15 research outputs found
Dynamic recruitment of microRNAs to their mRNA targets in the regenerating liver.
BACKGROUND: Validation of physiologic miRNA targets has been met with significant challenges. We employed HITS-CLIP to identify which miRNAs participate in liver regeneration, and to identify their target mRNAs.
RESULTS: miRNA recruitment to the RISC is highly dynamic, changing more than five-fold for several miRNAs. miRNA recruitment to the RISC did not correlate with changes in overall miRNA expression for these dynamically recruited miRNAs, emphasizing the necessity to determine miRNA recruitment to the RISC in order to fully assess the impact of miRNA regulation. We incorporated RNA-seq quantification of total mRNA to identify expression-weighted Ago footprints, and developed a microRNA regulatory element (MRE) prediction algorithm that represents a greater than 20-fold refinement over computational methods alone. These high confidence MREs were used to generate candidate \u27competing endogenous RNA\u27 (ceRNA) networks.
CONCLUSION: HITS-CLIP analysis provide novel insights into global miRNA:mRNA relationships in the regenerating liver
Studies on the structure and function of ribonucleoprotein complex of Dictyostelium discoideum RNase P
Ribonuclease Ρ is a ubiquitus ribonucleoprotein enzyme responsible for the production of the 5’ mature ends of all precursor tRNA molecules RNase Ρ endonucleolytic activity has been isolated from organisms representing the three domains of life namely Bacteria, Archaea, and Eukarya. It has been shown to contain an essential RNA subunit and one (Bacteria) or more (Archaea, Eukaryotes) proteins. The RNase Ρ RNA subunits from bacteria and some archaea are catalytically active in vitro, whereas those from eukaryotes and most archaea have lost most of their functionality and require protein subunits for activity. RNase Ρ has been characterized biochemically and genetically in several systems and structures for both RNA and protein subunits have emerged. The integration of structural and functional data is slowly forming a scenario for the evolution of RNase Ρ from an ancient enzyme to a highly organized ribonucleoprotein complex. Dictyostelium discoideum RNase Ρ harbors an essential RNA subunit, and has high protein content, as judged by its low boyant density. Nevertheless, our knowledge on the exact composition was limited. In the current study a gene showing significant similarity to human Rpp30 RNase Ρ protein subunit was identified in Dictyostelium genome. The gene encodes a protein (DRpp30) which is significantly larger than its homologues, due to an unusual C-terminus. The gene was cloned, overexpressed, and was used for the production of polyclonal antibodies. The participation of DRpp30 in the macromolecular complex of RNase Ρ was verified by an immunobiochemical approach. The recombinant protein was shown to bind specifically both the RNase Ρ RNA subunit and the pre-tRNA substrate in vitro, thus giving a first insight of its role in the holoenzyme complex. Homology modeling using as a template the archaeal Ph1887p, and molecular dynamics simulations of the modeled structure suggest that DRpp30 adopts a TIM-barrel fold. While our efforts to isolate the gene encoding the RNA subunit of D. discoideum RNase Ρ were in progress, Norman Pace and his group identified it through phylogenetic comparison. The full transcript of the gene was detected in active RNase Ρ samples along with a smaller transcript of the same gene. The exact 5’ and 3’ ends of both transcripts were identified and were cloned. Both these transcripts can substitute the endogenous RNA subunit in vitro, but no enzymatic activity associated with these RNA molecules could be detected so far.