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1.
  • Alalam, Hanna, et al. (author)
  • Global SLAM-Seq for accurate mRNA decay determination and identification of NMD targets.
  • 2022
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 28:7
  • Journal article (peer-reviewed)abstract
    • Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-Linked Alkylation for the Metabolic Sequencing of RNA (SLAM-Seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5 % of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-Seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the Nonsense-mediated decay (NMD) pathway by measuring the change in RNA half-lives; instead of steady-state RNA level changes. With SLAM-Seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 225 novel targets, and observe a strong agreement with previous reports of NMD targets, 61.2 % of our candidates being identified in previous studies. This indicates that SLAM-Seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.
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2.
  • Alkemar, Gunnar, et al. (author)
  • A possible tertiary rRNA interaction between expansion segments ES3 and ES6 in eukaryotic 40S ribosomal subunits
  • 2003
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 9:1, s. 20-24
  • Journal article (peer-reviewed)abstract
    • Eukaryotic 16S-like ribosomal RNAs contain 12 so-called expansion segments, i.e., sequences not included in the RNA secondary structure core common to eubacteria, archaea, and eukarya. Two of these expansion segments, ES3 and ES6, are juxtaposed in the recent three-dimensional model of the eukaryotic 40S ribosomal subunit. We have analyzed ES3 and ES6 sequences from more than 2900 discrete eukaryotic species, for possible sequence complementarity between the two expansion segments. The data show that ES3 and ES6 could interact by forming a helix consisting of seven to nine contiguous base pairs in almost all analyzed species. We, therefore, suggest that ES3 and ES6 form a direct RNA-RNA contact in the ribosome.
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3.
  • Alkemar, Gunnar, et al. (author)
  • Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits
  • 2004
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 10:3, s. 403-411
  • Journal article (peer-reviewed)abstract
    • The 18S rRNA of the small eukaryotic ribosomal subunit contains several expansion segments. Electron microscopy data indicate that two of the largest expansion segments are juxtaposed in intact 40S subunits, and data from phylogenetic sequence comparisons indicate that these two expansion segments contain complementary sequences that could form a direct tertiary interaction on the ribosome. We have investigated the secondary structure of the two expansion segments in the region around the putative tertiary interaction. Ribosomes from yeast, wheat, and mouse-three organisms representing separate eukaryotic kingdoms-were isolated, and the structure of ES3 and part of the ES6 region were analyzed using the single-strand-specific chemical reagents CMCT and DMS and the double-strand-specific ribonuclease V1. The modification patterns were analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The investigated sequences were relatively exposed to chemical and enzymatic modification. This is in line with their indicated location on the surface at the solvent side of the subunit. The complementary ES3 and ES6 sequences were clearly inaccessible to single-strand modification, but available for cleavage by double-strand-specific RNase V1. The results are compatible with a direct helical interaction between bases in ES3 and ES6. Almost identical results were obtained with ribosomes from the three organisms investigated.
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4.
  • Allner, O, et al. (author)
  • Loop-loop interaction in an adenine-sensing riboswitch: a molecular dynamics study
  • 2013
  • In: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 19:7, s. 916-926
  • Journal article (peer-reviewed)abstract
    • Riboswitches are mRNA-based molecules capable of controlling the expression of genes. They undergo conformational changes upon ligand binding, and as a result, they inhibit or promote the expression of the associated gene. The close connection between structural rearrangement and function makes a detailed knowledge of the molecular interactions an important step to understand the riboswitch mechanism and efficiency. We have performed all-atom molecular dynamics simulations of the adenine-sensing add A-riboswitch to study the breaking of the kissing loop, one key tertiary element in the aptamer structure. We investigated the aptamer domain of the add A-riboswitch in complex with its cognate ligand and in the absence of the ligand. The opening of the hairpins was simulated using umbrella sampling using the distance between two loops as the reaction coordinate. A two-step process was observed in all the simulated systems. First, a general loss of stacking and hydrogen bond interactions is seen. The last interactions that break are the two base pairs G37-C61 and G38-C60, but the break does not affect the energy profile, indicating their pivotal role in the tertiary structure formation but not in the structure stabilization. The junction area is partially organized before the kissing loop formation and residue A24 anchors together the loop helices. Moreover, when the distance between the loops is increased, one of the hairpins showed more flexibility by changing its orientation in the structure, while the other conserved its coaxial arrangement with the rest of the structure.
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5.
  • Allner, O, et al. (author)
  • Nucleotide modifications and tRNA anticodon-mRNA codon interactions on the ribosome
  • 2011
  • In: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 17:12, s. 2177-2188
  • Journal article (peer-reviewed)abstract
    • We have carried out molecular dynamics simulations of the tRNA anticodon and mRNA codon, inside the ribosome, to study the effect of the common tRNA modifications cmo5U34 and m6A37. In tRNAVal, these modifications allow all four nucleotides to be successfully read at the wobble position in a codon. Previous data suggest that entropic effects are mainly responsible for the extended reading capabilities, but detailed mechanisms have remained unknown. We have performed a wide range of simulations to elucidate the details of these mechanisms at the atomic level and quantify their effects: extensive free energy perturbation coupled with umbrella sampling, entropy calculations of tRNA (free and bound to the ribosome), and thorough structural analysis of the ribosomal decoding center. No prestructuring effect on the tRNA anticodon stem–loop from the two modifications could be observed, but we identified two mechanisms that may contribute to the expanded decoding capability by the modifications: The further reach of the cmo5U34 allows an alternative outer conformation to be formed for the noncognate base pairs, and the modification results in increased contacts between tRNA, mRNA, and the ribosome.
