TY - JOUR AB - RNA- and DNA-binding domains are essential building blocks for specific regulation of gene expression. While a number of canonical nucleic acid binding domains share sequence and structural conservation, others are less obviously linked by evolutionary traits. In this review, we describe a protein fold of about 150 aa in length, bearing a conserved beta-beta-beta-beta-alpha-linker-beta-beta-beta-beta-alpha topology and similar nucleic acid binding properties but no apparent sequence conservation. The same overall fold can also be achieved by dimerization of two proteins, each bearing a beta-beta-beta-beta-alpha topology. These proteins include but are not limited to the transcription factors PC4 and P24 from humans and plants, respectively, the human RNA-transport factor Pur-alpha (also termed PURA), as well as the ssDNA-binding SP_0782 protein fromStreptococcus pneumoniaand the bacteriophage coat proteins PP7 and MS2. Besides their common overall topology, these proteins share common nucleic acids binding surfaces and thus functional similarity. We conclude that these PC4-like domains include proteins from all kingdoms of life and are much more abundant than previously known. AU - Janowski, R. AU - Niessing, D. C1 - 59255 C2 - 48691 CY - 530 Walnut Street, Ste 850, Philadelphia, Pa 19106 Usa SP - 1228-1238 TI - The large family of PC4-like domains - similar folds and functions throughout all kingdoms of life. JO - RNA Biol. VL - 17 IS - 9 PB - Taylor & Francis Inc PY - 2020 SN - 1547-6286 ER - TY - JOUR AB - Nucleotide modifications constitute marks in RNA and DNA that contribute to gene regulation, development and other cellular processes. The understanding of their intricate molecular roles has been hampered by the high number of different modifications, the lack of effective methods and tools for their detection and quantification as well as by their complex structure-function relationship. The recent development of RNA and DNA immunoprecipitation followed by high-throughput sequencing (RIP- and DIP-seq) initiated detailed transcriptome- and genome-wide studies. Both techniques depend on highly specific and sensitive antibodies to specifically enrich the targeted modified nucleotides without background or potential biases. Here, we review the challenges and developments when generating and validating antibodies targeting modified nucleotides. We discuss antibody-antigen interactions, different strategies of antigen generation and compare different binder formats suitable for state-of-the-art high resolution mapping and imaging technologies. AU - Feederle, R. AU - Schepers, A. C1 - 50679 C2 - 42808 CY - Philadelphia SP - 1-10 TI - Antibodies specific for nucleic acid modifications. JO - RNA Biol. VL - 14 IS - 9 PB - Taylor & Francis Inc PY - 2017 SN - 1547-6286 ER - TY - JOUR AB - Asymmetric ASH1 mRNA transport during mitosis of budding yeast constitutes one of the best-studied examples of mRNA localization. Recently, 2 studies used in vitro motility assays to prove that motile ASH1 mRNA-transport complexes can be reconstituted entirely from recombinant factors. Both studies, however, differed in their conclusions on whether cargo RNA itself is required for particle assembly and thus activation of directional transport. Here we provide direct evidence that stable complexes do assemble in absence of RNA at physiologic conditions and even at ionic strengths above cellular levels. These results directly confirm the previous notion that the ASH1 transport machinery is not activated by the cargo RNA itself, but rather through protein-protein interactions. AU - Edelmann, F. AU - Niedner, A. AU - Niessing, D. C1 - 44088 C2 - 36880 CY - Philadelphia SP - 233-237 TI - ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions. JO - RNA Biol. VL - 12 IS - 3 PB - Taylor & Francis Inc PY - 2015 SN - 1547-6286 ER - TY - JOUR AB - microRNAs and microRNA-independent RNA-binding proteins are 2 classes of post-transcriptional regulators that have been shown to cooperate in gene-expression regulation. We compared the genome-wide target sets of microRNAs and RBPs identified by recent CLIP-Seq technologies, finding that RBPs have distinct target sets and favor gene interaction network hubs. To identify microRNAs and RBPs with a similar functional context, we developed simiRa, a tool that compares enriched functional categories such as pathways and GO terms. We applied simiRa to the known functional cooperation between Pumilio family proteins and miR-221/222 in the regulation of tumor supressor gene p27 and show that the cooperation is reflected by similar enriched categories but not by target genes. SimiRa also predicts possible cooperation of microRNAs and RBPs beyond direct interaction on the target mRNA for the nuclear RBP TAF15. To further facilitate research into cooperation of microRNAs and RBPs, we made simiRa available as a web tool that displays the functional neighborhood and similarity of microRNAs and RBPs: http://vsicb-simira.helmholtz-muenchen.de . AU - Preusse, M. AU - Marr, C. AU - Saunders, S.