Η ριβονουκλεάση Ρ είναι το ένζυμο το οποίο αναλαμβάνει την δημιουργία του 5’ ώριμου άκρου όλων των προδρόμων μορίων tRNA. Πρόκειται για ένα ριβονουκλεοπρωτεϊνικό σύμπλοκο το οποίο εντοπίζεται στα κύτταρα των οργανισμών και από τις τρεις κυρίες φυλογενετικές περιοχές, τα Βακτήρια, τα Αρχαία και τους Ευκαρυώτες. Αποτελείται από μια υπομονάδα RNA απαραίτητη για την κατάλυση, ενώ το μέγεθος και ο αριθμός των πρωτεϊνικών υπομονάδων ποικίλλει από μια μικρή στα βακτήρια έως δέκα πρωτεΐνες στο ολοένζυμο που απομονώνεται από τα ανθρωπινά κύτταρα. Οι υπομονάδες RNA των βακτηρίων καθώς επίσης και μερικών αρχαίων μπορούν να καταλύσουν την αντίδραση ωρίμανσης του tRNA απουσία της πρωτεΐνης in vitro είναι δηλαδή ριβοένζυμα. Η ανακάλυψη αυτή διεύρυνε τις αντιλήψεις μας για τις ιδιότητες των βιομορίων και επανέφερε στο προσκήνιο την θεωρία του κόσμου του RNA. Στο ευκαρυωτικό ριβοένζυμο ο ρόλος των πρωτεϊνών είναι πιο ουσιαστικός καθώς η υπομονάδα RNA φαίνεται ότι χάνει μεγάλο μέρος της λειτουργικής της ανεξαρτησίας. Η διαλεύκανση των λειτουργών της κάθε υπομονάδας θα δώσει σημαντικές πληροφορίες για την εξέλιξη της RNase Ρ από ένα αρχέγονο ένζυμο σε ένα πολύπλοκο ριβονουκλεοπρωτεϊνικό σύμπλοκο. Η RNase Ρ από το Dictyostelium discoideum διαθέτει μια απαραίτητη για την δραστικότητα υπομονάδα RNA όπως και όλα τα ένζυμα αυτού του είδους. Παράλληλα διαθέτει έντονο πρωτεϊνικό χαρακτήρα καθώς διαθέτει την χαμηλότερη πυκνότητα επιπολής σε σχέση με ένζυμα RNase Ρ από άλλους οργανισμούς. Οι πληροφορίες αυτές προέρχονται από τον αρχικό χαρακτηρισμό του ενζυμικού συμπλόκου, και δεν παρέχουν στοιχεία για την ακριβή σύστασή του. Στην παρούσα μελέτη πραγματοποιήθηκε κλωνοποίηση και χαρακτηρισμός ενός από τα γονίδια που εντοπίστηκαν στο γονιδίωμα του Dictyostelium, ομόλογα προς χαρακτηρισμένα γονίδια από τον άνθρωπο και άλλους ευκαρυώτες. Το γονίδιο drpp30 κωδικεύει μια πρωτεΐνη 40.7 kDa, σημαντικά μεγαλύτερη από τις ομόλογές της. Η πρωτεΐνη DRpp30 υπερεκφράστηκε σε βακτηριακά κύτταρα, και μετά τον χρωματογραφικό καθαρισμό της χρησιμοποιήθηκε για την παρασκευή πολυκλωνικών αντισωμάτων. Η συμμέτοχη της DRpp30 στο μακρομοριακό σύμπλοκο της RNase Ρ πιστοποιήθηκε με ανοσοβιοχημική προσέγγιση, ενώ η ανασυνδυασμένη πρωτεΐνη προσδένει το pre-tRNA υπόστρωμα του ένζυμου καθώς και την υπομονάδα RNA in vitro. Το μοντέλο ομολογίας της DRpp30 βάσει της κρυσταλλικής δομής της ορθόλογης Ρh1877 από τα αρχαία φανερώνει ότι η πρωτεΐνη αποκτά τη δομή αβ βαρελιού (TIM barrel fold). Κατά τη διάρκεια της διατριβής οι προσπάθειες για τον εντοπισμό του γονίδιου της RNA υπομονάδας ήταν σε εξέλιξη όταν το εν λόγω γονίδιο αναγνωρίστηκε μέσω φυλογενετικών συγκρίσεων από την ομάδα του Norman Pace. Το μετάγραφο του γονίδιου εντοπίστηκε σε ενεργά κλάσματα RNase Ρ, και παράλληλα εντοπίστηκε και ένα μικρότερο μετάγραφο του ίδιου γονίδιου. Προσδιορίστηκαν τα ακριβή 5’ και 3’ άκρα των δύο αυτών μορίων και ακολούθησε κλωνοποίησή τους. Τα in vitro μετάγραφα των δύο κλωνοποιημένων αλληλουχιών μπορούν να υποκαθιστούν την ενδογενή RNA υπομονάδα του ολοενζύμου in vitro, ενώ δεν εντοπίστηκε έως τώρα ενζυμική δραστικότητα που να σχετίζεται με τα δυο αυτά μόρια
Elective affinities: a Tudor–Aubergine tale of germline partnership
In Drosophila melanogaster and many other metazoans, the specification of germ cells requires cytoplasmic inheritance of maternally synthesized RNA and protein determinants, which are assembled in electron-dense cytoplasmic structures known as germ or polar granules, found at the posterior end of the oocytes. Recent studies have shown that the formation of germ granules is dependent on the interaction of proteins containing tudor domains with the piwi-interacting RNA (piRNA)-binding Piwi proteins, and such interactions are dependent on symmetrically dimethylated arginines (sDMAs) of Piwi proteins. Tudor–Piwi interactions are crucial and are conserved in the germ cells of sexually reproducing animals, including mammals. In the September 1, 2010, issue of Genes & Development, Liu and colleagues (pp. 1876–1881) use a combination of genetics, biochemistry, and crystallography to uncover the molecular and structural details of how Tudor recognizes and binds the sDMAs of the Piwi protein Aubergine
Activation of bacterial ribonuclease P by macrolides.
