Insights into the functions and RNA binding of Trypanosoma brucei ZC3H22, RBP9 and DRBD7

Abstract Trypanosoma brucei is unusually reliant on mRNA-binding proteins to control mRNA fate, because its protein-coding genes lack individual promoters. We here focus on three trypanosome RNA-binding proteins. ZC3H22 is specific to Tsetse fly forms, RBP9 is preferentially expressed in bloodstream forms; and DRBD7 is constitutively expressed. Depletion of RBP9 or DRBD7 did not affect bloodstream-form trypanosome growth. ZC3H22 depletion from procyclic forms caused cell clumping, decreased expression of genes required for cell growth and proliferation, and increased expression of some epimastigote markers. Apart from decreases in mRNAs encoding enzymes of glucose metabolism, levels of most ZC3H22-bound transcripts were unaffected by ZC3H22 depletion. We compared ZC3H22, RBP9 and DRBD7 RNA binding with that of 16 other RNA-binding proteins. ZC3H22, PUF3 and ERBP1 show a preference for ribosomal protein mRNAs. RBP9 preferentially binds mRNAs that are more abundant in bloodstream forms than in procyclic forms. RBP9, ZC3H5, ZC3H30 and DRBD7 prefer mRNAs with long coding regions; UBP1-associated mRNAs have long 3′-untranslated regions; and RRM1 prefers mRNAs with long 3′or 5′-untranslated regions. We suggest that proteins that prefer long mRNAs may have relatively short or degenerate binding sites, and that preferences for A or U increase binding in untranslated regions.

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The endoplasmic reticulum-associated mRNA-binding proteins ERBP1 and ERBP2 interact in bloodstream-form Trypanosoma brucei

10.1101/718031 ◽

2019 ◽

Author(s):

Kathrin Bajak ◽

Kevin Leiss ◽

Christine Clayton ◽

Esteban Erben

Keyword(s):

Endoplasmic Reticulum ◽

Ribosomal Protein ◽

Trypanosoma Brucei ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Bloodstream Form ◽

Control Of Gene Expression ◽

Mrna Binding Proteins ◽

Mrna Binding

AbstractKinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. Trypanosoma brucei ERBP1 (Tb927.10.14150) and ERBP2 (Tb927.9.9550) were previously identified as mRNA binding proteins that lack canonical RNA-binding domains. We here show that ERBP1 is associated with the endoplasmic reticulum, like ERBP2, and that the two proteins interact in vivo. Loss of ERBP1 from bloodstream-form T. brucei initially resulted in a growth defect but proliferation was restored after more prolonged cultivation. Results from a pull-down of tagged ERBP1 suggest that it preferentially binds to ribosomal protein mRNAs. The ERBP1 sequence resembles that of Saccharomyces cerevisiae Bfr1, which also localises to the endoplasmic reticulum and binds to ribosomal protein mRNAs. However, unlike Bfr1, ERBP1 does not bind to mRNAs encoding secreted proteins, and it is also not recruited to stress granules after starvation.

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RNA-Binding Proteins as Important Regulators of Long Non-Coding RNAs in Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms21082969 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2969 ◽

Cited By ~ 4

Author(s):

Katharina Jonas ◽

George A. Calin ◽

Martin Pichler

Keyword(s):

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Stability ◽

Protein Coding ◽

Cellular Processes ◽

Open Questions ◽

Non Coding Rnas ◽

The Stability ◽

Mrna Binding

The majority of the genome is transcribed into pieces of non-(protein) coding RNA, among which long non-coding RNAs (lncRNAs) constitute a large group of particularly versatile molecules that govern basic cellular processes including transcription, splicing, RNA stability, and translation. The frequent deregulation of numerous lncRNAs in cancer is known to contribute to virtually all hallmarks of cancer. An important regulatory mechanism of lncRNAs is the post-transcriptional regulation mediated by RNA-binding proteins (RBPs). So far, however, only a small number of known cancer-associated lncRNAs have been found to be regulated by the interaction with RBPs like human antigen R (HuR), ARE/poly(U)-binding/degradation factor 1 (AUF1), insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), and tristetraprolin (TTP). These RBPs regulate, by various means, two aspects in particular, namely the stability and the localization of lncRNAs. Importantly, these RBPs themselves are commonly deregulated in cancer and might thus play a major role in the deregulation of cancer-related lncRNAs. There are, however, still many open questions, for example regarding the context specificity of these regulatory mechanisms that, in part, is based on the synergistic or competitive interaction between different RBPs. There is also a lack of knowledge on how RBPs facilitate the transport of lncRNAs between different cellular compartments.

