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681 records returned

Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character-as is found in small interfering RNAs (siRNAs)-directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumula...
Source: RNA - November 16, 2009 Category: Genetics & Stem Cells Authors: Ghildiyal M, Xu J, Seitz H, Weng Z, Zamore PD Tags: RNA Source Type: journals

Prediction of geometrically feasible three-dimensional structures of pseudoknotted RNA through free energy estimation.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Accurate free energy estimation is essential for RNA structure prediction. The widely used Turner's energy model works well for nested structures. For pseudoknotted RNAs, however, there is no effective rule for estimation of loop entropy and free energy. In this work we present a new free energy estimation method, termed the pseudoknot predictor in three-dimensional space (pk3D), which goes beyond Turner's model. Our approach treats nested and pseudoknotted structures alike in one unifying physical framework, regardless of how complex the RNA structures are. We first test the ability of pk3D in selecting native structu...
Source: RNA - October 28, 2009 Category: Genetics & Stem Cells Authors: Zhang J, Dundas J, Lin M, Chen R, Wang W, Liang J Tags: RNA Source Type: journals

Structural features of the tmRNA-ribosome interaction.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA-ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using ch...
Source: RNA - October 27, 2009 Category: Genetics & Stem Cells Authors: Bugaeva EY, Surkov S, Golovin AV, Ofverstedt LG, Skoglund U, Isaksson LA, Bogdanov AA, Shpanchenko OV, Dontsova OA Tags: RNA Source Type: journals

The Arabidopsis thaliana double-stranded RNA binding protein DRB1 directs guide strand selection from microRNA duplexes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In Arabidopsis thaliana(Arabidopsis), DICER-LIKE1 (DCL1) functions together with the double-stranded RNA binding protein (dsRBP), DRB1, to process microRNAs (miRNAs) from their precursor transcripts prior to their transfer to the RNA-induced silencing complex (RISC). miRNA-loaded RISC directs RNA silencing of cognate mRNAs via ARGONAUTE1 (AGO1)-catalyzed cleavage. Short interefering RNAs (siRNAs) are processed from viral-derived or transgene-encoded molecules of double-stranded RNA (dsRNA) by the DCL/dsRBP partnership, DCL4/DRB4, and are also loaded to AGO1-catalyzed RISC for cleavage of complementary mRNAs. Here, we u...
Source: RNA - October 27, 2009 Category: Genetics & Stem Cells Authors: Eamens AL, Smith NA, Curtin SJ, Wang MB, Waterhouse PM Tags: RNA Source Type: journals

The tedious task of finding homologous noncoding RNA genes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
User-driven in silico RNA homology search is still a nontrivial task. In part, this is the consequence of a limited precision of the computational tools in spite of recent exciting progress in this area, and to a certain extent, computational costs are still problematic in practice. An important, and as we argue here, dominating issue is the dependence on good curated (secondary) structural alignments of the RNAs. These are often hard to obtain, not so much because of an inherent limitation in the available data, but because they require substantial manual curation, an effort that is rarely acknowledged. Here, we quali...
Source: RNA - October 27, 2009 Category: Genetics & Stem Cells Authors: Menzel P, Gorodkin J, Stadler PF Tags: RNA Source Type: journals

Free energy calculation of modified base-pair formation in explicit solvent: A predictive model.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The maturation of RNAs includes site-specific post-transcriptional modifications that contribute significantly to hydrogen bond formation within RNA and between different RNAs, especially in formation of mismatch base pairs. Thus, an understanding of the geometry and strength of the base-pairing of modified ribonucleoside 5'-monophosphates, previously not defined, is applicable to investigations of RNA structure and function and of the design of novel RNAs. The geometry and free energies of base-pairings were calculated in aqueous solution under neutral conditions with AMBER force fields and molecular dynamics simulati...
Source: RNA - October 27, 2009 Category: Genetics & Stem Cells Authors: Vendeix FA, Munoz AM, Agris PF Tags: RNA Source Type: journals

Transcription of the Dictyostelium discoideum mitochondrial genome occurs from a single initiation site.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Transcription of the mitochondrial genome in Dictyostelium discoideum gives rise to eight major polycistronic RNA species that can be detected by Northern hybridization. In order to determine whether these transcripts could possibly derive from processing of even larger transcripts, reverse transcriptase polymerase chain reactions (RT-PCRs) were performed in an attempt to amplify the intervening regions between the eight major transcripts. All but one intervening region were successfully reverse transcribed and amplified, indicating that even larger transcripts existed and that the eight major transcripts detected prev...
Source: RNA - October 27, 2009 Category: Genetics & Stem Cells Authors: Le P, Fisher PR, Barth C Tags: RNA Source Type: journals