The effect of macrolide antibiotic spiramycin on RNase P holoenzyme and M1 RNA from Escherichia coli was investigated. Ribonuclease P (RNase P) is a ribozyme that is responsible for the maturation of 5' termini of tRNA molecules. Spiramycin revealed a dose-dependent activation on pre-tRNA cleavage by E. coli RNase P holoenzyme and M1 RNA. The K s and V max, as well as the K s(app) and V max(app) values of RNase P holoenzyme and M1 RNA in the presence or absence of spiramycin, were calculated from primary and secondary kinetic plots. It was found that the activity status of RNase P holoenzyme and M1 RNA is improved by the presence of spiramycin 18- and 12-fold, respectively. Primer extension analysis revealed that spiramycin induces a conformational change of the P10/11 structural element of M1 RNA, which is involved in substrate recognition.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe
Insights into functional modulation of catalytic RNA activity.
RNA molecules play critical roles in cell biology, and novel findings continuously broaden their functional repertoires. Apart from their well-documented participation in protein synthesis, it is now apparent that several noncoding RNAs (i.e. micro-RNAs and riboswitches) also participate in the regulation of gene expression. The discovery of catalytic RNAs had profound implications on our views concerning the evolution of life on our planet at a molecular level. A characteristic attribute of RNA, probably traced back to its ancestral origin, is the ability to interact with and be modulated by several ions and molecules of different sizes. The inhibition of ribosome activity by antibiotics has been extensively used as a therapeutical approach, while activation and substrate-specificity alteration have the potential to enhance the versatility of ribozyme-based tools in translational research. In this review, we will describe some representative examples of such modulators to illustrate the potential of catalytic RNAs as tools and targets in research and clinical approaches.Journal ArticleResearch Support, Non-U.S. Gov'tReviewFLWINinfo:eu-repo/semantics/publishe
Domain architecture of the DRpp29 protein and its interaction with the RNA subunit of Dictyostelium discoideum RNase P.
Dictyostelium discoideum nuclear RNase P is a ribonucleoprotein complex that displays similarities with its counterparts from higher eukaryotes such as the human enzyme, but at the same time it retains distinctive characteristics. In the present study, we report the molecular cloning and interaction details of DRpp29 and RNase P RNA, two subunits of the RNase P holoenzyme from D. discoideum. Electrophoretic mobility shift assays exhibited that DRpp29 binds specifically to the RNase P RNA subunit, a feature that was further confirmed by the molecular modeling of the DRpp29 structure. Moreover, deletion mutants of DRpp29 were constructed in order to investigate the domains of DRpp29 that contribute to and/or are responsible for the direct interaction with the D. discoideum RNase P RNA. A eukaryotic specific, lysine- and arginine-rich region was revealed, which seems to facilitate the interaction between these two subunits. Furthermore, we tested the ability of wild-type and mutant DRpp29 to form active RNase P enzymatic particles with the Escherichia coli RNase P RNA.Journal Articleinfo:eu-repo/semantics/publishe
Partial purification and characterization of RNase P from human peripheral lymphocytes.
