RNA binding
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MRP1/2 is a heteromeric protein complex that functions in the trypanosomatid mitochondrion as part of the RNA editing machinery, which facilitates multiple targeted insertions and deletions of uridines. MRP1/2 was shown to interact with MRB8170, which initiates RNA editing by marking pre-edited mRNAs, while TbRGG2 is required for its efficient progression on pan-edited mRNAs. Both MRP1/2 and TbRGG2 are capable of modulating RNA-RNA interactions in vitro. As determined by using iCLIP and RIP-qPCR, RNAs bound to MRP1/2 are characterized and compared with those associated with MRB8170 and TbRGG2. We provide evidence that MRP1 and MRB8170 have correlated binding and similar RNA crosslinking peak profiles over minimally and never-edited mRNAs. Our results suggest that MRP1 assists MRB8170 in RNA editing on minimally edited mRNAs.
- Klíčová slova
- RNA binding proteins, RNA editing, iCLIP, mitochondrion, ribonuclear protein, trypanosome,
- MeSH
- editace RNA MeSH
- messenger RNA genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- proteiny vázající RNA metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA mitochondriální genetika metabolismus MeSH
- Trypanosoma genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- gBP21 protein, Trypanosoma brucei MeSH Prohlížeč
- messenger RNA MeSH
- mitochondrial messenger RNA MeSH Prohlížeč
- proteiny vázající RNA MeSH
- protozoální proteiny MeSH
- RNA mitochondriální MeSH
Reactive RNA probes are useful for studying and identifying RNA-binding proteins. To that end, we designed and synthesized chloroacetamide-linked 7-deaza-ATP which was a good substrate for T7 RNA polymerase in in vitro transcription assay to synthesize reactive RNA probes bearing one or several reactive modifications. Modified RNA probes reacted with thiol-containing molecules as well as with cysteine- or histidine-containing peptides to form stable covalent products. They also reacted selectively with RNA-binding proteins to form cross-linked conjugates in high conversions thanks to proximity effect. Our modified nucleotide and RNA probes are promising tools for applications in RNA (bio)conjugations or RNA proteomics.
- Klíčová slova
- Bioconjugations, Cross-Linking, Modified RNA, Proteins, RNA Polymerases,
- MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- DNA metabolismus MeSH
- nukleotidy * metabolismus MeSH
- proteiny vázající RNA MeSH
- reagencia zkříženě vázaná MeSH
- RNA sondy MeSH
- RNA * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chloroacetamide MeSH Prohlížeč
- DNA řízené RNA-polymerasy MeSH
- DNA MeSH
- nukleotidy * MeSH
- proteiny vázající RNA MeSH
- reagencia zkříženě vázaná MeSH
- RNA sondy MeSH
- RNA * MeSH
Expression of the nascent RNA transcript is regulated by its interaction with a number of proteins. The misregulation of such interactions can often result in impaired cellular functions that can lead to cancer and a number of diseases. Thus, our understanding of RNA-protein interactions within the cellular context is essential for the development of novel diagnostic and therapeutic tools. While there are many in vitro methods that analyze RNA-protein interactions in vivo approaches are scarce. Here we established a method based on fluorescence resonance energy transfer (FRET), which we term RNA-binding mediated FRET (RB-FRET), which determines RNA-protein interaction inside cells and tested it on hnRNP H protein binding to its cognate RNA. Using two different approaches, we provide evidence that RB-FRET is sensitive enough to detect specific RNA-protein interactions in the cell, providing a powerful tool to study spatial and temporal localization of specific RNA-protein complexes.
