TY  - JOUR
AB  - Ribosomes translate mRNA into proteins and are essential for every living organism. In eukaryotes both ribosomal subunits are rapidly assembled in a strict hierarchical order, starting in the nucleolus with transcription of a common precursor ribosomal RNA (pre-rRNA). This pre-rRNA encodes three of the four mature rRNAs which are formed by several, consecutive endonucleolytic and exonucleolytic processing steps. Historically, Northern Blots are used to analyze the variety of different pre-rRNA species, only allowing rough length estimations. Although this limitation can be overcome with Primer Extension, both approaches often use radioactivity and are time consuming and costly. Here we present "Riboprobing" a reverse transcription-based workflow extended by linker ligation for easy and fast detection and characterization of various pre-rRNA species and their 5` as well as 3` ends. Using standard molecular biology lab equipment, our technique allows reliable discrimination of pre-rRNA species not resolved by Northern Blotting (e.g.: 27SA2, 27SA3 and 27SB). The method can be successfully used for analysis of total cell extracts as well as purified pre-ribosomes for a straightforward evaluation of the impact of mutant gene versions or inhibitors. In the course of method development, we identified and characterized a hitherto undescribed aberrant pre-rRNA, arising from LiCl inhibition. This pre-rRNA fragment spans from processing site A1 to E, forming a small RNP that is lacking most early joining assembly factors. This finding expands our knowledge of how the cell deals with severe pre-rRNA processing defects and demonstrates the strict requirement for the 5'ETS for the assembly process.
AU  - Gerhalter, M.*
AU  - Kofler, L.*
AU  - Zisser, G.*
AU  - Merl-Pham, J.
AU  - Hauck, S.M.
AU  - Bergler, H.*
C1  - 70427
C2  - 55617
CY  - 1 Bungtown Rd, Cold Spring Harbor, Ny 11724 Usa
SP  - 807-823
TI  - The novel pre-rRNA detection workflow "Riboprobing" allows simple identification of undescribed RNA species.
JO  - RNA
VL  - 30
IS  - 7
PB  - Cold Spring Harbor Lab Press, Publications Dept
PY  - 2024
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - Chemical modifications are found on almost all RNAs and affect their coding and noncoding functions. The identification of m(6)A on mRNA and its important role in gene regulation stimulated the field to investigate whether additional modifications are present on mRNAs. Indeed, modifications including m(1)A, m(5)C, m(7)G, 2'-OMe, and Psi were detected. However, since their abundances are low and tools used for their corroboration are often not well characterized, their physiological relevance remains largely elusive. Antibodies targeting modified nucleotides are often used but have limitations such as low affinity or specificity. Moreover, they are not always well characterized and due to the low abundance of the modification, particularly on mRNAs, generated data sets might resemble noise rather than specific modification patterns. Therefore, it is critical that the affinity and specificity is rigorously tested using complementary approaches. Here, we provide an experimental toolbox that allows for testing antibody performance prior to their use.
AU  - Weichmann, F.*
AU  - Hett, R.*
AU  - Schepers, A.
AU  - Ito-Kureha, T.*
AU  - Flatley, A.
AU  - Slama, K.*
AU  - Hastert, F.D.*
AU  - Angstman, N.B.*
AU  - Cardoso, M.C.*
AU  - König, J.*
AU  - Hüttelmaier, S.*
AU  - Dieterich, C.*
AU  - Canzar, S.*
AU  - Helm, M.*
AU  - Heissmeyer, V.
AU  - Feederle, R.
AU  - Meister, G.*
C1  - 60137
C2  - 49267
CY  - 1 Bungtown Rd, Cold Spring Harbor, Ny 11724 Usa
SP  - 1489-1506
TI  - Validation strategies for antibodies targeting modified ribonucleotides.