* AU - Maticzka, D.* AU - Lickert, H. AU - Backofen, R.* AU - Theis, F.J. C1 - 46926 C2 - 39039 SP - 998-1009 TI - SimiRa: A tool to identify coregulation between microRNAs and RNA-binding proteins. JO - RNA Biol. VL - 12 IS - 9 PY - 2015 SN - 1547-6286 ER - TY - JOUR AB - Asymmetric, motor-protein dependent transport of mRNAs and subsequent localized translation is an important mechanism of gene regulation. Due to the high complexity of such motile particles, our mechanistic understanding of mRNA localization is limited. Over the last two decades, ASH1 mRNA localization in budding yeast has served as comparably simple and accessible model system. Recent advances have helped to draw an increasingly clear picture on the molecular mechanisms governing ASH1 mRNA localization from its co-transcriptional birth to its delivery at the site of destination. These new insights help to better understand the requirement of initial nuclear mRNPs, the molecular basis of specific mRNA-cargo recognition via cis-acting RNA elements, the different stages of RNP biogenesis and reorganization, as well as activation of the motile activity upon cargo binding. We discuss these aspects in context of published findings from other model organisms. AU - Niedner, A. AU - Edelmann, F* AU - Niessing, D. C1 - 32515 C2 - 35093 CY - Austin SP - 998-1009 TI - Of social molecules: The interactive assembly of ASH1 mRNA-transport complexes in yeast. JO - RNA Biol. VL - 11 IS - 8 PB - Landes Bioscience PY - 2014 SN - 1547-6286 ER - TY - JOUR AB - High concentrations (> 100 µM) of the ribonucleoside analog 4-thiouridine (4sU) is widely used in methods for RNA analysis like photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and nascent messenger (m)RNA labeling (4sU-tagging). Here, we show that 4sU-tagging at low concentrations ≤ 10 µM can be used to measure production and processing of ribosomal (r)RNA. However, elevated concentrations of 4sU (> 50 µM), which are usually used for mRNA labeling experiments, inhibit production and processing of 47S rRNA. The inhibition of rRNA synthesis is accompanied by nucleoplasmic translocation of nucleolar nucleophosmin (NPM1), induction of the tumor suppressor p53, and inhibition of proliferation. We conclude that metabolic labeling of RNA by 4sU triggers a nucleolar stress response, which might influence the interpretation of results. Therefore, functional ribosome biogenesis, nucleolar integrity, and cell cycle should be addressed in 4sU labeling experiments. AU - Burger, K. AU - Mühl, B. AU - Kellner, M. AU - Rohrmoser, M. AU - Gruber-Eber, A. AU - Windhager, L.* AU - Friedel, C.C.* AU - Dölken, L.* AU - Eick, D. C1 - 27792 C2 - 32815 SP - 1623-1630 TI - 4-thiouridine inhibits rRNA synthesis and causes a nucleolar stress response. JO - RNA Biol. VL - 10 IS - 10 PB - Landes Bioscience PY - 2013 SN - 1547-6286 ER - TY - JOUR AB - Development, growth and adult survival are coordinated with available metabolic resources, ascertaining that the organism responds appropriately to environmental conditions. MicroRNAs are short (21-23 nt) regulatory RNAs that confer specificity on the RNA-induced silencing complex (RISC) to inhibit a given set of mRNA targets. We profiled changes in miRNA expression during adult life in Drosophila melanogaster and determined that miR-277 is downregulated during adult life. Molecular analysis revealed that this miRNA controls branched-chain amino acid (BCAA) catabolism and as a result it can modulate the activity of the TOR kinase, a central growth regulator, in cultured cells. Metabolite analysis in cultured cells as well as flies suggests that the mechanistic basis may be an accumulation of branched-chain α-keto-acids (BCKA), rather than BCAAs, thus avoiding potentially detrimental consequences of increased branched chain amino acid levels on e.g., translational fidelity. Constitutive miR-277 expression shortens lifespan and is synthetically lethal with reduced insulin signaling, indicating that metabolic control underlies this phenotype. Transgenic inhibition with a miRNA sponge construct also shortens lifespan, in particular on protein-rich food. Thus, optimal metabolic adaptation appears to require tuning of cellular BCAA catabolism by miR-277. AU - Esslinger, S.M.* AU - Schwalb, B.* AU - Helfer, S.* AU - Michalik, K.M.* AU - Witte, H. AU - Maier, K.C.* AU - Martin, D.* AU - Michalke, B. AU - Tresch, A.* AU - Cramer, P.* AU - Forstemann, K.