Translation initiation complex formation in the crenarchaeon Sulfolobus solfataricus.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The function of initiation factors in and the sequence of events during translation initiation have been intensively studied in Bacteria and Eukaryotes, whereas in Archaea knowledge on these functions/processes is limited. By employing chemical probing, we show that translation initiation factor aIF1 of the model crenarchaeon Sulfolobus solfataricus binds to the same area on the ribosome as the bacterial and eukaryal orthologs. Fluorescence energy transfer assays (FRET) showed that aIF1, like its eukaryotic and bacterial orthologs, has a fidelity function in translation initiation complex formation, and that both aIF1 ...
Source: RNA - October 27, 2009 Category: Genetics & Stem Cells Authors: Hasenöhrl D, Fabbretti A, Londei P, Gualerzi CO, Bläsi U Tags: RNA Source Type: journals

Next-generation SELEX identifies sequence and structural determinants of splicing factor binding in human pre-mRNA sequence.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Many splicing factors interact with both mRNA and pre-mRNA. The identification of these interactions has been greatly improved by the development of in vivo cross-linking immunoprecipitation. However, the output carries a strong sampling bias in favor of RNPs that form on more abundant RNA species like mRNA. We have developed a novel in vitro approach for surveying binding on pre-mRNA, without cross-linking or sampling bias. Briefly, this approach entails specifically designed oligonucleotide pools that tile through a pre-mRNA sequence. The pool is then partitioned into bound and unbound fractions, which are quantified...
Source: RNA - October 27, 2009 Category: Genetics & Stem Cells Authors: Reid DC, Chang BL, Gunderson SI, Alpert L, Thompson WA, Fairbrother WG Tags: RNA Source Type: journals

Characterization of the functional role of nucleotides within the URE2 IRES element and the requirements for eIF2A-mediated repression.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we sought to investigate the mechanism of internal initiation in yeast by assessing the functional role of nucleotides within the minimal URE2 IRES element, and delineating the cis-sequences that modulate levels of internal initiation using a monocistronic reporter vector. Furthermore, we compared the eIF2A sensitivity of the URE2 IRES element with some of the invasive growth IRES elements using DeltaeIF2A yeast. We found that the stability of the stem-loop structure within the minimal URE2 IRES element is not a critical determinant of optimal IRES activity, and the downstream sequences that modulate URE2 IR...
Source: RNA - October 27, 2009 Category: Genetics & Stem Cells Authors: Reineke LC, Merrick WC Tags: RNA Source Type: journals

Absolute quantification of microRNAs by using a universal reference.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
MicroRNAs (miRNAs) are a species of small RNAs approximately 21-23-nucleotides long that have been shown to play an important role in many different cellular, developmental, and physiological processes. Accordingly, numerous PCR-, sequencing-, or hybridization-based methods have been established to identify and quantify miRNAs. Their short length results in a high dynamic range of melting temperatures and therefore impedes a proper selection of detection probes or optimized PCR primers. While miRNA microarrays allow for massive parallel and accurate relative measurement of all known miRNAs, they have so far been less u...
Source: RNA - October 27, 2009 Category: Genetics & Stem Cells Authors: Bissels U, Wild S, Tomiuk S, Holste A, Hafner M, Tuschl T, Bosio A Tags: RNA Source Type: journals

Tandem KH domains of Khd4 recognize AUACCC and are essential for regulation of morphology as well as pathogenicity in Ustilago maydis.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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...
Source: RNA - October 23, 2009 Category: Genetics & Stem Cells Authors: Vollmeister E, Haag C, Zarnack K, Baumann S, König J, Mannhaupt G, Feldbrügge M Tags: RNA Source Type: journals

Physical and genetic interactions of yeast Cwc21p, an ortholog of human SRm300/SRRM2, suggest a role at the catalytic center of the spliceosome.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In Saccharomyces cerevisiae, Cwc21p is a protein of unknown function that is associated with the NineTeen Complex (NTC), a group of proteins involved in activating the spliceosome to promote the pre-mRNA splicing reaction. Here, we show that Cwc21p binds directly to two key splicing factors-namely, Prp8p and Snu114p-and becomes the first NTC-related protein known to dock directly to U5 snRNP proteins. Using a combination of proteomic techniques we show that the N-terminus of Prp8p contains an intramolecular fold that is a Snu114p and Cwc21p interacting domain (SCwid). Cwc21p also binds directly to the C-terminus of Snu...
Source: RNA - October 23, 2009 Category: Genetics & Stem Cells Authors: Grainger RJ, Barrass JD, Jacquier A, Rain JC, Beggs JD Tags: RNA Source Type: journals