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6.
  • Andreev, Dmitri, et al. (author)
  • The bacterial toxin ReIE induces specific mRNA cleavage in the A site of the eukaryote ribosome
  • 2008
  • In: RNA. - : RNA Society. - 1355-8382 .- 1469-9001. ; 14:2, s. 233-239
  • Journal article (peer-reviewed)abstract
    • ReIE/ReIB is a well-characterized toxin-anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. ReIE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. ReIE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making "ReIE printing" a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site.
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7.
  • Avesson, Lotta, et al. (author)
  • MicroRNAs in Amoebozoa : Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs
  • 2012
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 18:10, s. 1771-1782
  • Journal article (peer-reviewed)abstract
    • The RNA interference machinery has served as a guardian of eukaryotic genomes since the divergence from prokaryotes. Although the basic components have a shared origin, silencing pathways directed by small RNAs have evolved in diverse directions in different eukaryotic lineages. Micro (mi)RNAs regulate protein-coding genes and play vital roles in plants and animals, but less is known about their functions in other organisms. Here, we report, for the first time, deep sequencing of small RNAs from the social amoeba Dictyostelium discoideum. RNA from growing single-cell amoebae as well as from two multicellular developmental stages was sequenced. Computational analyses combined with experimental data reveal the expression of miRNAs, several of them exhibiting distinct expression patterns during development. To our knowledge, this is the first report of miRNAs in the Amoebozoa supergroup. We also show that overexpressed miRNA precursors generate miRNAs and, in most cases, miRNA* sequences, whose biogenesis is dependent on the Dicer-like protein DrnB, further supporting the presence of miRNAs in D. discoideum. In addition, we find miRNAs processed from hairpin structures originating from an intron as well as from a class of repetitive elements. We believe that these repetitive elements are sources for newly evolved miRNAs.
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8.
  • Babina, Arianne M, et al. (author)
  • An S6 : S18 complex inhibits translation of E. coli rpsF.
  • 2015
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 21:12, s. 2039-46
  • Journal article (peer-reviewed)abstract
    • More than half of the ribosomal protein operons in Escherichia coli are regulated by structures within the mRNA transcripts that interact with specific ribosomal proteins to inhibit further protein expression. This regulation is accomplished using a variety of mechanisms and the RNA structures responsible for regulation are often not conserved across bacterial phyla. A widely conserved mRNA structure preceding the ribosomal protein operon containing rpsF and rpsR (encoding S6 and S18) was recently identified through comparative genomics. Examples of this RNA from both E. coli and Bacillus subtilis were shown to interact in vitro with an S6:S18 complex. In this work, we demonstrate that in E. coli, this RNA structure regulates gene expression in response to the S6:S18 complex. β-galactosidase activity from a lacZ reporter translationally fused to the 5' UTR and first nine codons of E. coli rpsF is reduced fourfold by overexpression of a genomic fragment encoding both S6 and S18 but not by overexpression of either protein individually. Mutations to the mRNA structure, as well as to the RNA-binding site of S18 and the S6-S18 interaction surfaces of S6 and S18, are sufficient to derepress β-galactosidase activity, indicating that the S6:S18 complex is the biologically active effector. Measurement of transcript levels shows that although reporter levels do not change upon protein overexpression, levels of the native transcript are reduced fourfold, suggesting that the mRNA regulator prevents translation and this effect is amplified on the native transcript by other mechanisms.
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9.
  • Babina, Arianne M., et al. (author)
  • Fitness advantages conferred by the L20-interacting RNA cis-regulator of ribosomal protein synthesis in Bacillus subtilis
  • 2018
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 24:9, s. 1133-1143
  • Journal article (peer-reviewed)abstract
    • In many bacteria, ribosomal proteins autogenously repress their own expression by interacting with RNA structures typically located in the 5'-UTRs of their mRNA transcripts. This regulation is necessary to maintain a balance between ribosomal proteins and rRNA to ensure proper ribosome production. Despite advances in noncoding RNA discovery and validation of RNA-protein regulatory interactions, the selective pressures that govern the formation and maintenance of such RNA cis-regulators in the context of an organism remain largely undetermined. To examine the impact disruptions to this regulation have on bacterial fitness, we introduced point mutations that abolish ribosomal protein binding and regulation into the RNA structure that controls expression of ribosomal proteins L20 and L35 within the Bacillus subtilis genome. Our studies indicate that removing this regulation results in reduced log phase growth, improper rRNA maturation, and the accumulation of a kinetically trapped or misassembled ribosomal particle at low temperatures, suggesting defects in ribosome synthesis. Such work emphasizes the important role regulatory RNAs play in the stoichiometric production of ribosomal components for proper ribosome composition and overall organism viability and reinforces the potential of targeting ribosomal protein production and ribosome assembly with novel antimicrobials.
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10.
