Purification of specific mRNP via the nascent polypeptide The RNA Binding Proteins ZC3H22 and ZC3H38

The main determinants of the cytoplasmic fate of an mRNA are the interactions between RNA-binding proteins (RBPs) and cis-regulatory motifs, present in the untranslated regions (UTRs) of the mRNAs. It is expected that translating mRNAs associate with many RBPs and other proteins that are recruited but do not necessarily interact directly with the mRNA, thus forming the messenger ribonucleo-protein particles (mRNPs). In the present dissertation, a method to detect key factors involved in the regulation of gene expression in trypanosomes was tested. The aim was to affinity purify specific ribosome-associated mRNPs and detect their protein components. The purification relies on three streptavidin binding peptides (3SBPs) at the N-terminus of the nascent polypeptide. These 3SBPs connected to the actively translating mRNAs on polyribosomes will bind to the streptavidin matrix. The average yield of the reporter mRNA was 16% relative to the input polysomal fraction. The reporter was also eight-fold enriched compared to the housekeeping gene. The method was validated using a known RNA-protein interaction in trypanosomes. A zinc finger protein, ZC3H11, binds to an AU-rich element present in the HSP70 3’-UTR and stabilizes this mRNA upon heat shock. Two independent purifications were made, one using a reporter containing the complete HSP70 3’-UTR and another without the AU-rich element, as a negative control. Indeed, ZC3H11 was detected in the purification when the AU-rich element was present in the HSP70 3’-UTR, and was absent from the control purification. The limitation of the method was the detection by mass spectrometry. Furthermore, two RBPs were studied, zinc finger proteins ZC3H22 and ZC3H38. ZC3H22 was found by quantitative mass spectrometry when purifying the EP 3’-UTR reporter using the affinity purification method described above. This protein seems to be present in the polyribosomes but down regulation of the gene by RNAi was only achieved to 30%, the protein was still produced and no changes in the growth phenotype observed. Only a single conditional knockout was obtained but not the double knockout. The role of this protein in procyclic trypanosomes is still not discovered. The protein ZC3H38 was previously found in our laboratory by Dr. Erben to increase target mRNA expression. My Tethering assay showed indeed, that the protein stabilized the reporter mRNA approximately 2-fold and increased 1.5-folds the amount of reporter protein produced. Analysing different parts of the protein by tethering assay also indicates that a region containing the HNPY domain might be responsible for the reporter mRNA stabilization. ZC3H38 protein is mainly localized in the cytoplasm. RNAi against ZC3H38 mRNA presents a change in the growth phenotype after 24 hours of tetracycline induction in bloodstream trypanosomes. Currently, more experiments are being carried out in order to further characterize this protein

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

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.Fil: Erben, Esteban Daniel. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentina. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Leiss, Kevin. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Liu, Bin. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Gil, Diana Inchaustegui. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Helbig, Claudia. Ruprecht Karls Universitat Heidelberg; AlemaniaFil: Clayton, Christine. Ruprecht Karls Universitat Heidelberg; Alemani

The Cell Cycle Regulated Transcriptome of Trypanosoma brucei

Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising questions about the extent of cell-cycle regulation in these organisms and the mechanisms whereby regulation is achieved. Here, we analyse gene expression over the Trypanosoma brucei cell cycle, measuring changes in mRNA abundance on a transcriptome-wide scale. We developed a “double-cut” elutriation procedure to select unperturbed, highly synchronous cell populations from log-phase cultures, and compared this to synchronization by starvation. Transcriptome profiling over the cell cycle revealed the regulation of at least 430 genes. While only a minority were homologous to known cell cycle regulated transcripts in yeast or human, their functions correlated with the cellular processes occurring at the time of peak expression. We searched for potential target sites of RNA-binding proteins in these transcripts, which might earmark them for selective degradation or stabilization. Over-represented sequence motifs were found in several co-regulated transcript groups and were conserved in other kinetoplastids. Furthermore, we found evidence for cell-cycle regulation of a flagellar protein regulon with a highly conserved sequence motif, bearing similarity to consensus PUF-protein binding motifs. RNA sequence motifs that are functional in cell-cycle regulation were more widespread than previously expected and conserved within kinetoplastids. These findings highlight the central importance of post-transcriptional regulation in the proliferation of parasitic kinetoplastids

Is There a Classical Nonsense-Mediated Decay Pathway in Trypanosomes?