Deadenylation of maternal mRNAs mediated by miR-427 in Xenopus laevis embryos.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We show that microRNA-427 (miR-427) mediates the rapid deadenylation of maternal mRNAs after the midblastula transition (MBT) of Xenopus laevis embryogenesis. By MBT, the stage when the embryonic cell cycle is remodeled and zygotic transcription of mRNAs is initiated, each embryo has accumulated approximately 10(9) molecules of miR-427 processed from multimeric pri-miR-427 transcripts synthesized after fertilization. We demonstrate that the maternal mRNAs for cyclins A1 and B2 each contain a single miR-427 target sequence, spanning less than 30 nucleotides, that is both necessary and sufficient for deadenylation, and t...
Source: RNA - October 23, 2009 Category: Genetics & Stem Cells Authors: Lund E, Liu M, Hartley RS, Sheets MD, Dahlberg JE Tags: RNA Source Type: journals

Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Deep sequencing technologies such as Illumina, SOLiD, and 454 platforms have become very powerful tools in discovering and quantifying small RNAs in diverse organisms. Sequencing small RNA fractions always identifies RNAs derived from abundant RNA species such as rRNAs, tRNAs, snRNA, and snoRNA, and they are widely considered to be random degradation products. We carried out bioinformatic analysis of deep sequenced HeLa RNA and after quality filtering, identified highly abundant small RNA fragments, derived from mature tRNAs that are likely produced by specific processing rather than from random degradation. Moreover, ...
Source: RNA - October 21, 2009 Category: Genetics & Stem Cells Authors: Cole C, Sobala A, Lu C, Thatcher SR, Bowman A, Brown JW, Green PJ, Barton GJ, Hutvagner G Tags: RNA Source Type: journals

Improved RNA preservation for immunolabeling and laser microdissection.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Microdissection techniques have the potential to allow for transcriptome analyses in specific populations of cells that are isolated from heterogeneous tissues such as the nervous system and certain cancers. Problematically, RNA is not stable under the labeling conditions usually needed to identify the cells of interest for microdissection. We have developed an immunolabeling method that utilizes a high salt buffer to stabilize RNA during prolonged antibody incubations. We first assessed RNA integrity by three methods and found that tissue incubated in high salt buffer for at least 20 h yielded RNA of similar quality t...
Source: RNA - October 21, 2009 Category: Genetics & Stem Cells Authors: Brown AL, Smith DW Tags: RNA Source Type: journals

CpG-island promoters drive transcription of human telomeres.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The longstanding dogma that telomeres, the heterochromatic extremities of linear eukaryotic chromosomes, are transcriptionally silent was overturned by the discovery that DNA-dependent RNA polymerase II (RNAPII) transcribes telomeric DNA into telomeric repeat-containing RNA (TERRA). Here, we show that CpG dinucleotide-rich DNA islands, shared among multiple human chromosome ends, promote transcription of TERRA molecules. TERRA promoters sustain cellular expression of reporter genes, are located immediately upstream of TERRA transcription start sites, and are bound by active RNAPII in vivo. Finally, the identified promo...
Source: RNA - October 21, 2009 Category: Genetics & Stem Cells Authors: Nergadze SG, Farnung BO, Wischnewski H, Khoriauli L, Vitelli V, Chawla R, Giulotto E, Azzalin CM Tags: RNA Source Type: journals

Splicing of mRNA mediated by tRNA sequences in mouse cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
tRNA splicing is essential for the formation of tRNAs and therefore for gene expression. A circularly permuted sequence of an amber-suppressor pre-tRNA gene was inserted into the sequence encoding the mouse NEMO protein. We demonstrated that, in mouse cells, the hybrid pre-tRNA/pre-mRNAs can be spliced precisely at the sites of the pre-tRNA intron. This splicing reaction produces functional tRNAs that suppress amber codons as well as translatable mRNAs that sustain the NF-kappaB activation pathway. The RNA molecules extracted from mouse cells were amplified by RT-PCR, and their sequences were determined, confirming the...
Source: RNA - October 21, 2009 Category: Genetics & Stem Cells Authors: Zamboni M, Scarabino D, Tocchini-Valentini GP Tags: RNA Source Type: journals

Sliced microRNA targets and precise loop-first processing of MIR319 hairpins revealed by analysis of the Physcomitrella patens degradome.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Expression profiling of the 5' ends of uncapped mRNAs ("degradome" sequencing) can be used to empirically catalog microRNA (miRNA) targets, to probe patterns of miRNA hairpin processing, to examine mRNA decay, and to analyze accumulation of endogenous short interfering RNA (siRNA) precursors. We sequenced and analyzed the degradome of the moss Physcomitrella patens, an important model system for functional genomic analyses in plant evolution. A total of 52 target mRNAs of 27 different Physcomitrella miRNA families were identified. Many targets of both more conserved and less conserved miRNA families encoded putative re...
Source: RNA - October 21, 2009 Category: Genetics & Stem Cells Authors: Addo-Quaye C, Snyder JA, Park YB, Li YF, Sunkar R, Axtell MJ Tags: RNA Source Type: journals

