The Tethering Assay: A Simple Method for the Characterization of mRNA-Fate Regulators

Mugo, Elisha; Erben, Esteban

doi:10.1007/978-1-0716-0294-2_18

Cited by 5 publications

(3 citation statements)

References 16 publications

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“…Tethering assays were done using cells expressing mRNAs with a chloramphenicol acetyltransferase open reading frame and a truncated version of the trypanosome actinA 3'-UTR, with 5 boxB sequences immediately upstream of the 3'-UTR (95). Lines with tetracycline-inducible expression of different versions of CFB2 (as shown in Figure 7) were made, and CAT was assayed with or without tetracycline addition.…”

Section: Tethering Assaysmentioning

confidence: 99%

The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein

Nascimento

Egler

Arnold

et al. 2020

Preprint

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Salivarian trypanosomes cause human sleeping sickness and economically important livestock diseases. The “bloodstream forms”, which replicate extracellularly in the blood and tissue fluids of mammals, are coated by a monolayer of Variant Surface Glycoprotein (VSG). Switching of the expressed VSG gene is central to parasite pathogenicity because it enables the parasites to evade adaptive immunity via antigenic variation. Adequate levels of VSG expression - 10% of total protein and 7% of mRNA - are attained through very active RNA polymerase I transcription, efficient mRNA processing (trans splicing of a capped leader and polyadenylation), and high mRNA stability. We here show how VSG mRNA stability is maintained. Purification of the VSG mRNA with associated proteins specifically selected CFB2, an F-box mRNA-binding protein that lacks known RNA-binding domains. CFB2 binds to a stabilizing complex (MKT1-PBP1-XAC1-LSM12) that recruits poly(A) binding protein and a specialized cap-binding translation initiation complex, EIF4E6-EIF4G5. The interaction of CFB2 with MKT1 is essential for CFB2’s expression-promoting activity, while the F-box auto-regulates CFB2 abundance via interaction with SKP1, a component of the ubiquitination machinery. The results of reporter experiments indicate that CFB2 acts via conserved sequences in the VSG mRNA 3’-untranslated region. Depletion of CFB2 leads to highly specific loss of VSG mRNA. VSG expression is essential not only for antigenic variation but also for trypanosome cell division. Correspondingly, depletion of CFB2 causes cell cycle arrest, dramatic morphological abnormalities and trypanosome death.

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Section: Tethering Assaysmentioning

confidence: 99%

The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein

Nascimento

Egler

Arnold

et al. 2020

Preprint

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“…Antibodies were against the V5 tag (AbD seroTec, 1:1000), the YFP tag (Roche, 1:1000), and aldolase (rabbit, 1:50,000). CAT was measured in a kinetic assay involving partition of 14 C-buturyl chloramphenicol from the aqueous to the organic phase of scintillation fluid (38). Coimmunoprecipitations of endogenously V5-tagged ERBP1 (7), Tb927.8.1500, Tb927.7.3040, and Tb927.11.4900 proteins were done as previously described (39).…”

Section: Protein Analysismentioning

confidence: 99%

A potential role for a novel ZC3H5 complex in regulating mRNA translation in Trypanosoma brucei

Bajak

Leiss

Clayton

et al. 2020

Journal of Biological Chemistry

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In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly post-transcriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood. Our previous research identified the RNA-binding protein ZC3H5 as possibly involved in gene repression, but its role in controlling gene expression was unknown. We here show that ZC3H5 is an essential cytoplasmic RNA-binding protein: RNAi targeting ZC3H5 causes accumulation of pre-cytokinetic cells followed by rapid cell death. Affinity purification and pair-wise yeast 2-hybrid analysis suggest that ZC3H5 forms a complex with three other proteins, encoded by genes Tb927.11.4900, Tb927.8.1500 and Tb927.7.3040. RNA immunoprecipitation revealed that ZC3H5 is preferentially associated with poorly translated, low-stability mRNAs, the 5'-untranslated regions and coding regions of which are enriched in the motif (U/A)UAG(U/A). As previously found in high-throughput analyses, artificial tethering of ZC3H5 to a reporter mRNA or other complex components repressed reporter expression. However, depletion of ZC3H5 in vivo caused only very minor decreases in a few targets, marked increases in the abundances of very stable mRNAs, an increase in monosomes at the expense of large polysomes, and appearance of "halfmer" disomes containing two 80S subunits and one 40S subunit. We speculate that the ZC3H5 complex might be implicated in quality control during the translation of sub-optimal open reading frames.

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“…One tool to study the effects of bound proteins on an RNA is the "tethering" assay [37,38]. In the most common arrangement, the protein of interest is expressed as a fusion with an RNA-binding domain, together with a reporter mRNA that includes the cognate recognition examples of BSD mRNAs using different PPT lengths.…”

Section: Introductionmentioning

confidence: 99%

Sequences and proteins that influence mRNA processing in Trypanosoma brucei: Evolutionary conservation of SR-domain and PTB protein functions

Waithaka

Maiakovska²,

Grimm³

et al. 2022

PLoS Negl Trop Dis

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Background Spliced leader trans splicing is the addition of a short, capped sequence to the 5’ end of mRNAs. It is widespread in eukaryotic evolution, but factors that influence trans splicing acceptor site choice have been little investigated. In Kinetoplastids, all protein-coding mRNAs are 5’ trans spliced. A polypyrimidine tract is usually found upstream of the AG splice acceptor, but there is no branch point consensus; moreover, splicing dictates polyadenylation of the preceding mRNA, which is a validated drug target. Methodology and principal findings We here describe a trans splicing reporter system that can be used for studies and screens concerning the roles of sequences and proteins in processing site choice and efficiency. Splicing was poor with poly(U) tracts less than 9 nt long, and was influenced by an intergenic region secondary structure. A screen for signals resulted in selection of sequences that were on average 45% U and 35% C. Tethering of either the splicing factor SF1, or the cleavage and polyadenylation factor CPSF3 within the intron stimulated processing in the correct positions, while tethering of two possible homologues of Opisthokont PTB inhibited processing. In contrast, tethering of SR-domain proteins RBSR1, RBSR2, or TSR1 or its interaction partner TSR1IP, promoted use of alternative signals upstream of the tethering sites. RBSR1 interacts predominantly with proteins implicated in splicing, whereas the interactome of RBSR2 is more diverse. Conclusions Our selectable constructs are suitable for screens of both sequences, and proteins that affect mRNA processing in T. brucei. Our results suggest that the functions of PTB and SR-domain proteins in splice site definition may already have been present in the last eukaryotic common ancestor tract binding protein, SR-domain protein.

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The Tethering Assay: A Simple Method for the Characterization of mRNA-Fate Regulators

Cited by 5 publications

References 16 publications

The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein

The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein

A potential role for a novel ZC3H5 complex in regulating mRNA translation in Trypanosoma brucei

Sequences and proteins that influence mRNA processing in Trypanosoma brucei: Evolutionary conservation of SR-domain and PTB protein functions

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