JO  - RNA
VL  - 26
IS  - 10
PB  - Cold Spring Harbor Lab Press, Publications Dept
PY  - 2020
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - Long noncoding RNAs (lncRNAs), which are longer than 200 nucleotides but often unstable, contribute a substantial and diverse portion to pervasive noncoding transcriptomes. Most lncRNAs are poorly annotated and understood, although several play important roles in gene regulation and diseases. Here we systematically uncover and analyze lncRNAs in Schizosaccharomyces pombe. Based on RNA-seq data from twelve RNA-processing mutants and nine physiological conditions, we identify 5775 novel lncRNAs, nearly 4× the previously annotated lncRNAs. The expression of most lncRNAs becomes strongly induced under the genetic and physiological perturbations, most notably during late meiosis. Most lncRNAs are cryptic and suppressed by three RNA-processing pathways: the nuclear exosome, cytoplasmic exonuclease, and RNAi. Double-mutant analyses reveal substantial coordination and redundancy among these pathways. We classify lncRNAs by their dominant pathway into cryptic unstable transcripts (CUTs), Xrn1-sensitive unstable transcripts (XUTs), and Dicer-sensitive unstable transcripts (DUTs). XUTs and DUTs are enriched for antisense lncRNAs, while CUTs are often bidirectional and actively translated. The cytoplasmic exonuclease, along with RNAi, dampens the expression of thousands of lncRNAs and mRNAs that become induced during meiosis. Antisense lncRNA expression mostly negatively correlates with sense mRNA expression in the physiological, but not the genetic conditions. Intergenic and bidirectional lncRNAs emerge from nucleosome-depleted regions, upstream of positioned nucleosomes. Our results highlight both similarities and differences to lncRNA regulation in budding yeast. This broad survey of the lncRNA repertoire and characteristics in S. pombe, and the interwoven regulatory pathways that target lncRNAs, provides a rich framework for their further functional analyses.
AU  - Atkinson, S.R.*
AU  - Marguerat, S.*
AU  - Bitton, D.A.*
AU  - Rodriguez-Lopez, M.*
AU  - Rallis, C.*
AU  - Lemay, J.*
AU  - Cotobal, C.*
AU  - Malecki, M.*
AU  - Smialowski, P.
AU  - Mata, J.*
AU  - Korber, P.*
AU  - Bachand, F.*
AU  - Bahler, J.*
C1  - 54202
C2  - 46013
CY  - 1 Bungtown Rd, Cold Spring Harbor, Ny 11724 Usa
SP  - 1195-1213
TI  - Long noncoding RNA repertoire and targeting by nuclear exosome, cytoplasmic exonuclease, and RNAi in fission yeast.
JO  - RNA
VL  - 24
IS  - 9
PB  - Cold Spring Harbor Lab Press, Publications Dept
PY  - 2018
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - N6-methyladenine (m6A) is found on many eukaryotic RNAs including mRNAs. m6A modification has been implicated in mRNA stability and turnover, localization, or translation efficiency. A heterodimeric enzyme complex composed of METTL3 and METTL14 generates m6A on mRNAs. METTL3/14 is found in the nucleus where it is localized to nuclear speckles and the splicing regulator WTAP is required for this distinct nuclear localization pattern. Although recent crystal structures revealed how the catalytic MT-A70 domains of METTL3 and METTL14 interact with each other, a more global architecture including WTAP and RNA interactions has not been reported so far. Here, we used recombinant proteins and mapped binding surfaces within the METTL3/14-WTAP complex. Furthermore, we identify nuclear localization signals and identify phosphorylation sites on the endogenous proteins. Using an in vitro methylation assay, we confirm that monomeric METTL3 is soluble and inactive while the catalytic center of METTL14 is degenerated and thus also inactive. In addition, we show that the C-terminal RGG repeats of METTL14 are required for METTL3/14 activity by contributing to RNA substrate binding. Our biochemical work identifies characteristic features of METTL3/14-WTAP and reveals novel insight into the overall architecture of this important enzyme complex.
AU  - Schöller, E.*
AU  - Weichmann, F.*
AU  - Treiber, T.*
AU  - Ringle, S.*
AU  - Treiber, N.*
AU  - Flatley, A.
AU  - Feederle, R.
AU  - Bruckmann, A.*
AU  - Meister, G.*
C1  - 52758
C2  - 44477
CY  - Cold Spring Harbor
SP  - 499-512
TI  - Interactions, localization, and phosphorylation of the m6A generating METTL3–METTL14–WTAP complex.