The flavivirus NS5 protein is a true RNA guanylyltransferase that catalyzes a two-step reaction to form the RNA cap structure.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The 5'-end of the flavivirus genome harbors a methylated (m7)GpppA(2'OMe) cap structure, which is generated by the virus-encoded RNA triphosphatase, RNA (guanine-N7) methyltransferase, nucleoside 2'-O-methyltransferase, and RNA guanylyltransferase. The presence of the flavivirus guanylyltransferase activity in NS5 has been suggested by several groups but has not been empirically proven. Here we provide evidence that the N-terminus of the flavivirus NS5 protein is a true RNA guanylyltransferase. We demonstrate that GTP can be used as a substrate by the enzyme to form a covalent GMP-enzyme intermediate via a phosphoamide...
Source: RNA - October 21, 2009 Category: Genetics & Stem Cells Authors: Issur M, Geiss BJ, Bougie I, Picard-Jean F, Despins S, Mayette J, Hobdey SE, Bisaillon M Tags: RNA Source Type: journals

Nonsense-mediated mRNA decay mutes the splicing defects of spliceosome component mutations.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The role of many splicing factors in pre-mRNA splicing and the involvement of these factors in the processing of specific transcripts have often been defined through the analysis of loss-of-function mutants in vivo. Here we show that inactivating the nonsense-mediated mRNA decay (NMD) results in an enhancement of splicing phenotypes associated with several S. cerevisiae splicing factor mutations. Tiling microarrays showed that inactivation of the NMD factor Upf1p in the prp17Delta and prp18Delta mutant strains results in a larger spectrum of splicing defects than what is observed in the single mutants, including new tr...
Source: RNA - October 21, 2009 Category: Genetics & Stem Cells Authors: Kawashima T, Pellegrini M, Chanfreau GF Tags: RNA Source Type: journals

Powering through ribosome assembly.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Ribosome assembly is required for cell growth in all organisms. Classic in vitro work in bacteria has led to a detailed understanding of the biophysical, thermodynamic, and structural basis for the ordered and correct assembly of ribosomal proteins on ribosomal RNA. Furthermore, it has enabled reconstitution of active subunits from ribosomal RNA and proteins in vitro. Nevertheless, recent work has shown that eukaryotic ribosome assembly requires a large macromolecular machinery in vivo. Many of these assembly factors such as ATPases, GTPases, and kinases hydrolyze nucleotide triphosphates. Because these enzymes are lik...
Source: RNA - October 21, 2009 Category: Genetics & Stem Cells Authors: Strunk BS, Karbstein K Tags: RNA Source Type: journals

Do conformational biases of simple helical junctions influence RNA folding stability and specificity?Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Structured RNAs must fold into their native structures and discriminate against a large number of alternative ones, an especially difficult task given the limited information content of RNA's nucleotide alphabet. The simplest motifs within structured RNAs are two helices joined by nonhelical junctions. To uncover the fundamental behavior of these motifs and to elucidate the underlying physical forces and challenges faced by structured RNAs, we computationally and experimentally studied a tethered duplex model system composed of two helices joined by flexible single- or double-stranded polyethylene glycol tethers, whose...
Source: RNA - October 21, 2009 Category: Genetics & Stem Cells Authors: Chu VB, Lipfert J, Bai Y, Pande VS, Doniach S, Herschlag D Tags: RNA Source Type: journals

Bacillus subtilis RNase J1 endonuclease and 5' exonuclease activities in the turnover of {Delta}ermC mRNA.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
RNase J1, a ribonuclease with 5' exonuclease and endonuclease activities, is an important factor in Bacillus subtilis mRNA decay. A model for RNase J1 endonuclease activity in mRNA turnover has RNase J1 binding to the 5' end and tracking to a target site downstream, where it makes a decay-initiating cleavage. The upstream fragment from this cleavage is degraded by 3' exonucleases; the downstream fragment is degraded by RNase J1 5' exonuclease activity. Previously, DeltaermC mRNA was used to show 5'-end dependence of mRNA turnover. Here we used DeltaermC mRNA to probe RNase J1-dependent degradation, and the results were...
Source: RNA - October 21, 2009 Category: Genetics & Stem Cells Authors: Yao S, Sharp JS, Bechhofer DH Tags: RNA Source Type: journals

Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin's active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V...
Source: RNA - October 20, 2009 Category: Genetics & Stem Cells Authors: Nimjee SM, Oney S, Volovyk Z, Bompiani KM, Long SB, Hoffman M, Sullenger BA Tags: RNA Source Type: journals