  • Banijamali, Elnaz, et al. (author)
  • RNA:RNA interaction in ternary complexes resolved by chemical probing
  • 2022
  • In: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 29:3, s. 317-329
  • Journal article (peer-reviewed)abstract
    • RNA regulation can be performed by a second targeting RNA molecule, such as in the microRNA regulation mechanism. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probes the structure of RNA molecules and can resolve RNA:protein interactions, but RNA:RNA interactions have not yet been addressed with this technique. Here, we apply SHAPE to investigate RNA-mediated binding processes in RNA:RNA and RNA:RNA-RBP complexes. We use RNA:RNA binding by SHAPE (RABS) to investigate microRNA-34a (miR-34a) binding its mRNA target, the silent information regulator 1 (mSIRT1), both with and without the Argonaute protein, constituting the RNA-induced silencing complex (RISC). We show that the seed of the mRNA target must be bound to the microRNA loaded into RISC to enable further binding of the compensatory region by RISC, while the naked miR-34a is able to bind the compensatory region without seed interaction. The method presented here provides complementary structural evidence for the commonly performed luciferase-assay-based evaluation of microRNA binding-site efficiency and specificity on the mRNA target site and could therefore be used in conjunction with it. The method can be applied to any nucleic acid-mediated RNA- or RBP-binding process, such as splicing, antisense RNA binding, or regulation by RISC, providing important insight into the targeted RNA structure.
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11.
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12.
  • Bateman, Alex, et al. (author)
  • RNAcentral: A vision for an international database of RNA sequences.
  • 2011
  • In: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 17:11, s. 1941-6
  • Journal article (peer-reviewed)abstract
    • During the last decade there has been a great increase in the number of noncoding RNA genes identified, including new classes such as microRNAs and piRNAs. There is also a large growth in the amount of experimental characterization of these RNA components. Despite this growth in information, it is still difficult for researchers to access RNA data, because key data resources for noncoding RNAs have not yet been created. The most pressing omission is the lack of a comprehensive RNA sequence database, much like UniProt, which provides a comprehensive set of protein knowledge. In this article we propose the creation of a new open public resource that we term RNAcentral, which will contain a comprehensive collection of RNA sequences and fill an important gap in the provision of biomedical databases. We envision RNA researchers from all over the world joining a federated RNAcentral network, contributing specialized knowledge and databases. RNAcentral would centralize key data that are currently held across a variety of databases, allowing researchers instant access to a single, unified resource. This resource would facilitate the next generation of RNA research and help drive further discoveries, including those that improve food production and human and animal health. We encourage additional RNA database resources and research groups to join this effort. We aim to obtain international network funding to further this endeavor.
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13.
  • Björk, Glenn, et al. (author)
  • A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast.
  • 2007
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 13:8, s. 1245-55
  • Journal article (peer-reviewed)abstract
    • Transfer RNAs specific for Gln, Lys, and Glu from all organisms (except Mycoplasma) and organelles have a 2-thiouridine derivative (xm(5)s(2)U) as wobble nucleoside. These tRNAs read the A- and G-ending codons in the split codon boxes His/Gln, Asn/Lys, and Asp/Glu. In eukaryotic cytoplasmic tRNAs the conserved constituent (xm(5)-) in position 5 of uridine is 5-methoxycarbonylmethyl (mcm(5)). A protein (Tuc1p) from yeast resembling the bacterial protein TtcA, which is required for the synthesis of 2-thiocytidine in position 32 of the tRNA, was shown instead to be required for the synthesis of 2-thiouridine in the wobble position (position 34). Apparently, an ancient member of the TtcA family has evolved to thiolate U34 in tRNAs of organisms from the domains Eukarya and Archaea. Deletion of the TUC1 gene together with a deletion of the ELP3 gene, which results in the lack of the mcm(5) side chain, removes all modifications from the wobble uridine derivatives of the cytoplasmic tRNAs specific for Gln, Lys, and Glu, and is lethal to the cell. Since excess of the unmodified form of these three tRNAs rescued the double mutant elp3 tuc1, the primary function of mcm(5)s(2)U34 seems to be to improve the efficiency to read the cognate codons rather than to prevent mis-sense errors. Surprisingly, overexpression of the mcm(5)s(2)U-lacking tRNA(Lys) alone was sufficient to restore viability of the double mutant.
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14.
  • Borg, Anneli, et al. (author)
  • Complete kinetic mechanism for recycling of the bacterial ribosome
  • 2016
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 22:1, s. 10-21
  • Journal article (peer-reviewed)abstract
    • How EF-G and RRF act together to split a post-termination ribosomal complex into its subunits has remained obscure. Here, using stopped-flow experiments with Rayleigh light scattering detection and quench-flow experiments with radio-detection of GTP hydrolysis, we have clarified the kinetic mechanism of ribosome recycling and obtained precise estimates of its kinetic parameters. Ribosome splitting requires that EF-G binds to an already RRF-containing ribosome. EF-G binding to RRF-free ribosomes induces futile rounds of GTP hydrolysis and inhibits ribosome splitting, implying that while RRF is purely an activator of recycling, EF-G acts as both activator and competitive inhibitor of RRF in recycling of the post-termination ribosome. The ribosome splitting rate and the number of GTPs consumed per splitting event depend strongly on the free concentrations of EF-G and RRF. The maximal recycling rate, here estimated as 25 sec(-1), is approached at very high concentrations of EF-G and RRF with RRF in high excess over EF-G. The present in vitro results, suggesting an in vivo ribosome recycling rate of 5 sec(-1), are discussed in the perspective of rapidly growing bacterial cells.
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15.