Ribonuclease P (RNase P) is ubiquitous and essential Mg(2+)-dependent endoribonuclease that catalyzes the 5'-maturation of transfer RNAs. RNase P and the ribosome are so far the only ribozymes known to be conserved in all kingdoms of life. Eukaryotic RNase P activity has been detected in nuclei, mitochondria and chloroplasts and demonstrates great variability in sequence and subunit composition. In the last few years we have developed methodologies and pursued projects addressing the occurrence, distribution and the potential physiological role of RNase P in human epidermal keratinocytes. In view of the vital importance of lymphocytes for an effective immune system and their successful application after transfection with RNase P-associated external guide sequences in gene therapy, we concerned ourselves with the isolation and characterization of RNase P of peripheral human lymphocytes. We developed a method described herein, that will enable the study of the possible involvement of this ribozyme in the pathogenetic mechanisms of diverse autoimmune, inflammatory and neoplastic cutaneous disorders and may facilitate the further development of RNase P-based technology for gene therapy of infectious and neoplastic dermatoses.Journal ArticleResearch Support, Non-U.S. Gov'tFLWINinfo:eu-repo/semantics/publishe
The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing
Piwi–piRNA (Piwi-interacting RNA) ribonucleoproteins (piRNPs) enforce retrotransposon silencing, a function critical for preserving the genome integrity of germ cells. The molecular functions of most of the factors that have been genetically implicated in primary piRNA biogenesis are still elusive. Here we show that MOV10L1 exhibits 5′-to-3′ directional RNA-unwinding activity in vitro and that a point mutation that abolishes this activity causes a failure in primary piRNA biogenesis in vivo. We demonstrate that MOV10L1 selectively binds piRNA precursor transcripts and is essential for the generation of intermediate piRNA processing fragments that are subsequently loaded to Piwi proteins. Multiple analyses suggest an intimate coupling of piRNA precursor processing with elements of local secondary structures such as G quadruplexes. Our results support a model in which MOV10L1 RNA helicase activity promotes unwinding and funneling of the single-stranded piRNA precursor transcripts to the endonuclease that catalyzes the first cleavage step of piRNA processing.</p
Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization
Piwi proteins such as Drosophila Aubergine (Aub) and mouse Miwi are essential for germline development and for primordial germ cell (PGC) specification. They bind piRNAs and contain symmetrically dimethylated arginines (sDMAs), catalyzed by dPRMT5. PGC specification in Drosophila requires maternal inheritance of cytoplasmic factors, including Aub, dPRMT5, and Tudor (Tud), that are concentrated in the germ plasm at the posterior end of the oocyte. Here we show that Miwi binds to Tdrd6 and Aub binds to Tudor, in an sDMA-dependent manner, demonstrating that binding of sDMA-modified Piwi proteins with Tudor-domain proteins is an evolutionarily conserved interaction in germ cells. We report that in Drosophila tud1 mutants, the piRNA pathway is intact and most transposons are not de-repressed. However, the localization of Aub in the germ plasm is severely reduced. These findings indicate that germ plasm assembly requires sDMA modification of Aub by dPRMT5, which, in turn, is required for binding to Tudor. Our study also suggests that the function of the piRNA pathway in PGC specification may be independent of its role in transposon control
The MOV10 RNA helicase is a dosage-dependent host restriction factor for LINE1 retrotransposition in mice.
Transposable elements constitute nearly half of the mammalian genome and play important roles in genome evolution. While a multitude of both transcriptional and post-transcriptional mechanisms exist to silence transposable elements, control of transposition in vivo remains poorly understood. MOV10, an RNA helicase, is an inhibitor of mobilization of retrotransposons and retroviruses in cell culture assays. Here we report that MOV10 restricts LINE1 retrotransposition in mice. Although MOV10 is broadly expressed, its loss causes only incomplete penetrance of embryonic lethality, and the surviving MOV10-deficient mice are healthy and fertile. Biochemically, MOV10 forms a complex with UPF1, a key component of the nonsense-mediated mRNA decay pathway, and primarily binds to the 3' UTR of somatically expressed transcripts in testis. Consequently, loss of MOV10 results in an altered transcriptome in testis. Analyses using a LINE1 reporter transgene reveal that loss of MOV10 leads to increased LINE1 retrotransposition in somatic and reproductive tissues from both embryos and adult mice. Moreover, the degree of LINE1 retrotransposition inhibition is dependent on the Mov10 gene dosage. Furthermore, MOV10 deficiency reduces reproductive fitness over successive generations. Our findings demonstrate that MOV10 attenuates LINE1 retrotransposition in a dosage-dependent manner in mice