* C1 - 26138 C2 - 32088 SP - 1042-1056 TI - Drosophila miR-277 controls branched-chain amino acid catabolism and affects lifespan. JO - RNA Biol. VL - 10 IS - 6 PB - Landes Bioscience PY - 2013 SN - 1547-6286 ER - TY - JOUR AB - Heat denaturation of native phages SD suspensions, phage "shadows", and isolated phage DNA solutions were studied by scanning microcalorimetry and viscosimetry. Energetic parameters of cooperative transitions of protein fraction and DNA were measured. DNA melting was shown to be preceded by the destruction of capsid and protein denaturation. The melting curve of isolated DNA and DNA in the presence of protein component is characterized by a fine structure which is completely restored at repeated denaturation only in the presence of the protein component. "Creeping" of DNA out of the capsid in heated suspensions at 50-52 degrees C was shown to proceed with "zero" enthalpy without significant endo- and exo-thermal effects. No change of specific heat capacity of the suspension was also observed. It is emphasized that the mechanism of DNA going out of the capsid can be understood by studying DNA hydration inside the phage and its change in the course of liberation of the phage genome from the protein capsid.MiRNAs are short, non-coding RNAs that regulate gene expression post-transcriptionally through specific binding to mRNA. Deregulation of miRNAs is associated with various diseases and interference with miRNA function has proven therapeutic potential. Most mRNAs are thought to be regulated by multiple miRNAs and there is some evidence that such joint activity is enhanced if a short distance between sites allows for cooperative binding. Until now, however, the concept of cooperativity among miRNAs has not been addressed in a transcriptome-wide approach. Here, we computationally screened human mRNAs for distances between miRNA binding sites that are expected to promote cooperativity. We find that sites with a maximal spacing of 26 nucleotides are enriched for naturally occurring miRNAs compared with control sequences. Furthermore, miRNAs with similar characteristics as indicated by either co-expression within a specific tissue or co-regulation in a disease context are predicted to target a higher number of mRNAs cooperatively than unrelated miRNAs. These bioinformatic data were compared with genome-wide sets of biochemically validated miRNA targets derived by Argonaute crosslinking and immunoprecipitation (HITS-CLIP and PAR-CLIP). To ease further research into combined and cooperative miRNA function, we developed miRco, a database connecting miRNAs and respective targets involved in distance-defined cooperative regulation (mips.helmholtz-muenchen.de/mirco). In conclusion, our findings suggest that cooperativity of miRNA-target interaction is a widespread phenomenon that may play an important role in miRNA-mediated gene regulation. AU - Rinck, A. AU - Preusse, M. AU - Laggerbauer, B.* AU - Lickert, H. AU - Engelhardt, S.* AU - Theis, F.J. C1 - 26595 C2 - 32293 SP - 1125-1135 TI - The human transcriptome is enriched for miRNA-binding sites located in cooperativity-permitting distance. JO - RNA Biol. VL - 10 IS - 7 PB - Landes Bioscience PY - 2013 SN - 1547-6286 ER - TY - JOUR AB - Pur-alpha was identified as a DNA-binding protein with high affinity for the single-stranded PUR-motif (GGN)(n). Bound to DNA, Pur-alpha can both activate and repress transcription. In addition, Pur-alpha binds to RNA and may participate in nuclear RNA export as well as transport of cytoplasmic neuronal mRNP granules. The heritable trinucleotide-repeat expansion disease fragile X associated tremor and ataxia syndrome (FXTAS) leads to interaction of Pur-alpha with mutant, abnormally long r(CGG)(n) stretches, which appears to titrate the protein away from its physiologic mRNA targets into nuclear RNA-protein aggregates. We examined the function of Drosophila Pur-alpha and demonstrate that the protein accumulates in the growing oocyte early in oogenesis. Co-purifying proteins reveal that Pur-alpha is part of transported mRNP complexes, analogous to its reported role in nerve cells. We analyzed the subcellular localization of mutant GFP-Pur-alpha fusion proteins where either nucleic acid binding or dimerization, or both, were prevented. We propose that association with mRNAs occurs in the nucleus and is required for nuclear export of the complex. Furthermore, efficient translocation into the oocyte also requires RNA binding as well as dimerization. RNA binding assays demonstrate that recombinant Drosophila Pur-alpha can bind r(CGG)(4) with higher affinity than previously thought. Related sequences, such as r(CAG)(4) and the consensus sequence of the opa-repeat r(CAG)(3) CAA, can also associate with Pur-alpha in vitro and in vivo. The mRNA target spectrum of Pur-alpha may therefore be larger than previously anticipated. AU - Aumiller, V.