  • Bratt, E, et al. (author)
  • Coordination of editing and splicing of glutamate receptor pre-mRNA
  • 2003
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 9, s. 309-318
  • Journal article (peer-reviewed)abstract
    • Adenosine deaminase that acts on RNA, ADAR, catalyzes the conversion of adenosine into inosine within double-stranded RNA. This type of editing has mainly been found in genes involved in neurotransmission. Site-specific A to I modifications often require intronic sequences to create the double-stranded structure necessary for editing. A system was developed to investigate if editing and splicing of pre-mRNA are coordinated. We have focused on a selectively edited site (R/G) in the glutamate receptor subunit B pre-mRNA. This editing site is situated in close proximity to a 5′ splice site. To ensure efficient splicing, the editing site, together with its natural 5′ splice site, was fused to a 3′ splice site of the major late transcript from adenovirus. In vitro, on a premade transcript, ADAR2 editing and splicing were found to interfere with each other. The stable stem-loop required for ADAR2 editing had a negative effect on in vitro splicing, possibly by sequestering the 5′ splice site. Further, RNA helicase A was shown to overcome the splicing inhibition caused by ADAR2. In vivo, allowing cotranscriptional processing, the same construct was found to efficiently edit and splice without interference, suggesting that the two RNA processing events are coordinated.
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16.
  • Bugaeva, Elizaveta Y, et al. (author)
  • Structural features of the tmRNA-ribosome interaction
  • 2009
  • In: RNA. - : RNA Society. - 1355-8382 .- 1469-9001. ; 15:12, s. 2312-2320
  • Journal article (peer-reviewed)abstract
    • 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 chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.
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17.
  • Cavallin, I, et al. (author)
  • HITS-CLIP analysis of human ALKBH8 reveals interactions with fully processed substrate tRNAs and with specific noncoding RNAs
  • 2022
  • In: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 28:12, s. 1568-1581
  • Journal article (peer-reviewed)abstract
    • Transfer RNAs acquire a large plethora of chemical modifications. Among those, modifications of the anticodon loop play important roles in translational fidelity and tRNA stability. Four human wobble U containing tRNAs obtain 5-methoxycarbonylmethyluridine (mcm5U34) or 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34), which play a role in decoding. This mark involves a cascade of enzymatic activities. The last step is mediated by Alkylation Repair Homolog 8 (ALKBH8). In this study, we performed a transcriptome-wide analysis of the repertoire of ALKBH8 RNA targets. Using a combination of HITS-CLIP and RIP-seq analyses, we uncover ALKBH8-bound RNAs. We show that ALKBH8 targets fully processed and CCA modified tRNAs. Our analyses uncovered the previously known set of wobble-U containing tRNAs. In addition, our both approaches revealed ALKBH8 binding to several other types of non-coding RNAs, in particular C/D box snoRNAs.
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18.
  • Chavali, Sreenivas, et al. (author)
  • MicroRNAs act complementarily to regulate disease-related mRNA modules in human diseases
  • 2013
  • In: Rna-a Publication of the Rna Society. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 19:11, s. 1552-1562
  • Journal article (peer-reviewed)abstract
    • MicroRNAs (miRNAs) play a key role in regulating mRNA expression, and individual miRNAs have been proposed as diagnostic and therapeutic candidates. The identification of such candidates is complicated by the involvement of multiple miRNAs and mRNAs as well as unknown disease topology of the miRNAs. Here, we investigated if disease-associated miRNAs regulate modules of disease-associated mRNAs, if those miRNAs act complementarily or synergistically, and if single or combinations of miRNAs can be targeted to alter module functions. We first analyzed publicly available miRNA and mRNA expression data for five different diseases. Integrated target prediction and network-based analysis showed that the miRNAs regulated modules of disease-relevant genes. Most of the miRNAs acted complementarily to regulate multiple mRNAs. To functionally test these findings, we repeated the analysis using our own miRNA and mRNA expression data from CD4+ T cells from patients with seasonal allergic rhinitis. This is a good model of complex diseases because of its well-defined phenotype and pathogenesis. Combined computational and functional studies confirmed that miRNAs mainly acted complementarily and that a combination of two complementary miRNAs, miR-223 and miR-139-3p, could be targeted to alter disease-relevant module functions, namely, the release of type 2 helper T-cell (Th2) cytokines. Taken together, our findings indicate that miRNAs act complementarily to regulate modules of disease-related mRNAs and can be targeted to alter disease-relevant functions.
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19.
  • Cruz, Jose Almeida, et al. (author)
  • RNA-Puzzles : A CASP-like evaluation of RNA three-dimensional structure prediction
  • 2012
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 18:4, s. 610-625
  • Journal article (peer-reviewed)abstract
    • We report the results of a first, collective, blind experiment in RNA three-dimensional (3D) structure prediction, encompassing three prediction puzzles. The goals are to assess the leading edge of RNA structure prediction techniques; compare existing methods and tools; and evaluate their relative strengths, weaknesses, and limitations in terms of sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in ongoing efforts to improve prediction tools. We also report the creation of an automated evaluation pipeline to facilitate the analysis of future RNA structure prediction exercises.
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20.
  • Dao, E. Han, et al. (author)
  • Structure of the 30S ribosomal decoding complex at ambient temperature
  • 2018
  • In: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 24:12, s. 1667-1676
  • Journal article (peer-reviewed)abstract
    • The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New light sources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Therm us thermophilus 30S ribosomal subunit decoding complex to 3.45 angstrom resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA-protein complexes at near-physiological temperatures.
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21.
