control the fate of these non-productive transcripts is still incomplete. Trypanosomes are single-celled eukaryotes that show constitutive RNA polymerase II transcription and in which initiation and termination of transcription occur at a limited number of sites per chromosome. It is not known whether pervasive transcription exists in organisms with unregulated RNA polymerase II activity, and which factors could be involved in the process. We show here that depletion of RBP33 results in overexpression of ∼40% of all annotated genes in the genome, with a marked accumulation of sense and antisense transcripts derived from silenced regions. RBP33 loss does not result in a significant increase in chromatin accessibility. Finally, we have found that transcripts that increase in abundance upon RBP33 knockdown are significantly more stable in RBP33-depleted trypanosomes, and that the exosome complex is responsible for their degradation. Our results provide strong evidence that RBP33 dampens non-productive transcription in trypanosomes.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The RNA-binding protein RBP33 dampens non-productive transcription in trypanosomes

Gómez-Liñán

Gómez-Díaz

Ceballos-Pérez

et al. 2022

Nucleic Acids Research

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“…Regulation of mRNA processing, degradation, and translation are therefore central to parasite homeostasis, and for changes in gene expression during differentiation [ 14 – 17 ]. The sequences required for regulation of mRNA stability and translation often lie in the 3’-UTRs of the mRNAs, and most regulation so far has been found to depend on RNA-binding proteins [ 37 , 38 ].…”

Section: Introductionmentioning

confidence: 99%

Several different sequences are implicated in bloodstream-form-specific gene expression in Trypanosoma brucei

et al. 2022

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The parasite Trypanosoma brucei grows as bloodstream forms in mammalian hosts, and as procyclic forms in tsetse flies. In trypanosomes, gene expression regulation depends heavily on post-transcriptional mechanisms. Both the RNA-binding protein RBP10 and glycosomal phosphoglycerate kinase PGKC are expressed only in mammalian-infective forms. RBP10 targets procyclic-specific mRNAs for destruction, while PGKC is required for bloodstream-form glycolysis. Developmental regulation of both is essential: expression of either RBP10 or PGKC in procyclic forms inhibits their proliferation. We show that the 3’-untranslated region of the RBP10 mRNA is extraordinarily long—7.3kb—and were able to identify six different sequences, scattered across the untranslated region, which can independently cause bloodstream-form-specific expression. The 3’-untranslated region of the PGKC mRNA, although much shorter, still contains two different regions, of 125 and 153nt, that independently gave developmental regulation. No short consensus sequences were identified that were enriched either within these regulatory regions, or when compared with other mRNAs with similar regulation, suggesting that more than one regulatory RNA-binding protein is important for repression of mRNAs in procyclic forms. We also identified regions, including an AU repeat, that increased expression in bloodstream forms, or suppressed it in both forms. Trypanosome mRNAs that encode RNA-binding proteins often have extremely extended 3’-untranslated regions. We suggest that one function of this might be to act as a fail-safe mechanism to ensure correct regulation even if mRNA processing or expression of trans regulators is defective.

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“…The sequences required for regulation of mRNA stability and translation often lie in the 3'-UTRs of the mRNAs, and most regulation so far has been found to depend on RNA-binding proteins (21). Examples include PUF9, which stabilises S-phase mRNAs (22); ZC3H11, which stabilises chaperone mRNAs (23); RBP6, which promotes differentiation of procyclic forms to epimastigotes (24,25); two PuREBPs, which are implicated in purine-dependent repression of a small number of mRNAs (26); and CFB2, which is required for stabilization of the VSG mRNA in bloodstream-form trypanosomes (27). RBP10 (Tb927.8.2780), which is exclusively expressed in bloodstream forms and metacyclic forms (10,11,(28)(29)(30) specifically associates with procyclic-specific mRNAs that contain the motif UA(U)6 in their 3'-UTRs, targeting them for destruction (31).…”

Section: Introductionmentioning

confidence: 99%

The RNA-binding protein DRBD18 regulates processing and export of the mRNA encoding Trypanosoma brucei RNA-binding protein 10

Tshitenge

Liu

Clayton

2021

Preprint

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The parasite Trypanosoma brucei grows as bloodstream forms in mammalian hosts, and as procyclic forms in tsetse flies. Trypanosome protein coding genes are arranged in polycistronic transcription units, so gene expression regulation depends heavily on post-transcriptional mechanisms. The essential RNA-binding protein RBP10 is expressed only in mammalian-infective forms, where it targets procyclic-specific mRNAs for destruction. We show that developmental regulation of RBP10 expression is mediated by the exceptionally long 7.3 Kb 3'-UTR of its mRNA. Different regulatory sequences that can independently enhance mRNA stability and translation in bloodstream forms, or destabilize and repress translation in procyclic forms, are scattered throughout the 3'-UTR. The RNA-binding protein DRBD18 is implicated in the export of a subset of mRNAs from the nucleus in procyclic forms. We confirmed that in bloodstream forms, DRBD18 copurifies the outer ring of the nuclear pore, mRNA export proteins and exon junction complex proteins. Loss of DRBD18 in bloodstream forms caused accumulation of several shortened RBP10 mRNA isoforms, with loss of longer species, but RNAi targeting the essential export factor MEX67 did not cause such changes, demonstrating specificity. Long RBP10 mRNAs accumulated in the nucleus, while shorter ones reached the cytoplasm. We suggest that DRBD18 binds to processing signals in the RBP10 3'-UTR, simultaneously preventing their use and recruiting mRNA export factors. DRBD18 depletion caused truncation of the 3'-UTRs of more than 100 other mRNAs, suggesting that it has an important role in regulating use of alternative processing sites.

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An RNA-binding protein complex regulates the purine-dependent expression of a nucleobase transporter in trypanosomes

Cited by 14 publications

References 52 publications

The RNA-binding protein RBP33 dampens non-productive transcription in trypanosomes

The RNA-binding protein RBP33 dampens non-productive transcription in trypanosomes

Several different sequences are implicated in bloodstream-form-specific gene expression in Trypanosoma brucei

The RNA-binding protein DRBD18 regulates processing and export of the mRNA encoding Trypanosoma brucei RNA-binding protein 10

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