- MeSH
- genetické vektory genetika MeSH
- HeLa buňky MeSH
- lidé MeSH
- proteiny vázající RNA genetika metabolismus MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- RNA analýza metabolismus MeSH
- sekvence nukleotidů MeSH
- substrátová specifita MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- proteiny vázající RNA MeSH
- RNA MeSH
The mitochondrial RNA binding complex 1 (MRB1) is a recently discovered complex of proteins associated with the TbRGG1 and TbRGG2 proteins in Trypanosoma brucei. Based on the phenotype caused by down-regulation of these two proteins, it was proposed to play an unspecified role in RNA editing. RNAi silencing of three newly characterized protein subunits, guide RNA associated proteins (GAPs) 1 and 2 as well as a predicted DExD/H-box RNA helicase, show they are essential for cell growth in the procyclic stage. Furthermore, their down-regulation leads to inhibition of editing in only those mRNAs for which minicircle-encoded guide (g) RNAs are required. However, editing remains unaffected when the maxicircle-encoded cis-acting gRNA is employed. Interestingly, all three proteins are necessary for the expression of the minicircle-encoded gRNAs. Moreover, down-regulation of a fourth assayed putative MRB1 subunit, Nudix hydrolase, does not appear to destabilize gRNAs, and down-regulation of this protein has a general impact on the stability of maxicircle-encoded RNAs. GAP1 and 2 are also essential for the survival of the bloodstream stage, in which the gRNAs become eliminated upon depletion of either protein. Immunolocalization revealed that GAP1 and 2 are concentrated into discrete spots along the mitochondrion, usually localized in the proximity of the kinetoplast. Finally, we demonstrate that the same mtRNA polymerase known to transcribe the maxicircle mRNAs may also have a role in expression of the minicircle-encoded gRNAs.
- MeSH
- DEAD-box RNA-helikasy metabolismus MeSH
- DNA řízené RNA-polymerasy metabolismus MeSH
- guide RNA, Kinetoplastida genetika MeSH
- mitochondriální proteiny metabolismus MeSH
- NUDIX hydrolasy MeSH
- proteiny vázající RNA metabolismus MeSH
- protozoální proteiny metabolismus MeSH
- pyrofosfatasy metabolismus MeSH
- RNA mitochondriální MeSH
- RNA protozoální genetika MeSH
- RNA genetika MeSH
- Trypanosoma brucei brucei genetika růst a vývoj MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DEAD-box RNA-helikasy MeSH
- DNA řízené RNA-polymerasy MeSH
- guide RNA, Kinetoplastida MeSH
- mitochondriální proteiny MeSH
- proteiny vázající RNA MeSH
- protozoální proteiny MeSH
- pyrofosfatasy MeSH
- RNA mitochondriální MeSH
- RNA protozoální MeSH
- RNA MeSH
DNA and RNA binding proteins (DRBPs) are a broad class of molecules that regulate numerous cellular processes across all living organisms, creating intricate dynamic multilevel networks to control nucleotide metabolism and gene expression. These interactions are highly regulated, and dysregulation contributes to the development of a variety of diseases, including cancer. An increasing number of proteins with DNA and/or RNA binding activities have been identified in recent years, and it is important to understand how their activities are related to the molecular mechanisms of cancer. In addition, many of these proteins have overlapping functions, and it is therefore essential to analyze not only the loss of function of individual factors, but also to group abnormalities into specific types of activities in regard to particular cancer types. In this review, we summarize the classes of DNA-binding, RNA-binding, and DRBPs, drawing particular attention to the similarities and differences between these protein classes. We also perform a cross-search analysis of relevant protein databases, together with our own pipeline, to identify DRBPs involved in cancer. We discuss the most common DRBPs and how they are related to specific cancers, reviewing their biochemical, molecular biological, and cellular properties to highlight their functions and potential as targets for treatment.
- Klíčová slova
- DNA/RNA binding protein, biomarkers, cancer, mutation, targeted treatment,
- MeSH
- DNA vazebné proteiny metabolismus MeSH
- DNA MeSH
- lidé MeSH
- nádory * genetika metabolismus MeSH
- proteiny vázající RNA * metabolismus MeSH
- RNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- DNA vazebné proteiny MeSH
- DNA MeSH
- proteiny vázající RNA * MeSH
- RNA MeSH
Repression of msl-2 mRNA translation is essential for viability of Drosophila melanogaster females to prevent hypertranscription of both X chromosomes. This translational control event is coordinated by the female-specific protein Sex-lethal (Sxl) which recruits the RNA binding proteins Unr and Hrp48 to the 3' untranslated region (UTR) of the msl-2 transcript and represses translation initiation. The mechanism exerted by Hrp48 during translation repression and its interaction with msl-2 are not well understood. Here we investigate the RNA binding specificity and affinity of the tandem RNA recognition motifs of Hrp48. Using NMR spectroscopy, molecular dynamics simulations and isothermal titration calorimetry, we identified the exact region of msl-2 3' UTR recognized by Hrp48. Additional biophysical experiments and translation assays give further insights into complex formation of Hrp48, Unr, Sxl and RNA. Our results show that Hrp48 binds independent of Sxl and Unr downstream of the E and F binding sites of Sxl and Unr to msl-2.