  • Daub, Jennifer, et al. (author)
  • The RNA WikiProject : Community annotation of RNA families
  • 2008
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 14:12, s. 2462-2464
  • Journal article (peer-reviewed)abstract
    • The online encyclopedia Wikipedia has become one of the most important online references in the world and has a substantial and growing scientific content. A search of Google with many RNA-related keywords identifies a Wikipedia article as the top hit. We believe that the RNA community has an important and timely opportunity to maximize the content and quality of RNA information in Wikipedia. To this end, we have formed the RNA WikiProject (http://en.wikipedia.org/wiki/Wikipedia: WikiProject_RNA) as part of the larger Molecular and Cellular Biology WikiProject. We have created over 600 new Wikipedia articles describing families of noncoding RNAs based on the Rfam database, and invite the community to update, edit, and correct these articles. The Rfam database now redistributes this Wikipedia content as the primary textual annotation of its RNA families. Users can, therefore, for the first time, directly edit the content of one of the major RNA databases. We believe that this Wikipedia/Rfam link acts as a functioning model for incorporating community annotation into molecular biology databases.
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22.
  • Davidsson, Marcus, et al. (author)
  • Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing
  • 2018
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 24:5, s. 673-687
  • Journal article (peer-reviewed)abstract
    • Genome editing has proven to be highly potent in the generation of functional gene knockouts in dividing cells. In the CNS however, efficient technologies to repair sequences are yet to materialize. Reprogramming on the mRNA level is an attractive alternative as it provides means to perform in situ editing of coding sequences without nuclease dependency. Furthermore, de novo sequences can be inserted without the requirement of homologous recombination. Such reprogramming would enable efficient editing in quiescent cells (e.g., neurons) with an attractive safety profile for translational therapies. In this study, we applied a novel molecular-barcoded screening assay to investigate RNA trans-splicing in mammalian neurons. Through three alternative screening systems in cell culture and in vivo, we demonstrate that factors determining trans-splicing are reproducible regardless of the screening system. With this screening, we have located the most permissive trans-splicing sequences targeting an intron in the Synapsin I gene. Using viral vectors, we were able to splice full-length fluorophores into the mRNA while retaining very low off-target expression. Furthermore, this approach also showed evidence of functionality in the mouse striatum. However, in its current form, the trans-splicing events are stochastic and the overall activity lower than would be required for therapies targeting loss-of-function mutations. Nevertheless, the herein described barcode-based screening assay provides a unique possibility to screen and map large libraries in single animals or cell assays with very high precision.
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23.
  • Davila Lopez, Marcela, et al. (author)
  • Early evolution of histone mRNA 3' end processing.
  • 2008
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 14:1, s. 1-10
  • Journal article (peer-reviewed)abstract
    • The replication-dependent histone mRNAs in metazoa are not polyadenylated, in contrast to the bulk of mRNA. Instead, they contain an RNA stem-loop (SL) structure close to the 3' end of the mature RNA, and this 3' end is generated by cleavage using a machinery involving the U7 snRNP and protein factors such as the stem-loop binding protein (SLBP). This machinery of 3' end processing is related to that of polyadenylation as protein components are shared between the systems. It is commonly believed that histone 3' end processing is restricted to metazoa and green algae. In contrast, polyadenylation is ubiquitous in Eukarya. However, using computational approaches, we have now identified components of histone 3' end processing in a number of protozoa. Thus, the histone mRNA stem-loop structure as well as the SLBP protein are present in many different protozoa, including Dictyostelium, alveolates, Trypanosoma, and Trichomonas. These results show that the histone 3' end processing machinery is more ancient than previously anticipated and can be traced to the root of the eukaryotic phylogenetic tree. We also identified histone mRNAs from both metazoa and protozoa that are polyadenylated but also contain the signals characteristic of histone 3' end processing. These results provide further evidence that some histone genes are regulated at the level of 3' end processing to produce either polyadenylated RNAs or RNAs with the 3' end characteristic of replication-dependent histone mRNAs.
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24.
  • Destefanis, Eliana, et al. (author)
  • A mark of disease : How mRNA modifications shape genetic and acquired pathologies
  • 2021
  • In: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 27:4, s. 367-389
  • Research review (peer-reviewed)abstract
    • RNA modifications have recently emerged as a widespread and complex facet of gene expression regulation. Counting more than 170 distinct chemical modifications with far-reaching implications for RNA fate, they are collectively referred to as the epitranscriptome. These modifications can occur in all RNA species, including messenger RNAs (mRNAs) and noncoding RNAs (ncRNAs). In mRNAs the deposition, removal, and recognition of chemical marks by writers, erasers and readers influence their structure, localization, stability, and translation. In turn, this modulates key molecular and cellular processes such as RNA metabolism, cell cycle, apoptosis, and others. Unsurprisingly, given their relevance for cellular and organismal functions, alterations of epitranscriptomic marks have been observed in a broad range of human diseases, including cancer, neurological and metabolic disorders. Here, we will review the major types of mRNA modifications and editing processes in conjunction with the enzymes involved in their metabolism and describe their impact on human diseases. We present the current knowledge in an updated catalog. We will also discuss the emerging evidence on the crosstalk of epitranscriptomic marks and what this interplay could imply for the dynamics of mRNA modifications. Understanding how this complex regulatory layer can affect the course of human pathologies will ultimately lead to its exploitation toward novel epitranscriptomic therapeutic strategies.
  •  
25.