In many eukaryotes, messenger RNAs with premature termination codons are destroyed by a process called “nonsense-mediated decay”, which requires the RNA helicase Upf1 and also, usually, an interacting factor, Upf2. Recognition of premature termination codons may rely on their distance from either a splice site or the polyadenylation site, and long 3′-untranslated regions can trigger mRNA decay. The protist Trypanosoma brucei relies heavily on mRNA degradation to determine mRNA levels, and 3′-untranslated regions play a major role in control of mRNA decay. We show here that trypanosomes have a homologue of Upf1, TbUPF1, which interacts with TbUPF2 and (in an RNA-dependent fashion) with poly(A) binding protein 1, PABP1. Introduction of a premature termination codon in either an endogenous gene or a reporter gene decreased mRNA abundance, as expected for nonsense-mediated decay, but a dependence of this effect on TbUPF1 could not be demonstrated, and depletion of TbUPF1 by over 95% had no effect on parasite growth or the mRNA transcriptome. Further investigations of the reporter mRNA revealed that increases in open reading frame length tended to increase mRNA abundance. In contrast, inhibition of translation, either using 5′-secondary structures or by lengthening the 5′-untranslated region, usually decreased reporter mRNA abundance. Meanwhile, changing the length of the 3′-untranslated region had no consistent effect on mRNA abundance. We suggest that in trypanosomes, translation per se may inhibit mRNA decay, and interactions with multiple RNA-binding proteins preclude degradation based on 3′-untranslated region length alone

Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide.

The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA

ZC3H11 co-purifies with the <i>HSP70</i> 3’ -UTR.

<p>(A) Schematic representation of the reporters used for the HSP70 mRNP purification. The control reporter (right) lacks the AUU repeat that is bound by ZC3H11. (B) Northern blot showing the purification of both <i>3SBP</i>-<i>CAT</i> mRNAs; details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148131#pone.0148131.g002" target="_blank">Fig 2</a>. (C) Western blot showing specific co-purification of V5-ZC3H11 with <i>3SBP</i>-<i>CAT</i>-HSP70 mRNA. The figures below the lanes show the relative loading in cell-equivalents. For the beads, about 10⁹ cell-equivalents were loaded. TR: trypanothione reductase (with an additional cross-reacting band); S9: ribosomal protein S9 (which detects two bands). A single V5-tagged band was observed. (D) Summary for four independent purifications. Results for the <i>CAT</i> baits are for four experiments each; those for TR and S9 are for all eight experiments. (E) The method cannot be used to identify proteins that bind to many other mRNAs in addition to the target mRNA. The same experimental set-up as in (B), but in cells expressing V5-MKT1. For the beads, about 10⁹ cell-equivalents were loaded.</p

Purification of the <i>3SBP-CAT-SKL-EP</i> mRNP.

<p>(A) The mRNAs. Multi-tag reporter (upper left) composed of 3SBPs at the N-terminus of the ORF and the control reporter (upper right), without the 3SBPs. SBP: streptavidin binding peptide; UTR: untranslated region; CAT: chloramphenicol acetyltransferase; <i>SL</i>: spliced leader; RBP: RNA-binding protein. The black portion is the <i>CAT</i> gene. (B) Northern blot showing purification of the <i>3SBP-CAT-SKL-EP</i> mRNA and failure to purify <i>CAT-SKL-EP</i> mRNA. <i>TUBA</i>: alpha tubulin. The numbers below the blots are the relative amounts of the mRNA measured, relative to the polysomal RNA input. These numbers are already correcting for loading. In: input cells (6x10⁷ cell-equivalents): pol: polysomes (6x10⁷ cell-equivalents); UB: unbound (6x10⁷ cell-equivalents); W: wash (6x10⁷ cell-equivalents); E: eluate (8x10⁷ cell-equivalents). Each probe detects a single band. (C) Box plot for all purifications similar to those in this Figure and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148131#pone.0148131.g003" target="_blank">Fig 3</a>. The centre line is the median, the boxes extend over the 25th to 75th percentiles, and the whiskers show the 95% confidence limits. The number of independent experiments for each construct is shown beneath the boxes.</p

Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins.

We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites

Locations of V5-RBP29 and V5-ZC3H29.

<p>(A) An <i>RBP29</i> gene was tagged <i>in situ</i> with a sequence encoding a V5 tag, in bloodstream forms. The tag was detected using anti-V5 antibody, with or without DNA staining with DAPI. Cells were shown as differential interference contrast (DIC) images. (B) Location of V5-ZC3H29 by immunofluorescence. Details as for (A).</p