* AU - Graebsch, A. AU - Kremmer, E. AU - Niessing, D. AU - Forstemann, K.* C1 - 8394 C2 - 30094 SP - 633-643 TI - Drosophila Pur-α binds to trinucleotide-repeat containing cellular RNAs and translocates to the early oocyte. JO - RNA Biol. VL - 9 IS - 5 PB - Landes Bioscience PY - 2012 SN - 1547-6286 ER - TY - JOUR AB - For decades, cold-adapted, temperature-sensitive (ca/ts) strains of influenza A virus have been used as live attenuated vaccines. Due to their great public health importance it is crucial to understand the molecular mechanism(s) of cold adaptation and temperature sensitivity that are currently unknown. For instance, secondary RNA structures play important roles in influenza biology. Thus, we hypothesized that a relatively minor change in temperature (32-39 degrees C) can lead to perturbations in influenza RNA structures and, that these structural perturbations may be different for mRNAs of the wild type (wt) and ca/ts strains. To test this hypothesis, we developed a novel in silico method that enables assessing whether two related RNA molecules would undergo (dis)similar structural perturbations upon temperature change. The proposed method allows identifying those areas within an RNA chain where dissimilarities of RNA secondary structures at two different temperatures are particularly pronounced, without knowing particular RNA shapes at either temperature. We identified such areas in the NS2, PA, PB2 and NP mRNAs. However, these areas are not identical for the wt and ca/ts mutants. Differences in temperature-induced structural changes of wt and ca/ts mRNA structures may constitute a yet unappreciated molecular mechanism of the cold adaptation/temperature sensitivity phenomena. AU - Chursov, A.* AU - Kopetzky, S.J.* AU - Leshchiner, I.* AU - Kondofersky, I. AU - Theis, F.J. AU - Frishman, D. AU - Shneider, A.* C1 - 11270 C2 - 30588 SP - 1266-1274 TI - Specific temperature-induced perturbations of secondary mRNA structures are associated with the cold-adapted temperature-sensitive phenotype of influenza A virus. JO - RNA Biol. VL - 9 IS - 10 PB - Landes Bioscience PY - 2012 SN - 1547-6286 ER - TY - JOUR AB - Eukaryotic RNA polymerase II (RNAP II) has evolved an array of heptad repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 at the carboxy-terminal domain (CTD) of its largest subunit (Rpb1). Dynamic phosphorylation of Ser2, Ser5 and Ser7 residues orchestrates the binding of transcription and RNA processing factors to the transcription machinery. Recent studies show that the two remaining potential phosphorylation sites, tyrosine-1 and threonine-4, are phosphorylated as well and contribute to the previously proposed "CTD code". With the impairment of binding of CTD interacting factors, these novel phosphorylation marks add an accessory layer of regulation to the RNAP II transcription cycle. AU - Heidemann, M. AU - Eick, D. C1 - 10887 C2 - 30435 SP - 1144-1146 TI - Tyrosine-1 and threonine-4 phosphorylation marks complete the RNA polymerase II CTD phospho-code. JO - RNA Biol. VL - 9 IS - 9 PB - Landes Bioscience PY - 2012 SN - 1547-6286 ER - TY - JOUR AB - MicroRNAs (miRNAs) constitute a class of small non-coding RNAs that regulate gene expression on the level of translation and/or mRNA stability. Mammalian miRNAs associate with members of the Argonaute (Ago) protein family and bind to partially complementary sequences in the 3' untranslated region (UTR) of specific target mRNAs. Computer algorithms based on factors such as free binding energy or sequence conservation have been used to predict miRNA target mRNAs. Based on such predictions, up to one third of all mammalian mRNAs seem to be under miRNA regulation. However, due to the low degree of complementarity between the miRNA and its target, such computer programs are often imprecise and therefore not very reliable. Here we report the first biochemical identification approach of miRNA targets from human cells. Using highly specific monoclonal antibodies against members of the Ago protein family, weco-immunoprecipitate Ago-bound mRNAs and identify them by cloning. Interestingly, most of the identified targets are also predicted by different computer programs. Moreover, we randomly analyzed six different target candidates and were able to experimentally validate five as miRNA targets. Our data clearly indicate that miRNA targets can be experimentally identified from Ago complexes and therefore provide a new tool to directly analyze miRNA function. AU - Beitzinger, M.* AU - Peters, L. AU - Zhu, J.Y.* AU - Kremmer, E. AU - Meister, G.* C1 - 2702 C2 - 24826 SP - 76-84 TI - Identification of human microRNA targets from isolated argonaute protein complexes. JO - RNA Biol. VL - 4 IS - 2 PB - Landes Bioscience PY - 2007 SN - 1547-6286 ER -