  • Dunham, Christine M., et al. (author)
  • Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit
  • 2007
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 13:6, s. 817-823
  • Journal article (peer-reviewed)abstract
    • During translation, some +1 frameshift mRNA sites are decoded by frameshift suppressor tRNAs that contain an extra base in their anticodon loops. Similarly engineered tRNAs have been used to insert nonnatural amino acids into proteins. Here, we report crystal structures of two anticodon stem–loops (ASLs) from tRNAs known to facilitate +1 frameshifting bound to the 30S ribosomal subunit with their cognate mRNAs. ASLCCCG and ASLACCC (5'–3' nomenclature) form unpredicted anticodon–codon interactions where the anticodon base 34 at the wobble position contacts either the fourth codon base or the third and fourth codon bases. In addition, we report the structure of ASLACGA bound to the 30S ribosomal subunit with its cognate mRNA. The tRNA containing this ASL was previously shown to be unable to facilitate +1 frameshifting in competition with normal tRNAs (Hohsaka et al. 2001), and interestingly, it displays a normal anticodon–codon interaction. These structures show that the expanded anticodon loop of +1 frameshift promoting tRNAs are flexible enough to adopt conformations that allow three bases of the anticodon to span four bases of the mRNA. Therefore it appears that normal triplet pairing is not an absolute constraint of the decoding center.
  •  
26.
  • Farazi, Thalia A, et al. (author)
  • Identification of the RNA recognition element of the RBPMS family of RNA-binding proteins and their transcriptome-wide mRNA targets.
  • 2014
  • In: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 20, s. 1090-1102
  • Journal article (peer-reviewed)abstract
    • Recent studies implicated the RNA-binding protein with multiple splicing (RBPMS) family of proteins in oocyte, retinal ganglion cell, heart, and gastrointestinal smooth muscle development. These RNA-binding proteins contain a single RNA recognition motif (RRM), and their targets and molecular function have not yet been identified. We defined transcriptome-wide RNA targets using photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) in HEK293 cells, revealing exonic mature and intronic pre-mRNA binding sites, in agreement with the nuclear and cytoplasmic localization of the proteins. Computational and biochemical approaches defined the RNA recognition element (RRE) as a tandem CAC trinucleotide motif separated by a variable spacer region. Similar to other mRNA-binding proteins, RBPMS family of proteins relocalized to cytoplasmic stress granules under oxidative stress conditions suggestive of a support function for mRNA localization in large and/or multinucleated cells where it is preferentially expressed.
  •  
27.
  •  
28.
  •  
29.
  • Freihofer, Pietro, et al. (author)
  • Nonmutational compensation of the fitness cost of antibiotic resistance in mycobacteria by overexpression of tlyA rRNA methylase
  • 2016
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 22:12, s. 1836-1843
  • Journal article (peer-reviewed)abstract
    • Several studies over the last few decades have shown that antibiotic resistance mechanisms frequently confer a fitness cost and that these costs can be genetically ameliorated by intra- or extragenic second-site mutations, often without loss of resistance. Another, much less studied potential mechanism by which the fitness cost of antibiotic resistance could be reduced is via a regulatory response where the deleterious effect of the resistance mechanism is lowered by a physiological alteration that buffers the mutational effect. In mycobacteria, resistance to the clinically used tuberactinomycin antibiotic capreomycin involves loss-of-function mutations in rRNA methylase TIyA or point mutations in 16S rRNA (in particular the A1408G mutation). Both of these alterations result in resistance by reducing drug binding to the ribosome. Here we show that alterations of tlyA gene expression affect both antibiotic drug susceptibility and fitness cost of drug resistance. In particular, we demonstrate that the common resistance mutation A1408G is accompanied by a physiological change that involves increased expression of the tlyA gene. This gene encodes an enzyme that methylates neighboring 16S rRNA position C1409, and as a result of increased TIyA expression the fitness cost of the A1408G mutation is significantly reduced. Our findings suggest that in mycobacteria, a nonmutational mechanism (i.e., gene regulatory) can restore fitness to genetically resistant bacteria. Our results also point to a new and clinically relevant treatment strategy to combat evolution of resistance in multidrug-resistant tuberculosis. Thus, by utilizing antagonistic antibiotic interactions, resistance evolution could be reduced.
  •  
30.
  • Fromm, Bastian, et al. (author)
  • Ancient microRNA profiles of 14,300-yr-old canid samples confirm taxonomic origin and provide glimpses into tissue-specific gene regulation from the Pleistocene
  • 2021
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 27:3, s. 324-334
  • Journal article (peer-reviewed)abstract
    • DNA sequencing is the current key technology for historic or ancient biological samples and has led to many exciting discoveries in the field of paleogenomics. However, functional insights into tissue identity, cellular composition, or gene regulation cannot be gained from DNA. Recent analyses have shown that, under favorable conditions, RNA can also be sequenced from ancient samples, enabling studies at the transcriptomic and regulatory level. Analyzing ancient RNA data from a Pleistocene canid, we find hundreds of intact microRNAs that are taxonomically informative, show tissue specificity and have functionally predictive characteristics. With an extraordinary age of 14,300 yr, these microRNA sequences are by far the oldest ever reported. The authenticity of the sequences is further supported by (i) the presence of canid/Caniformia-specific sequences that never evolved outside of this Glade, (ii) tissue-specific expression patterns (cartilage, liver, and muscle) that resemble those of modern dogs, and (iii) RNA damage patterns that are clearly distinct from those of fresh samples. By performing computational microRNA-target enrichment analyses on the ancient sequences, we predict microRNA functions consistent with their tissue pattern of expression. For instance, we find a liver-specific microRNA that regulates carbohydrate metabolism and starvation responses in canids. In summary, we show that straightforward paleotranscriptomic microRNA analyses can give functional glimpses into tissue identity, cellular composition, and gene regulatory activity of ancient samples and biological processes that took place in the Pleistocene, thus holding great promise for deeper insights into gene regulation in extinct animals based on ancient RNA sequencing.