A systematic characterization of Cwc21, the yeast ortholog of the human spliceosomal protein SRm300.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this report, we employ quantitative genetic interaction mapping, mass spectrometry of tandem affinity-purified complexes, and microarray profiling to investigate genetic, physical, and functional interactions involving Cwc21. Combined data from these assays support multiple roles for Cwc21 in the formation and function of splicing complexes. Consistent with a role for Cwc21 at the core of the spliceosome, we observe strong genetic, physical, and functional interactions with Isy1, a protein previously implicated in the first catalytic step of splicing and splicing fidelity. Together, the results suggest multiple function...
Source: RNA - September 28, 2009 Category: Genetics & Stem Cells Authors: Khanna M, Van Bakel H, Tang X, Calarco JA, Babak T, Guo G, Emili A, Greenblatt JF, Hughes TR, Krogan NJ, Blencowe BJ Tags: RNA Source Type: journals

A variant riboswitch aptamer class for S-adenosylmethionine common in marine bacteria.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Riboswitches that sense S-adenosylmethionine (SAM) are widely distributed throughout a variety of bacterial lineages. Four classes of SAM-binding riboswitches have been reported to date, constituting the most diverse collection of riboswitch classes that sense the same compound. Three of these classes, termed SAM-I, SAM-II, and SAM-III represent unique structures that form distinct binding pockets for the ligand. SAM-IV riboswitches carry different conserved sequence and structural features compared to other SAM riboswitches, but nucleotides and substructures corresponding to the ligand binding pocket are identical to ...
Source: RNA - September 22, 2009 Category: Genetics & Stem Cells Authors: Poiata E, Meyer MM, Ames TD, Breaker RR Tags: RNA Source Type: journals

Domain-elongation NMR spectroscopy yields new insights into RNA dynamics and adaptive recognition.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
By simplifying the interpretation of nuclear magnetic resonance spin relaxation and residual dipolar couplings data, recent developments involving the elongation of RNA helices are providing new atomic insights into the dynamical properties that allow RNA structures to change functionally and adaptively. Domain elongation, in concert with spin relaxation measurements, has allowed the detailed characterization of a hierarchical network of local and collective motional modes occurring at nanosecond timescale that mirror the structural rearrangements that take place following adaptive recognition. The combination of domai...
Source: RNA - September 22, 2009 Category: Genetics & Stem Cells Authors: Zhang Q, Al-Hashimi HM Tags: RNA Source Type: journals

Distinct extremely abundant siRNAs associated with cosuppression in petunia.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Cosuppression is a classical form of eukaryotic post-transcriptional gene silencing. It was first reported in transgenic petunia, where a sense transgene meant to overexpress the host Chalcone Synthase-A (CHS-A) gene caused the degradation of the homologous transcripts and the loss of flower pigmentation. In this work, we used deep sequencing technology to characterize in detail the small RNA population generated from the CHS-A sequence in cosuppressed transgenic petunia. Unexpectedly, two distinct small interfering RNAs (siRNAs) were found to vastly predominate. Our demonstration that they guide prominent cleavage eve...
Source: RNA - September 22, 2009 Category: Genetics & Stem Cells Authors: De Paoli E, Dorantes-Acosta A, Zhai J, Accerbi M, Jeong DH, Park S, Meyers BC, Jorgensen RA, Green PJ Tags: RNA Source Type: journals

Combined proteomic-RNAi screen for host factors involved in human hepatitis delta virus replication.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Human hepatitis delta virus (HDV) is the only animal virus known to replicate its RNA genome using a host polymerase because its only virally encoded proteins, the small and large hepatitis delta antigens (HDAg-S and HDAg-L), lack polymerase activity. Although this makes HDV an ideal model system to study RNA-directed transcription in mammalian cells, little is known about the host factors involved in its replication. To comprehensively identify such host factors, we created a stable cell line carrying a functional FLAG-HDAg-S. Anti-Flag immunopurification and mass spectrometry identified >100 proteins associated wi...
Source: RNA - September 22, 2009 Category: Genetics & Stem Cells Authors: Cao D, Haussecker D, Huang Y, Kay MA Tags: RNA Source Type: journals

Direct selection for ribozyme cleavage activity in cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Selection may prove to be a powerful tool for the generation of functional RNAs for in vivo genetic regulation. However, traditional in vitro selection schemes do not mimic physiological conditions, and in vivo selection schemes frequently use small pool sizes. Here we describe a hybrid in vitro/in vivo selection scheme that overcomes both of these disadvantages. In this new method, PCR-amplified expression templates are transfected into mammalian cells, transcribed hammerhead RNAs self-cleave, and the extracted, functional hammerhead ribozyme species are specifically amplified for the next round of selection. Using th...
Source: RNA - September 22, 2009 Category: Genetics & Stem Cells Authors: Chen X, Denison L, Levy M, Ellington AD Tags: RNA Source Type: journals