- Klíčová slova
- Dosage compensation, Hrp48, RNA binding protein, RNA recognition motif, Translation regulation,
- MeSH
- 3' nepřekládaná oblast * MeSH
- DNA vazebné proteiny MeSH
- Drosophila melanogaster * metabolismus genetika MeSH
- heterogenní jaderné ribonukleoproteiny MeSH
- messenger RNA metabolismus genetika chemie MeSH
- proteiny Drosophily * metabolismus chemie genetika MeSH
- proteiny vázající RNA * metabolismus chemie genetika MeSH
- proteosyntéza MeSH
- simulace molekulární dynamiky MeSH
- transkripční faktory metabolismus chemie genetika MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 3' nepřekládaná oblast * MeSH
- DNA vazebné proteiny MeSH
- heterogenní jaderné ribonukleoproteiny MeSH
- Hrb27C protein, Drosophila MeSH Prohlížeč
- messenger RNA MeSH
- msl-2 protein, Drosophila MeSH Prohlížeč
- proteiny Drosophily * MeSH
- proteiny vázající RNA * MeSH
- transkripční faktory MeSH
In Saccharomyces cerevisiae, the Nrd1-dependent termination and processing pathways play an important role in surveillance and processing of non-coding ribonucleic acids (RNAs). The termination and subsequent processing is dependent on the Nrd1 complex consisting of two RNA-binding proteins Nrd1 and Nab3 and Sen1 helicase. It is established that Nrd1 and Nab3 cooperatively recognize specific termination elements within nascent RNA, GUA[A/G] and UCUU[G], respectively. Interestingly, some transcripts do not require GUA[A/G] motif for transcription termination in vivo and binding in vitro, suggesting the existence of alternative Nrd1-binding motifs. Here we studied the structure and RNA-binding properties of Nrd1 using nuclear magnetic resonance (NMR), fluorescence anisotropy and phenotypic analyses in vivo. We determined the solution structure of a two-domain RNA-binding fragment of Nrd1, formed by an RNA-recognition motif and helix-loop bundle. NMR and fluorescence data show that not only GUA[A/G] but also several other G-rich and AU-rich motifs are able to bind Nrd1 with affinity in a low micromolar range. The broad substrate specificity is achieved by adaptable interaction surfaces of the RNA-recognition motif and helix-loop bundle domains that sandwich the RNA substrates. Our findings have implication for the role of Nrd1 in termination and processing of many non-coding RNAs arising from bidirectional pervasive transcription.
- MeSH
- dimerizace MeSH
- molekulární modely MeSH
- mutace MeSH
- proteiny vázající RNA chemie genetika metabolismus MeSH
- RNA chemie metabolismus MeSH
- Saccharomyces cerevisiae - proteiny chemie genetika metabolismus MeSH
- terciární struktura proteinů MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- NRD1 protein, S cerevisiae MeSH Prohlížeč
- proteiny vázající RNA MeSH
- RNA MeSH
- Saccharomyces cerevisiae - proteiny MeSH
RNA-binding proteins (RBPs) are critical to posttranscriptional gene regulation. Therefore, characterization of the RNA molecules bound by RBPs in vivo represent a key step in elucidating their function. The recently developed iCLIP technique allows single nucleotide resolution of the RNA binding footprints of RBPs. We present the iCLIP technique modified for its application to Trypanosoma brucei and most likely other kinetoplastid flagellates. By using the immuno- or affinity purification approach, it was successfully applied to the analysis of several RBPs. Furthermore, we also provide a detailed description of the iCLIP/iCLAP protocol that shall be particularly suitable for the studies of trypanosome RBPs.