JO  - RNA
VL  - 24
IS  - 4
PB  - Cold Spring Harbor Lab Press, Publications Dept
PY  - 2018
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - The nuclear exosome and the associated RNA helicase Mtr4 participate in the processing of several ribonucleoprotein particles (RNP), including the maturation of the large ribosomal subunit (60S). S. cerevisiae Mtr4 interacts directly with Nop53, a ribosomal biogenesis factor present in late pre-60S particles containing precursors of the 5.8S rRNA. The Mtr4-Nop53 interaction plays a pivotal role in the maturation of the 5.8S rRNA, providing a physical link between the nuclear exosome and the pre-60S RNP. An analogous interaction between Mtr4 and another ribosome biogenesis factor, Utp18, directs the exosome to an earlier pre-ribosomal particle. Nop53 and Utp18 contain a similar Mtr4-binding motif known as the arch-interacting motif (AIM). Here, we report the 3.2 Å resolution crystal structure of S.cerevisiae Mtr4 bound to the interacting region of Nop53, revealing how the KOW domain of the helicase recognizes the AIM sequence of Nop53 with a network of hydrophobic and electrostatic interactions. The AIM-interacting residues are conserved in Mtr4 and are not present in the related cytoplasmic helicase Ski2, rationalizing the specificity and versatility of Mtr4 in the recognition of different AIM-containing proteins. Using nuclear magnetic resonance (NMR), we show that the KOW domain of Mtr4 can simultaneously bind an AIM-containing protein and a structured RNA at adjacent surfaces, suggesting how it can dock onto RNPs. The KOW domains of exosome-associated helicases thus appear to have evolved from the KOW domains of ribosomal proteins and to function as RNP-binding modules in the context of the nuclear exosome.
AU  - Falk, S.*
AU  - Tants, J.-N.
AU  - Basquin, J.*
AU  - Thoms, M.*
AU  - Hurt, E.*
AU  - Sattler, M.
AU  - Conti, E.*
C1  - 51839
C2  - 43498
CY  - Cold Spring Harbor
SP  - 1780-1787
TI  - Structural insights into the interaction of the nuclear exosome helicase Mtr4 with the pre-ribosomal protein Nop53.
JO  - RNA
VL  - 23
IS  - 12
PB  - Cold Spring Harbor Lab Press, Publications Dept
PY  - 2017
SN  - 1355-8382
ER  - 

TY  - JOUR
AU  - Sattler, M.
C1  - 43857
C2  - 36619
CY  - Cold Spring Harbor
SP  - 727-728
TI  - Dynamic RNA world.
JO  - RNA
VL  - 21
IS  - 4
PB  - Cold Spring Harbor Lab Press, Publications Dept
PY  - 2015
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - Piwi-interacting RNAs (piRNAs) are small noncoding RNAs expressed in the germline of animals. They associate with Argonaute proteins of the Piwi subfamily, forming ribonucleoprotein complexes that are involved in maintaining genome integrity. The N-terminal region of some Piwi proteins contains symmetrically dimethylated arginines. This modification is thought to enable recruitment of Tudor domain-containing proteins (TDRDs), which might serve as platforms mediating interactions between various proteins in the piRNA pathway. We measured the binding affinity of the four individual extended Tudor domains (TDs) of murine TDRD1 protein for three different methylarginine-containing peptides from murine Piwi protein MILI. The results show a preference of TD2 and TD3 for consecutive MILI peptides, whereas TD4 and TD1 have, respectively, lower and very weak affinity for any peptide. The affinity of TD1 for methylarginine peptides can be restored by a single-point mutation back to the consensus aromatic cage sequence. These observations were confirmed by pull-down experiments with endogenous Piwi and Piwi-associated proteins. The crystal structure of TD3 bound to a methylated MILI peptide shows an unexpected orientation of the bound peptide, with additional contacts of nonmethylated residues being made outside of the aromatic cage, consistent with solution NMR titration experiments. Finally, the molecular envelope of the four tandem Tudor domains of TDRD1, derived from small angle scattering data, reveals a flexible, elongated shape for the protein. Overall, the results show that TDRD1 can accommodate different peptides from different proteins, and can therefore act as a scaffold protein for complex assembly in the piRNA pathway.
AU  - Mathioudakis, N.*
AU  - Palencia, A.*
AU  - Kadlec, J.*
AU  - Round, A.*
AU  - Tripsianes, K.
AU  - Sattler, M.
AU  - Pillai, R.S.*
AU  - Cusack, S.*
C1  - 11340
C2  - 30622
SP  - 2056-2072
TI  - The multiple Tudor domain-containing protein TDRD1 is a molecular scaffold for mouse piwi proteins and piRNA biogenesis factors.