In vivo detection of RNA-binding protein interactions with cognate RNA sequences by fluorescence resonance energy transfer.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 (...
Source: RNA - September 17, 2009 Category: Genetics & Stem Cells Authors: Huranová M, Jablonski JA, Benda A, Hof M, Stanek D, Caputi M Tags: RNA Source Type: journals

Complex architecture and regulated expression of the Sox2ot locus during vertebrate development.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The Sox2 gene is a key regulator of pluripotency embedded within an intron of a long noncoding RNA (ncRNA), termed Sox2 overlapping transcript (Sox2ot), which is transcribed in the same orientation. However, this ncRNA remains uncharacterized. Here we show that Sox2ot has multiple transcription start sites associated with genomic features that indicate regulated expression, including highly conserved elements (HCEs) and chromatin marks characteristic of gene promoters. To identify biological processes in which Sox2ot may be involved, we analyzed its expression in several developmental systems, compared to expression of...
Source: RNA - September 17, 2009 Category: Genetics & Stem Cells Authors: Amaral PP, Neyt C, Wilkins SJ, Askarian-Amiri ME, Sunkin SM, Perkins AC, Mattick JS Tags: RNA Source Type: journals

Real-time fluorescence detection of exoribonucleases.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The identification of RNases or RNase effectors is a continuous challenge, particularly given the current importance of RNAs in the control of genome expression. Here, we show that a fluorogenic RNA-DNA hybrid is a powerful tool for a real-time fluorescence detection and assay of exoribonucleases (RT-FeDEx). This RT-FeDEx assay provides a new strategy for the isolation, purification, and assay of known and unknown exoribonucleases. PMID: 19767421 [PubMed - as supplied by publisher] (Source: RNA)
Source: RNA - September 17, 2009 Category: Genetics & Stem Cells Authors: Sinturel F, Pellegrini O, Xiang S, Tong L, Condon C, Bénard L Tags: RNA Source Type: journals

Quantitative miRNA expression analysis: Comparing microarrays with next-generation sequencing.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Recently, next-generation sequencing has been introduced as a promising, new platform for assessing the copy number of transcripts, while the existing microarray technology is considered less reliable for absolute, quantitative expression measurements. Nonetheless, so far, results from the two technologies have only been compared based on biological data, leading to the conclusion that, although they are somewhat correlated, expression values differ significantly. Here, we use synthetic RNA samples, resembling human microRNA samples, to find that microarray expression measures actually correlate better with sample RNA ...
Source: RNA - September 9, 2009 Category: Genetics & Stem Cells Authors: Willenbrock H, Salomon J, Søkilde R, Barken KB, Hansen TN, Nielsen FC, Møller S, Litman T Tags: RNA Source Type: journals

Three-way RNA junctions with remote tertiary contacts: A recurrent and highly versatile fold.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Three-way junction RNAs adopt a recurrent Y shape when two of the helices form a coaxial stack and the third helix establishes one or more tertiary contacts several base pairs away from the junction. In this review, the structure, distribution, and functional relevance of these motifs are examined. Structurally, the folds exhibit conserved junction topologies, and the distal tertiary interactions play a crucial role in determining the final shape of the structures. The junctions and remote tertiary contacts behave as flexible hinge motifs that respond to changes in the other region, providing these folds with switching...
Source: RNA - September 8, 2009 Category: Genetics & Stem Cells Authors: de la Peña M, Dufour D, Gallego J Tags: RNA Source Type: journals

Messenger RNA processing in Methanocaldococcus (Methanococcus) jannaschii.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Messenger RNA (mRNA) processing plays important roles in gene expression in all domains of life. A number of cases of mRNA cleavage have been documented in Archaea, but available data are fragmentary. We have examined RNAs present in Methanocaldococcus (Methanococcus) jannaschii for evidence of RNA processing upstream of protein-coding genes. Of 123 regions covered by the data, 31 were found to be processed, with 30 including a cleavage site 12-16 nucleotides upstream of the corresponding translation start site. Analyses with 3'-RACE (rapid amplification of cDNA ends) and 5'-RACE indicate that the processing is endonuc...
Source: RNA - August 27, 2009 Category: Genetics & Stem Cells Authors: Zhang J, Olsen GJ Tags: RNA Source Type: journals