- Klíčová slova
- Posttranscriptional gene regulation, RNA-binding proteins (RBPs), iCLAP, iCLIP,
- MeSH
- imunoprecipitace metody MeSH
- nukleotidy genetika metabolismus MeSH
- parazitologie metody MeSH
- proteiny vázající RNA analýza genetika metabolismus MeSH
- protozoální proteiny analýza genetika metabolismus MeSH
- RNA protozoální genetika metabolismus MeSH
- RNA genetika metabolismus MeSH
- Trypanosoma brucei brucei genetika MeSH
- ultrafialové záření MeSH
- vazba proteinů genetika účinky záření MeSH
- vazebná místa genetika MeSH
- zobrazení jednotlivé molekuly metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- nukleotidy MeSH
- proteiny vázající RNA MeSH
- protozoální proteiny MeSH
- RNA protozoální MeSH
- RNA MeSH
Trypanosoma brucei is the causative agent of the human and veterinarian diseases African sleeping sickness and nagana. A majority of its mitochondrial-encoded transcripts undergo RNA editing, an essential process of post-transcriptional uridine insertion and deletion to produce translatable mRNA. Besides the well-characterized RNA editing core complex, the mitochondrial RNA-binding 1 (MRB1) complex is one of the key players. It comprises a core complex of about six proteins, guide RNA-associated proteins (GAPs) 1/2, which form a heterotetramer that binds and stabilizes gRNAs, plus MRB5390, MRB3010, and MRB11870, which play roles in initial stages of RNA editing, presumably guided by the first gRNA:mRNA duplex in the case of the latter two proteins. To better understand all functions of the MRB1 complex, we performed a functional analysis of the MRB8620 core subunit, the only one not characterized so far. Here we show that MRB8620 plays a role in RNA editing in both procyclic and bloodstream stages of T. brucei, which reside in the tsetse fly vector and mammalian circulatory system, respectively. While RNAi silencing of MRB8620 does not affect procyclic T. brucei fitness when grown in glucose-containing media, it is somewhat compromised in cells grown in the absence of this carbon source. MRB8620 is crucial for integrity of the MRB1 core, such as its association with GAP1/2, which presumably acts to deliver gRNAs to this complex. In contrast, GAP1/2 is not required for the fabrication of the MRB1 core. Disruption of the MRB1 core assembly is followed by the accumulation of mRNAs associated with GAP1/2.
- Klíčová slova
- RNA editing, mitochondrion, trypanosome,
- MeSH
- buněčné linie MeSH
- editace RNA * MeSH
- messenger RNA genetika metabolismus MeSH
- mitochondriální proteiny fyziologie MeSH
- mitochondrie MeSH
- protozoální proteiny fyziologie MeSH
- Trypanosoma brucei brucei genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- messenger RNA MeSH
- mitochondriální proteiny MeSH
- protozoální proteiny MeSH
A majority of Trypanosoma brucei proteins have unknown functions, a consequence of its independent evolutionary history within the order Kinetoplastida that allowed for the emergence of several unique biological properties. Among these is RNA editing, needed for expression of mitochondrial-encoded genes. The recently discovered mitochondrial RNA binding complex 1 (MRB1) is composed of proteins with several functions in processing organellar RNA. We characterize two MRB1 subunits, referred to herein as MRB8170 and MRB4160, which are paralogs arisen from a large chromosome duplication occurring only in T. brucei. As with many other MRB1 proteins, both have no recognizable domains, motifs, or orthologs outside the order. We show that they are both novel RNA binding proteins, possibly representing a new class of these proteins. They associate with a similar subset of MRB1 subunits but not directly with each other. We generated cell lines that either individually or simultaneously target the mRNAs encoding both proteins using RNAi. Their dual silencing results in a differential effect on moderately and pan-edited RNAs, suggesting a possible functional separation of the two proteins. Cell growth persists upon RNAi silencing of each protein individually in contrast to the dual knockdown. Yet, their apparent redundancy in terms of cell viability is at odds with the finding that only one of these knockdowns results in the general degradation of pan-edited RNAs. While MRB8170 and MRB4160 share a considerable degree of conservation, our results suggest that their recent sequence divergence has led to them influencing mitochondrial mRNAs to differing degrees.
- MeSH
- biologické modely MeSH
- klonování DNA MeSH
- konzervovaná sekvence MeSH
- makromolekulární látky metabolismus MeSH
- messenger RNA metabolismus MeSH
- podjednotky proteinů genetika metabolismus MeSH
- proteiny vázající RNA chemie genetika metabolismus fyziologie MeSH
- protozoální proteiny chemie genetika metabolismus fyziologie MeSH
- RNA mitochondriální MeSH
- RNA metabolismus MeSH
- sekvenční homologie MeSH
- substrátová specifita MeSH
- Trypanosoma brucei brucei genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- makromolekulární látky MeSH
- messenger RNA MeSH
- podjednotky proteinů MeSH
- proteiny vázající RNA MeSH
- protozoální proteiny MeSH
- RNA mitochondriální MeSH
- RNA MeSH