JO  - RNA
VL  - 18
IS  - 11
PB  - Cold Spring Harbor Lab Press
PY  - 2012
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - X-chromosome inactivation (XCI) in female mammals depends on the noncoding RNA X inactivation specific transcript (Xist). The mechanism of chromosome-wide silencing by Xist is poorly understood. While it is established that the 5' region of Xist RNA, comprising the A-repeats and holding 7.5-8.5 copies of a conserved 26-mer sequence, is essential for Xist-mediated silencing, high-resolution structural information for the A-repeats is not available. Here, we report the three-dimensional solution structure of a 14-mer hairpin in the 5' region of a human A-repeat. This hairpin is remarkably stable and adopts a novel AUCG tetraloop fold, the integrity of which is required for silencing. We show that, contrary to previous predictions, the 3' region of single or tandem A-repeats mediates duplex formation in vitro. Significantly, mutations in this region disrupt the inter-repeat duplex formation in vitro and abrogate the silencing function of Xist A-repeats in vivo. Our data suggest that the complete A-repeat region may be stabilized by inter-repeat duplex formation and, as such, may provide a platform for multimerization and specific recognition of the AUCG tetraloops by trans-acting factors.
AU  - Duszczyk, M.M.
AU  - Wutz, A.*
AU  - Rybin, V.*
AU  - Sattler, M.
C1  - 6642
C2  - 29022
CY  - New York, NY
SP  - 1973-1982
TI  - The Xist RNA A-repeat comprises a novel AUCG tetraloop fold and a platform for multimerization.
JO  - RNA
VL  - 17
IS  - 11
PB  - Cambridge Univ. Press
PY  - 2011
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - MicroRNAs (miRNAs) are an important class of post-transcriptional regulators of gene expression that are involved in various cellular and phenotypic processes. A number of studies have shown that miRNA expression is induced by signaling pathways. Moreover, miRNAs emerge as regulators of signaling pathways. Here, we present the miTALOS web resource, which provides insight into miRNA-mediated regulation of signaling pathways. As a novel feature, miTALOS considers the tissue-specific expression signatures of miRNAs and target transcripts to improve the analysis of miRNA regulation in biological pathways. MiTALOS identifies potential pathway regulation by (i) an enrichment analysis of miRNA targets genes and (ii) by using a proximity score to evaluate the functional role of miRNAs in biological pathways by their network proximity. Moreover, miTALOS integrates five different miRNA target prediction tools and two different signaling pathway resources (KEGG and NCI). A graphical visualization of miRNA targets in both KEGG and NCI PID signaling pathways is provided to illustrate their respective pathway context. We perform a functional analysis on prostate cancer-related miRNAs and are able to infer a model of miRNA-mediated regulation on tumor proliferation, mobility and anti-apoptotic behavior. miTALOS provides novel features that accomplish a substantial support to systematically infer regulation of signaling pathways mediated by miRNAs.
AU  - Kowarsch, A.
AU  - Preusse, M.
AU  - Marr, C.
AU  - Theis, F.J.
C1  - 6257
C2  - 29080
CY  - Cold Spring Harbor
SP  - 809-819
TI  - miTALOS: Analyzing the tissue-specific regulation of signaling pathways by human and mouse microRNAs.
JO  - RNA
VL  - 17
IS  - 5
PB  - Cold Spring Harbor Lab Press
PY  - 2011
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - Argonaute (Ago) proteins form the core of RNA-induced silencing complexes (RISCs) and mediate small RNA-guided gene silencing. In RNAi, short interfering RNAs (siRNAs) guide RISCs to complementary target RNAs, leading to cleavage by the endonuclease Ago2. Noncatalytic Ago proteins, however, contribute to RNAi as well but cannot cleave target RNA and often generate off-target effects. Here we show that synthetic siRNA duplexes interact with all Ago proteins, but a functional RISC rapidly assembles only around Ago2. By stabilizing the siRNA duplex, we show that the noncatalytic Ago proteins Ago1, -3, and -4 can be selectively blocked and do not form functional RISCs. In addition, stabilized siRNAs form an Ago2-RISC more efficiently, leading to increased silencing activity. Our data suggest novel parameters for the design of siRNAs with selective activation of the endonuclease Ago2.
AU  - Petri, S.*
AU  - Dueck, A.*
AU  - Lehmann, G.*
AU  - Putz, N.*
AU  - Rüdel, S.*
AU  - Kremmer, E.
AU  - Meister, G.*
C1  - 6239
C2  - 28654
SP  - 737-749
TI  - Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects.