The XIAP IRES activates 3' cistron expression by inducing production of monocistronic mRNA in the {beta}gal/CAT bicistronic reporter system.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
X-chromosome linked inhibitor of apoptosis (XIAP) mRNA has been proposed to bear a stress-activated internal ribosome entry site (IRES) that stimulates translation under conditions that inhibit cap-dependent initiation. However, several reports have indicated that the strong activity of the XIAP IRES in certain bicistronic reporter assay systems stems from production of unintended monocistronic transcripts through splicing or cryptic promoter activity. Here we extend these findings by providing evidence that the XIAP IRES similarly provokes the production of monocistronic mRNA encompassing the 3' cistron in the betagal...
Source: RNA - August 26, 2009 Category: Genetics & Stem Cells Authors: Saffran HA, Smiley JR Tags: RNA Source Type: journals

The Thermus thermophilus DEAD box helicase Hera contains a modified RNA recognition motif domain loosely connected to the helicase core.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DEAD box family helicases consist of a helicase core that is formed by two flexibly linked RecA-like domains. The helicase activity can be regulated by N- or C-terminal extensions flanking the core. Thermus thermophilus heat resistant RNA-dependent ATPase (Hera) is the first DEAD box helicase that forms a dimer using a unique dimerization domain. In addition to the dimerization domain, Hera contains a C-terminal RNA binding domain (RBD) that shares sequence homology only to uncharacterized proteins of the Deinococcus/Thermus group. The crystal structure of Hera_RBD reveals the fold of an altered RNA recognition motif (...
Source: RNA - August 25, 2009 Category: Genetics & Stem Cells Authors: Rudolph MG, Klostermeier D Tags: RNA Source Type: journals

Slow formation of a pseudoknot structure is rate limiting in the productive co-transcriptional folding of the self-splicing Candida intron.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Pseudoknots play critical roles in packing the active structure of various functional RNAs. The importance of the P3-P7 pseudoknot in refolding of group I intron ribozymes has been recently appreciated, while little is known about the pseudoknot function in co-transcriptional folding. Here we used the Candida group I intron as a model to address the question. We show that co-transcriptional folding of the active self-splicing intron is twice as fast as refolding. The P3-P7 pseudoknot folds slowly during co-transcriptional folding at a rate constant similar to the folding of the active ribozyme, and folding of both P3-P...
Source: RNA - August 25, 2009 Category: Genetics & Stem Cells Authors: Zhang L, Bao P, Leibowitz MJ, Zhang Y Tags: RNA Source Type: journals

New metrics for comparing and assessing discrepancies between RNA 3D structures and models.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
To benchmark progress made in RNA three-dimensional modeling and assess newly developed techniques, reliable and meaningful comparison metrics and associated tools are necessary. Generally, the average root-mean-square deviations (RMSDs) are quoted. However, RMSD can be misleading since errors are spread over the whole molecule and do not account for the specificity of RNA base interactions. Here, we introduce two new metrics that are particularly suitable to RNAs: the deformation index and deformation profile. The deformation index is calibrated by the interaction network fidelity, which considers base-base-stacking a...
Source: RNA - August 25, 2009 Category: Genetics & Stem Cells Authors: Parisien M, Cruz JA, Westhof E, Major F Tags: RNA Source Type: journals

Formation of She2p tetramers is required for mRNA binding, mRNP assembly, and localization.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
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 complex...
Source: RNA - August 25, 2009 Category: Genetics & Stem Cells Authors: Müller M, Richter K, Heuck A, Kremmer E, Buchner J, Jansen RP, Niessing D Tags: RNA Source Type: journals

Improved RNA secondary structure prediction by maximizing expected pair accuracy.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, a program, MaxExpect, that predicts RNA secondary structure by maximizing the expected base-pair accuracy, is reported. This approach was first pioneered in the program CONTRAfold, using pair probabilities predicted with a statistical learning method. Here, a partition function calculation that utilizes the free energy change nearest-neighbor parameters is used to predict base-pair probabilities as well as probabilities of nucleotides being single-stranded. MaxExpect predicts both the optimal structure (having highest expected pair accuracy) and suboptimal structures to serve as alternative hypotheses for th...
Source: RNA - August 23, 2009 Category: Genetics & Stem Cells Authors: Lu ZJ, Gloor JW, Mathews DH Tags: RNA Source Type: journals

Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report that unadulterated cytoplasmic RNA uncovers differentially expressed mRNAs that otherwise would not have been detected when using whole cell RNA and that the inclusion of nuclear RNA has a large impact on whole cell gene expression microarray results by distorting the mRNA profile to the extent that a substantial number of false positives are generated. We conclude that to produce a valid profile of the steady-state mRNA population, the nuclear component must be excluded, and to arrive at a more realistic view of a cell's gene expression profile, the nuclear and cytoplasmic RNA fractions should be analyzed separa...
Source: RNA - August 23, 2009 Category: Genetics & Stem Cells Authors: Trask HW, Cowper-Sal-Lari R, Sartor MA, Gui J, Heath CV, Renuka J, Higgins AJ, Andrews P, Korc M, Moore JH, Tomlinson CR Tags: RNA Source Type: journals