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The endoplasmic reticulum-associated mRNA-binding proteins ERBP1 and ERBP2 interact in bloodstream-form Trypanosoma brucei

PeerJ ◽

10.7717/peerj.8388 ◽

2020 ◽

Vol 8 ◽

pp. e8388 ◽

Cited By ~ 3

Author(s):

Kathrin Bajak ◽

Kevin Leiss ◽

Christine E. Clayton ◽

Esteban Erben

Keyword(s):

Endoplasmic Reticulum ◽

Ribosomal Protein ◽

Trypanosoma Brucei ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Bloodstream Form ◽

Control Of Gene Expression ◽

Mrna Binding Proteins ◽

Mrna Binding

Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. Trypanosoma brucei ERBP1 (Tb927.10.14150) and ERBP2 (Tb927.9.9550) were previously identified as mRNA binding proteins that lack canonical RNA-binding domains. We show here that ERBP1 is associated with the endoplasmic reticulum, like ERBP2, and that the two proteins interact in vivo. Loss of ERBP1 from bloodstream-form T. brucei initially resulted in a growth defect but proliferation was restored after more prolonged cultivation. Pull-down analysis of tagged ERBP1 suggests that it preferentially binds to ribosomal protein mRNAs. The ERBP1 sequence resembles that of Saccharomyces cerevisiae Bfr1, which also localises to the endoplasmic reticulum and binds to ribosomal protein mRNAs. However, unlike Bfr1, ERBP1 does not bind to mRNAs encoding secreted proteins, and it is also not recruited to stress granules after starvation.

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Interactions of the Trypanosoma brucei brucei zinc-finger-domain protein ZC3H28

10.1101/2021.08.09.455650 ◽

2021 ◽

Author(s):

Tania Bishola ◽

Christine Clayton

Keyword(s):

Trypanosoma Brucei ◽

Zinc Finger ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Level ◽

Critical Role ◽

Regulation Of Gene Expression ◽

Low Complexity ◽

Mrna Binding

In Trypanosoma brucei and related Kinetoplastids, regulation of gene expression occurs mostly post-transcriptionally, and RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Trypanosoma brucei ZC3H28 is a 114 KDa cytoplasmic mRNA-binding protein with a single C(x)7C(x)5C(x)sH zinc finger at the C-terminus and numerous proline-, histine- or glutamine-rich regions. We here show that N-terminally tagged ZC3H28 copurifies ribosomes, various RNA-binding proteins, and the translation initiation complex EIF4E4/EIF4G3. ZC3H28 is preferentially associated with long RNAs that have low complexity sequences in their 3'-untranslated regions. When tethered to a reporter mRNA, ZC3H28 increased the mRNA level without a corresponding increase in protein expression; this suggests that it stabilized the reporter but at the same time suppressed its translation. Indeed, there was a clear negative correlation between ZC3H28 mRNA binding and ribosome density. After ZC3H28 depletion, the relative levels of ribosomal protein mRNAs increased while levels of some long mRNAs decreased, but there is no overall correlation between binding and RNAi effects on mRNA abundance. We speculate that ZC3H28 might be implicated in stabilizing poorly-translated mRNAs.

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PlncRNADB: A Repository of Plant lncRNAs and lncRNA-RBP Protein Interactions

Current Bioinformatics ◽

10.2174/1574893614666190131161002 ◽

2019 ◽

Vol 14 (7) ◽

pp. 621-627 ◽

Cited By ~ 3

Author(s):

Youhuang Bai ◽

Xiaozhuan Dai ◽

Tiantian Ye ◽

Peijing Zhang ◽

Xu Yan ◽

...

Keyword(s):

Protein Interactions ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Populus Trichocarpa ◽

Noncoding Rnas ◽

Reference Database ◽

Protein Coding ◽

Arabidopsis Lyrata ◽

User Friendly

Background: Long noncoding RNAs (lncRNAs) are endogenous noncoding RNAs, arbitrarily longer than 200 nucleotides, that play critical roles in diverse biological processes. LncRNAs exist in different genomes ranging from animals to plants. Objective: PlncRNADB is a searchable database of lncRNA sequences and annotation in plants. Methods: We built a pipeline for lncRNA prediction in plants, providing a convenient utility for users to quickly distinguish potential noncoding RNAs from protein-coding transcripts. Results: More than five thousand lncRNAs are collected from four plant species (Arabidopsis thaliana, Arabidopsis lyrata, Populus trichocarpa and Zea mays) in PlncRNADB. Moreover, our database provides the relationship between lncRNAs and various RNA-binding proteins (RBPs), which can be displayed through a user-friendly web interface. Conclusion: PlncRNADB can serve as a reference database to investigate the lncRNAs and their interaction with RNA-binding proteins in plants. The PlncRNADB is freely available at http://bis.zju.edu.cn/PlncRNADB/.