JO  - RNA
VL  - 17
IS  - 4
PB  - Cold Spring Harbor
PY  - 2011
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - Small nuclear and small nucleolar RNAs (snRNAs and snoRNAs) are critical components of snRNPs and snoRNPs and play an essential role in the maturation of, respectively, mRNAs and rRNAs within the nucleus of eukaryotic cells. Complex and specific pathways exist for the assembly of snRNPs and snoRNPs, involving, for instance, nucleocytoplasmic transport of snRNAs and intranuclear transport between compartments of snoRNAs. The phosphorylated adaptor for nuclear export (PHAX) is required for nuclear export of snRNAs in metazoans and also involved in the intranuclear transport of snoRNAs to Cajal bodies. PHAX contains a conserved single-stranded nucleic acid binding domain (RNA_GG_bind domain) with no sequence homology with any other known RNA-binding module. Here, we report NMR and X-ray crystallography studies that elucidate the structural basis for RNA recognition by the PHAX RNA-binding domain (PHAX-RBD). The crystal structure of the RNA_GG_bind domain from the parasite Cryptosporidium parvum (Cp RBD) forms well-folded dimers in solution in the absence of any ligand. The human PHAX-RBD is monomeric and only adopts a tertiary fold upon RNA binding. The PHAX-RBD represents a novel helical fold and binds single-stranded RNA with micromolar affinity without sequence specificity. RNA recognition by human PHAX-RBD is consistent with mutational analysis that affects RNA binding and PHAX-mediated nuclear export. Our data suggest that the PHAX-RBD mediates auxiliary RNA contacts with the snRNA and snoRNA substrates that are required for transport and/or substrate release.
AU  - Mourao, A.
AU  - Varrot, A.*
AU  - Mackereth, C.D.*
AU  - Cusack, S.*
AU  - Sattler, M.
C1  - 5661
C2  - 27930
SP  - 1205-1216
TI  - Structure and RNA recognition by the snRNA and snoRNA transport factor PHAX.
JO  - RNA
VL  - 16
IS  - 6
PB  - Cold Spring Harbor Lab Press
PY  - 2010
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - The CCR4-NOT complex is the main enzyme catalyzing the deadenylation of mRNA. We have investigated the composition of this complex in Drosophila melanogaster by immunoprecipitation with a monoclonal antibody directed against NOT1. The CCR4, CAF1 (=POP2), NOT1, NOT2, NOT3, and CAF40 subunits were associated in a stable complex, but NOT4 was not. Factors known to be involved in mRNA regulation were prominent among the other proteins coprecipitated with the CCR4-NOT complex, as analyzed by mass spectrometry. The complex was localized mostly in the cytoplasm but did not appear to be a major component of P bodies. Of the known CCR4 paralogs, Nocturnin was found associated with the subunits of the CCR4-NOT complex, whereas Angel and 3635 were not. RNAi experiments in Schneider cells showed that CAF1, NOT1, NOT2, and NOT3 are required for bulk poly(A) shortening and hsp70 mRNA deadenylation, but knock-down of CCR4, CAF40, and NOT4 did not affect these processes. Overexpression of catalytically dead CAF1 had a dominant-negative effect on mRNA decay. In contrast, overexpression of inactive CCR4 had no effect. We conclude that CAF1 is the major catalytically important subunit of the CCR4-NOT complex in Drosophila Schneider cells. Nocturnin may also be involved in mRNA deadenylation, whereas there is no evidence for a similar role of Angel and 3635.
AU  - Temme, C.*
AU  - Zhang, L.B.*
AU  - Kremmer, E.
AU  - Ihling, C.*
AU  - Chartier, A.*
AU  - Sinz, A.*
AU  - Simonelig, M.*
AU  - Wahle, E.*
C1  - 5417
C2  - 27486
SP  - 1356-1370
TI  - Subunits of the Drosophila CCR4-NOT complex and their roles in mRNA deadenylation.
JO  - RNA
VL  - 16
IS  - 7
PB  - Cold Spring Harbor Lab Press
PY  - 2010
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - In eukaryotic cells, dozens to hundreds of different mRNAs are localized by specialized motor-dependent transport complexes. One of the best-studied examples for directional mRNA transport is the localization of ASH1 mRNA in Saccharomyces cerevisiae. For transport, ASH1 mRNA is bound by the unusual RNA-binding protein She2p. Although previous results indicated that She2p forms dimers required for RNA binding and transcript localization, it remained unclear if the dimer constitutes the minimal RNA-binding unit assembling in vivo. By using analytical ultracentrifugation we found that She2p forms larger oligomeric complexes in solution. We also identified a point mutant that shows impaired oligomer formation. Size-exclusion chromatography suggests that She2p forms defined tetramers at physiological concentrations. Subsequent structural studies by small-angle X-ray scattering confirmed this finding and demonstrated that the previously observed She2p dimers interact in a head-to-head conformation to form an elongated tetrameric complex. This She2p tetramer suggests the generation of large continuous RNA-binding surfaces at both sides of the complex. Biochemical studies and immunostaining of cells confirmed that She2p tetramer formation is required for RNA binding, efficient mRNP assembly, and mRNA localization in vivo. Our finding on She2p tetramerization resolves previously raised questions on complex formation and mRNP function.