  •  
31.
  •  
32.
  • Fromm, Bastian, et al. (author)
  • The limits of human microRNA annotation have been met
  • 2022
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 28:6, s. 781-785
  • Journal article (peer-reviewed)abstract
    • Over the last few years, the number of microRNAs in the human genome has become a controversially debated issue. Several publications reported thousands of putative novel microRNAs not included in the curated microRNA gene database MirGeneDB and the repository miRBase. Recently, by using sequencing of ∼300 human tissues and cell lines, the human RNA atlas, an expanded inventory of human RNA annotations, was published, reporting thousands of putative microRNAs. We, the developers of established microRNA prediction tools and hosts of MirGeneDB, raise concerns about the frequently applied prediction and functional validation strategies, briefly discussing the drawbacks of false positive detections. By means of quantifying well-established biogenesis-derived features, we show that the reported novel microRNAs essentially represent false-positives and argue that the human microRNA complement, at about 550 microRNA genes, is already near complete. Output of available tools must be curated as false predictions will misguide scientists looking for biomarkers or therapeutic targets.
  •  
33.
  • Gonzalez, Grecia M., et al. (author)
  • Structure of the Escherichia coli ProQ RNA-binding protein
  • 2017
  • In: RNA. - : COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. - 1355-8382 .- 1469-9001. ; 23:5, s. 696-711
  • Journal article (peer-reviewed)abstract
    • The protein ProQ has recently been identified as a global small noncoding RNA-binding protein in Salmonella, and a similar role is anticipated for its numerous homologs in divergent bacterial species. We report the solution structure of Escherichia call ProQ, revealing an N-terminal FinO-like domain, a C-terminal domain that unexpectedly has a Tudor domain fold commonly found in eukaryotes, and an elongated bridging intradomain linker that is flexible but nonetheless incompressible. Structure-based sequence analysis suggests that the Tudor domain was acquired through horizontal gene transfer and gene fusion to the ancestral FinO-like domain. Through a combination of biochemical and biophysical approaches, we have mapped putative RNA-binding surfaces on all three domains of ProQ and modeled the protein's conformation in the apo and RNA-bound forms. Taken together, these data suggest how the FinO, Tudor, and linker domains of ProQ cooperate to recognize complex RNA structures and serve to promote RNA-mediated regulation.
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34.
  • Gustavsson, Marie, et al. (author)
  • Evidence that tRNA modifying enzymes are important in vivo targets for 5-fluorouracil in yeast
  • 2008
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 14:4, s. 666-674
  • Journal article (peer-reviewed)abstract
    • We have screened a collection of haploid yeast knockout strains for increased sensitivity to 5-fluorouracil (5-FU). A total of 138 5-FU sensitive strains were found. Mutants affecting rRNA and tRNA maturation were particularly sensitive to 5-FU, with the tRNA methylation mutant trm10 being the most sensitive mutant. This is intriguing since trm10, like many other tRNA modification mutants, lacks a phenotype under normal conditions. However, double mutants for nonessential tRNA modification enzymes are frequently temperature sensitive, due to destabilization of hypomodified tRNAs. We therefore tested if the sensitivity of our mutants to 5-FU is affected by the temperature. We found that the cytotoxic effect of 5-FU is strongly enhanced at 38 degrees C for tRNA modification mutants. Furthermore, tRNA modification mutants show similar synthetic interactions for temperature sensitivity and sensitivity to 5-FU. A model is proposed for how 5-FU kills these mutants by reducing the number of tRNA modifications, thus destabilizing tRNA. Finally, we found that also wild-type cells are temperature sensitive at higher concentrations of 5-FU. This suggests that tRNA destabilization contributes to 5-FU cytotoxicity in wild-type cells and provides a possible explanation why hyperthermia can enhance the effect of 5-FU in cancer therapy.
  •  
35.
  • Hart, K, et al. (author)
  • Molecular dynamics simulations and free energy calculations of base flipping in dsRNA
  • 2005
  • In: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 11:5, s. 609-618
  • Journal article (peer-reviewed)abstract
    • The family of adenosine deaminases acting on RNA (ADARs) targets adenosines in RNA that is mainly double stranded. Some substrates are promiscuously deaminated whereas others, such as the mammalian glutamate receptor B (gluR-B) pre-mRNA, are more selectively deaminated. Many DNA/RNA-base modification enzymes use a base flipping mechanism to be able to reach their target base and it is believed that ADARs function in a similar way. In this study we used molecular dynamics (MD) simulations to describe two sites on the gluR-B pre-mRNA, the selectively targeted R/G site and the nontargeted 46 site, in an attempt to explain the substrate specificity. We used regular MD and also a forced base flipping method with umbrella sampling to calculate the free energy of base opening. Spontaneous opening of the mismatched adenosine was observed for the R/G site but not for the 46 site.
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36.
  • Hessle, Viktoria, et al. (author)
  • Rrp6 is recruited to transcribed genes and accompanies the spliced mRNA to the nuclear pore
  • 2012
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 18:8, s. 1466-1474
  • Journal article (peer-reviewed)abstract
    • Rrp6 is an exoribonuclease involved in the quality control of mRNA biogenesis. We have analyzed the association of Rrp6 with the Balbiani ring pre-mRNPs of Chironomus tentans to obtain insight into the role of Rrp6 in splicing surveillance. Rrp6 is recruited to transcribed genes and its distribution along the genes does not correlate with the positions of exons and introns. In the nucleoplasm, Rrp6 is bound to both unspliced and spliced transcripts. Rrp6 is released from the mRNPs in the vicinity of the nuclear pore before nucleo-cytoplasmic translocation. We show that Rrp6 is associated with newly synthesized transcripts during all the nuclear steps of gene expression and is associated with the transcripts independently of their splicing status. These observations suggest that the quality control of pre-mRNA splicing is not based on the selective recruitment of the exoribonuclease Rrp6 to unprocessed mRNAs.