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014346 ◽

2020 ◽

Vol 295 (42) ◽

pp. 14291-14304

Author(s):

Kathrin Bajak ◽

Kevin Leiss ◽

Christine Clayton ◽

Esteban Erben

Keyword(s):

Gene Expression ◽

Trypanosoma Brucei ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Affinity Purification ◽

Critical Role ◽

Rna Binding Protein ◽

Mrna Translation

In Trypanosoma brucei and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. 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 precytokinetic cells followed by rapid cell death. Affinity purification and pairwise yeast two-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 suboptimal open reading frames.

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Molecular cloning, characterization, and stress-responsive expression of genes encoding glycine-rich RNA-binding proteins in Camelina sativa L.

Plant Physiology and Biochemistry ◽

10.1016/j.plaphy.2013.03.023 ◽

2013 ◽

Vol 68 ◽

pp. 44-51 ◽

Cited By ~ 10

Author(s):

Kyung Jin Kwak ◽

Hunseung Kang ◽

Kyung-Hwan Han ◽

Sung-Ju Ahn

Keyword(s):

Molecular Cloning ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Camelina Sativa ◽

Expression Of Genes ◽

Genes Encoding

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Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains

Biological Chemistry ◽

10.1515/hsz-2013-0111 ◽

2013 ◽

Vol 394 (8) ◽

pp. 1077-1090 ◽

Cited By ~ 45

Author(s):

Kristin Wächter ◽

Marcel Köhn ◽

Nadine Stöhr ◽

Stefan Hüttelmaier

Keyword(s):

Subcellular Localization ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Nuclear Accumulation ◽

Structural Constraints ◽

Cellular Functions ◽

Dependent Protein ◽

Mrna Binding

Abstract The IGF2 mRNA-binding protein family (IGF2BPs) directs the cytoplasmic fate of various target mRNAs and controls essential cellular functions. The three IGF2BP paralogues expressed in mammals comprise two RNA-recognition motifs (RRM) as well as four KH domains. How these domains direct IGF2BP paralogue-dependent protein function remains largely elusive. In this study, we analyze the role of KH domains in IGF2BPs by the mutational GXXG-GEEG conversion of single KH domain loops in the context of full-length polypeptides. These analyses reveal that all four KH domains of IGF2BP1 and IGF2BP2 are essentially involved in RNA-binding in vitro and the cellular association with RNA-binding proteins (RBPs). Moreover the KH domains prevent the nuclear accumulation of these two paralogues and facilitate their recruitment to stress granules. The role of KH domains appears less pronounced in IGF2BP3, because GxxG-GEEG conversion in all four KH domains only modestly affects RNA-binding, subcellular localization and RNA-dependent protein association of this paralogue. These findings indicate paralogue-dependent RNA-binding properties of IGF2BPs which likely direct distinct cellular functions. Our findings suggest that IGF2BPs contact target RNAs via all four KH domains. This implies significant structural constraints, which presumably allow the formation of exceedingly stable protein-RNA complexes.

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An Interaction between Two RNA Binding Proteins, Nab2 and Pub1, Links mRNA Processing/Export and mRNA Stability

Molecular and Cellular Biology ◽

10.1128/mcb.00881-07 ◽

2007 ◽

Vol 27 (18) ◽

pp. 6569-6579 ◽

Cited By ~ 16

Author(s):

Luciano H. Apponi ◽

Seth M. Kelly ◽

Michelle T. Harreman ◽

Alexander N. Lehner ◽

Anita H. Corbett ◽

...

Keyword(s):

Mrna Stability ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Tail Length ◽

Functional Link ◽

Mrna Binding Protein ◽

Mrna Binding

ABSTRACT mRNA stability is modulated by elements in the mRNA transcript and their cognate RNA binding proteins. Poly(U) binding protein 1 (Pub1) is a cytoplasmic Saccharomyces cerevisiae mRNA binding protein that stabilizes transcripts containing AU-rich elements (AREs) or stabilizer elements (STEs). In a yeast two-hybrid screen, we identified nuclear poly(A) binding protein 2 (Nab2) as being a Pub1-interacting protein. Nab2 is an essential nucleocytoplasmic shuttling mRNA binding protein that regulates poly(A) tail length and mRNA export. The interaction between Pub1 and Nab2 was confirmed by copurification and in vitro binding assays. The interaction is mediated by the Nab2 zinc finger domain. Analysis of the functional link between these proteins reveals that Nab2, like Pub1, can modulate the stability of specific mRNA transcripts. The half-life of the RPS16B transcript, an ARE-like sequence-containing Pub1 target, is decreased in both nab2-1 and nab2-67 mutants. In contrast, GCN4, an STE-containing Pub1 target, is not affected. Similar results were obtained for other ARE- and STE-containing Pub1 target transcripts. Further analysis reveals that the ARE-like sequence is necessary for Nab2-mediated transcript stabilization. These results suggest that Nab2 functions together with Pub1 to modulate mRNA stability and strengthen a model where nuclear events are coupled to the control of mRNA turnover in the cytoplasm.

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