The nuclear RNA binding protein RBP33 influences mRNA and spliced leader RNA abundance in Trypanosoma brucei

Cirovic, Olivera; Trikin, Roman; Hoffmann, Anneliese; Doiron, Nicholas; Jakob, Martin; Ochsenreiter, Torsten

doi:10.1016/j.molbiopara.2016.12.008

Cited by 4 publications

(10 citation statements)

References 18 publications

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“…3). Overexpression of RBP33 also resulted in a decrease of total mRNA [29], an observation we noted previously in metacyclics when compared to procyclics [7]. Thus, RBP33 could play a role in the establishment of metacyclics and in particular in the generation of metacyclics uncharacteristically arrested at the G2/M checkpoint.…”

Section: Resultsmentioning

confidence: 58%

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A single-point mutation in the RNA-binding protein 6 generates Trypanosoma brucei metacyclics that are able to progress to bloodstream forms in vitro

Shi

Butler

Tschudi

2018

Molecular and Biochemical Parasitology

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We previously established an in vitro differentiation system based on the inducible expression of the RNA binding protein 6 (RBP6), which initiated differentiation of Trypanosoma brucei non-infectious procyclics to infectious metacyclics (MFs). However, further differentiation to bloodstream forms (BFs) required infection of mice. Here we report the serendipitous isolation of a single point mutation in RBP6 (Q109K), whose expression not only generated MFs, but purified MFs continued the developmental cycle in vitro to BFs expressing variant surface glycoprotein-2 (VSG-2), formerly known as VSG 221. This transition occurred over a period of 11 days and by RNA-Seq, VSG-2 was first measureable on day 1, whereas metacyclic VSGs were detected up to 8 days. We further showed that inducible expression of mutant RBP6 appeared to skip the intermediate epimastigote stage and we highlight the potential involvement of RBP33 in the establishment of metacyclics and in particular in the generation of metacyclics uncharacteristically arrested at the G2/M checkpoint.

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Section: Resultsmentioning

confidence: 58%

“…This is an intriguing result, since it was previously shown that overexpression of RBP33 leads to a rapid growth arrest in G2/M, i.e. accumulation of 2K2N cells [29], which is one of the configurations enriched in metacyclics (13%; Fig. 3).…”

Section: Resultsmentioning

confidence: 66%

A single-point mutation in the RNA-binding protein 6 generates Trypanosoma brucei metacyclics that are able to progress to bloodstream forms in vitro

Shi

Butler

Tschudi

2018

Molecular and Biochemical Parasitology

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“…RBP33 is an essential nuclear protein, so its association with DRBD18 but not ZC3H28 corresponds to the localisations of the two proteins. RBP33 associates with non-coding RNAs, especially from antisense transcription (Fernandez-Moya et al 2014), but its depletion led to loss of mRNA and spliced leader RNA (Cirovic et al 2017), suggesting a link to splicing. Both DRBD18 and ZC3H28 enriched various ribosomal proteins and RNA-binding proteins, including both poly(A) binding proteins (PABP1 and PABP2), ALBA2, ALBA3, DRBD2, DRBD3, ZC3H9, ZC3H34, ZC3H39, ZC3H40, UPF1, HNRNPH/F and TRRM1.…”

Section: Resultsmentioning

confidence: 99%

The Trypanosoma brucei RNA-binding protein DRBD18 ensures correct mRNA trans splicing and polyadenylation patterns

Tshitenge

Clayton

2022

Preprint

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The parasite Trypanosoma brucei grows as bloodstream forms in mammals, and as procyclic forms in tsetse flies. Transcription is polycistronic, all mRNAs are trans spliced, and polyadenylation sites are defined by downstream splicing signals. Expression regulation therefore depends heavily on post-transcriptional mechanisms. The RNA-binding protein DRBD18 was previously implicated in the export of some mRNAs from the nucleus in procyclic forms. It copurifies the outer ring of the nuclear pore, mRNA export factors and exon-junction-complex proteins. We show that for >200 mRNAs, DRBD18 depletion caused preferential accumulation of versions with shortened 3'-untranslated regions, arising from use of polyadenylation sites that were either undetectable or rarely seen in non-depleted cells. The shortened mRNAs were often, but not always, more abundant in depleted cells than the corresponding longer versions in normal cells. Their appearance was linked to the appearance of trans spliced, polyadenylated RNAs containing only downstream 3'-untranslated-region-derived sequences. Experiments with one mRNA suggested that nuclear retention alone, through depletion of MEX67, did not affect mRNA length, suggesting a specific effect of DRBD18 on processing. Since DRBD18-bound mRNAs were enriched in polypyrimidine tract motifs, and it is found in both the nucleus and the cytoplasm, we suggest that DRBD18 acts in the nucleus by binding to polypyrimidine tracts in 3'-UTRs. DRBD18 binding might both prevent polypyrimidine tract recognition by splicing factors, and promote export of the bound RNAs to the cytosol.

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“…RBP33 is an essential nuclear protein, so its association with DRBD18 but not ZC3H28 corresponds to the localisations of the two proteins. RBP33 associates with non-coding RNAs, especially from antisense transcription (77), but its depletion led to loss of mRNA and spliced leader RNA (78), suggesting a link to splicing. Both DRBD18 and ZC3H28 enriched various ribosomal proteins and RNA-binding proteins, including both poly(A) binding proteins, ALBA2, ALBA3, DRBD2, DRBD3, ZC3H9, ZC3H34, ZC3H39, ZC3H40, UPF1, HNRNPH/F and TRRM1.…”

Section: Bmentioning

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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The nuclear RNA binding protein RBP33 influences mRNA and spliced leader RNA abundance in Trypanosoma brucei

Cited by 4 publications

References 18 publications

A single-point mutation in the RNA-binding protein 6 generates Trypanosoma brucei metacyclics that are able to progress to bloodstream forms in vitro

A single-point mutation in the RNA-binding protein 6 generates Trypanosoma brucei metacyclics that are able to progress to bloodstream forms in vitro

The Trypanosoma brucei RNA-binding protein DRBD18 ensures correct mRNA trans splicing and polyadenylation patterns

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

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