Formation of an active site in trans by interaction of two complete Varkud Satellite ribozymes.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The complete VS ribozyme comprises seven helical segments, connected by three three-way RNA junctions. In the presence of Mg(2+) ions, cleavage occurs within the internal loop of helix I. This requires the participation of a guanine (G638) within the helix I loop, and a remote adenine (A756) within an internal loop of helix VI. Previous structural studies have suggested that helix I docks into the fold of the remaining part of the ribozyme, bringing A756 and G638 close to the scissile phosphate to allow the cleavage reaction to proceed. We show here that while either A756C or G638A individually exhibit very low cleavag...
Source: RNA - August 23, 2009 Category: Genetics & Stem Cells Authors: Ouellet J, Byrne M, Lilley DM Tags: RNA Source Type: journals

Distinct kinetic determinants for the stepwise CCA addition to tRNA.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The universally conserved CCA sequence is present at the 3' terminal 74-76 positions of all active tRNA molecules as a functional tag to participate in ribosome protein synthesis. The CCA enzyme catalyzes CCA synthesis in three sequential steps of nucleotide addition at rapid and identical rates. However, the kinetic determinant of each addition is unknown, thus limiting the insights into the kinetic basis of CCA addition. Using our recently developed single turnover kinetics of Escherichia coli CCA enzyme as a model, we show here that the identical rate of the stepwise CCA addition is determined by distinct kinetic pa...
Source: RNA - August 19, 2009 Category: Genetics & Stem Cells Authors: Kim S, Liu C, Halkidis K, Gamper HB, Hou YM Tags: RNA Source Type: journals

An electrochemiluminescent aptamer switch for a high-throughput assay of an RNA editing reaction.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
An RNA editing reaction that is both essential and specific to the trypanosomatid parasites is an attractive target for new drug development. Although high-throughput screening of chemical libraries is a powerful strategy often used to identify new drugs, the available in vitro editing assays do not have the necessary sensitivity and format for this approach to be feasible. A ruthenium labeled reporter RNA is described here that overcomes these limitations as it can both detect edited product in the low femtomole range and is ideal for high-throughput format. The reporter RNA consists of an RNA editing substrate linked...
Source: RNA - August 19, 2009 Category: Genetics & Stem Cells Authors: Liang S, Connell GJ Tags: RNA Source Type: journals

Structure-activity relationships in human RNA 3'-phosphate cyclase.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
RNA 3'-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2',3' cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP(i); transfer of AMP from Rtc1 to an RNA 3'-phosphate to form an RNA(3')pp(5')A intermediate; and attack by the terminal nucleoside O2' on the 3'-phosphate to form an RNA 2',3' cyclic phosphate product and release AMP. Here we used the crystal structure of Escherichia coli RtcA to guide a mutational analysis of the human R...
Source: RNA - August 17, 2009 Category: Genetics & Stem Cells Authors: Tanaka N, Shuman S Tags: RNA Source Type: journals

Methodological obstacles in knocking down small noncoding RNAs.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In the recent past, several thousand noncoding RNA (ncRNA) genes have been predicted within eukaryal genomes. However, for their functional analysis only a few high-throughput methods are currently available to knock down selected ncRNA species, such as microRNAs, which are targeted by antisense probes, termed antagomirs. We thus compared the efficiencies of four knockdown strategies, previously mainly employed for the analysis of protein-coding genes, to study the function of ncRNAs, in particular, small nucleolar RNAs (snoRNAs). Thereby, the class of snoRNAs represents one of the most abundant ncRNA species. The majo...
Source: RNA - August 17, 2009 Category: Genetics & Stem Cells Authors: Ploner A, Ploner C, Lukasser M, Niederegger H, Hüttenhofer A Tags: RNA Source Type: journals

Metazoan stress granule assembly is mediated by P-eIF2{alpha}-dependent and -independent mechanisms.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report that Drosophila cells form SGs in response to arsenite and heat shock. Drosophila SGs, like mammalian SGs, are distinct from but adjacent to processing bodies (PBs, sites of mRNA silencing and decay), require polysome disassembly, and are in dynamic equilibrium with polysomes. We further examine the role of the two Drosophila eIF2alpha kinases, PEK and GCN2, in regulating SG formation in response to heat and arsenite stress. While arsenite-induced SGs are dependent upon eIF2alpha phosphorylation, primarily via PEK, heat-induced SGs are phospho-eIF2alpha-independent. In contrast, heat-induced SGs require eIF2alpha...
Source: RNA - August 5, 2009 Category: Genetics & Stem Cells Authors: Farny NG, Kedersha NL, Silver PA Tags: RNA Source Type: journals