AU  - Müller, M.
AU  - Richter, K.*
AU  - Heuck, A.*
AU  - Kremmer, E.
AU  - Buchner, J.*
AU  - Jansen, R.P.*
AU  - Niessing, D.
C1  - 1351
C2  - 26705
CY  - U.S.
SP  - 2002-2012
TI  - Formation of She2p tetramers is required for mRNA binding, mRNP assembly, and localization.
JO  - RNA
VL  - 15
IS  - 11
PB  - Cold Spring Harbor Laboratory Press.
PY  - 2009
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - RNA-binding proteins constitute key factors of the post-transcriptional machinery. These regulatory proteins recognize specific elements within target transcripts to promote, for example, maturation, translation, or stability of mRNAs. In Ustilago maydis, evidence is accumulating that post-transcriptional processes are important to determine pathogenicity. Deletion of khd4, encoding a predicted RNA-binding protein with five K homology (KH) domains, causes aberrant cell morphology and reduced virulence. Here, we demonstrate that Khd4 recognizes the sequence AUACCC in vivo via its tandem KH domains 3 and 4. This sequence most likely functions as a regulatory RNA element in U. maydis, since it accumulates in 3' untranslated regions. Consistently, an independent mRNA expression profiling approach revealed that the binding motif is significantly enriched in transcripts showing altered expression levels in khd4 Delta strains. Since the vast majority of potential Khd4 target mRNAs exhibit increased amounts in deletion mutants, Khd4 might promote mRNA instability. Mutants that fail to bind AUACCC resemble deletion mutants, which exhibit altered cell morphology, disturbed filamentous growth, and severely reduced virulence. Hence, RNA binding is essential for function of Khd4, stressing the importance of post-transcriptional control in regulating morphology and pathogenicity.
AU  - Vollmeister, E.*
AU  - Haag, C.*
AU  - Zarnack, K.*
AU  - Baumann, S.*
AU  - König, J.*
AU  - Mannhaupt, G.
AU  - Feldbrügge, M.*
C1  - 1049
C2  - 26525
CY  - WOODBURY
SP  - 2206-2218
TI  - Tandem KH domains of Khd4 recognize AUACCC and are essential for regulation of morphology as well as pathogenicity in Ustilago maydis.
JO  - RNA
VL  - 15
IS  - 12
PB  - COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
PY  - 2009
SN  - 1355-8382
ER  - 

TY  - JOUR
AB  - Small regulatory RNAs including small interfering RNAs (siRNAs), microRNAs (miRNAs), or Piwi interacting RNAs (piRNAs) guide regulation of gene expression in many different organisms. The Argonaute (Ago) protein family constitutes the cellular binding partners of such small RNAs and regulates gene expression on the levels of transcription, mRNA stability, or translation. Due to the lack of highly specific and potent monoclonal antibodies directed against the different Ago proteins, biochemical analyses such as Ago complex purification and characterization rely on overexpression of tagged Ago proteins. Here, we report the generation and functional characterization of a highly specific monoclonal anti-Ago2 antibody termed anti-Ago2(11A9). We show that anti-Ago2(11A9) is specific for human Ago2 and detects Ago2 in Western blots as well as in immunoprecipitation experiments. We further demonstrate that Ago2 can be efficiently eluted from our antibody by a competing peptide. Finally, we show that anti-Ago2(11A9) recognizes Ago2 in immunofluorescence experiments, and we find that Ago2 not only localizes to cytoplasmic processing bodies (P-bodies) and the diffuse cytoplasm but also to the nucleus. With the anti-Ago2(11A9) antibody we have generated a potent tool that is useful for many biochemical or cell biological applications.
AU  - Rüdel, S.*
AU  - Flatley, A.
AU  - Weinmann, L.*
AU  - Kremmer, E.
AU  - Meister, G.*
C1  - 3149
C2  - 25779
SP  - 1244-1253
TI  - A multifunctional human Argonaute2-specific monoclonal antibody.
JO  - RNA
VL  - 14
IS  - 6
PB  - Cold Spring Harbor Laboratory Press
PY  - 2008
SN  - 1355-8382
ER  -