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37.
  • Holmqvist, Isak, et al. (author)
  • FLAME: long-read bioinformatics tool for comprehensive spliceome characterization
  • 2021
  • In: Rna. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 27:10, s. 1127-1139
  • Journal article (peer-reviewed)abstract
    • Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide long-read sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this work-flow circumvents issues related to inadequate reference annotations and allows for incorporation of short-read sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long noncoding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing data sets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.
  •  
38.
  • Huang, Bo, et al. (author)
  • A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae.
  • 2008
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 14:10, s. 2183-2194
  • Journal article (peer-reviewed)abstract
    • We recently showed that the gamma-subunit of Kluyveromyces lactis killer toxin (gamma-toxin) is a tRNA endonuclease that cleaves tRNA(mcm5s2UUC Glu), tRNA(mcm5s2UUU Lys), and tRNA(mcm5s2UUG Gln) 3' of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). The 5-methoxycarbonylmethyl (mcm(5)) side chain was important for efficient cleavage by gamma-toxin, and defects in mcm(5) side-chain synthesis correlated with resistance to gamma-toxin. Based on this correlation, a genome-wide screen was performed to identify gene products involved in the formation of the mcm(5) side chain. From a collection of 4826 homozygous diploid Saccharomyces cerevisiae strains, each with one nonessential gene deleted, 63 mutants resistant to Kluyveromyces lactis killer toxin were identified. Among these, eight were earlier identified to have a defect in formation of the mcm(5) side chain. Analysis of the remaining mutants and other known gamma-toxin resistant mutants revealed that sit4, kti14, and KTI5 mutants also have a defect in the formation of mcm(5). A mutant lacking two of the Sit4-associated proteins, Sap185 and Sap190, displays the same modification defect as a sit4-null mutant. Interestingly, several mutants were found to be defective in the synthesis of the 2-thio (s(2)) group of the mcm(5)s(2)U nucleoside. In addition to earlier described mutants, formation of the s(2) group was also abolished in urm1, uba4, and ncs2 mutants and decreased in the yor251c mutant. Like the absence of the mcm(5) side chain, the lack of the s(2) group renders tRNA(mcm5s2UUC Glu) less sensitive to gamma-toxin, reinforcing the importance of the wobble nucleoside mcm(5)s(2)U for tRNA cleavage by gamma-toxin.
  •  
39.
  • Huang, Bo, et al. (author)
  • An early step in wobble uridine tRNA modification requires the Elongator complex
  • 2005
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 11:4, s. 424-436
  • Journal article (peer-reviewed)abstract
    • Elongator has been reported to be a histone acetyltransferase complex involved in elongation of RNA polymerase II transcription. In Saccharomyces cerevisiae, mutations in any of the six Elongator protein subunit (ELP1–ELP6) genes or the three killer toxin insensitivity (KTI11–KTI13) genes cause similar pleiotropic phenotypes. By analyzing modified nucleosides in individual tRNA species, we show that the ELP1–ELP6 and KTI11–KTI13 genes are all required for an early step in synthesis of 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) groups present on uridines at the wobble position in tRNA. Transfer RNA immunoprecipitation experiments showed that the Elp1 and Elp3 proteins specifically coprecipitate a tRNA susceptible to formation of an mcm5 side chain, indicating a direct role of Elongator in tRNA modification. The presence of mcm5U, ncm5U, or derivatives thereof at the wobble position is required for accurate and efficient translation, suggesting that the phenotypes of elp1–elp6 and kti11–kti13 mutants could be caused by a translational defect. Accordingly, a deletion of any ELP1–ELP6 or KTI11–KTI13 gene prevents an ochre suppressor tRNA that normally contains mcm5U from reading ochre stop codons.
  •  
40.
  • Huntley, RP, et al. (author)
  • Guidelines for the functional annotation of microRNAs using the Gene Ontology
  • 2016
  • In: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 22:5, s. 667-676
  • Journal article (peer-reviewed)abstract
    • MicroRNA regulation of developmental and cellular processes is a relatively new field of study, and the available research data have not been organized to enable its inclusion in pathway and network analysis tools. The association of gene products with terms from the Gene Ontology is an effective method to analyze functional data, but until recently there has been no substantial effort dedicated to applying Gene Ontology terms to microRNAs. Consequently, when performing functional analysis of microRNA data sets, researchers have had to rely instead on the functional annotations associated with the genes encoding microRNA targets. In consultation with experts in the field of microRNA research, we have created comprehensive recommendations for the Gene Ontology curation of microRNAs. This curation manual will enable provision of a high-quality, reliable set of functional annotations for the advancement of microRNA research. Here we describe the key aspects of the work, including development of the Gene Ontology to represent this data, standards for describing the data, and guidelines to support curators making these annotations. The full microRNA curation guidelines are available on the GO Consortium wiki (http://wiki.geneontology.org/index.php/MicroRNA_GO_annotation_manual).
  •  
41.
  • Ieong, Ka-Weng, et al. (author)
  • A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids
  • 2014
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 20:5, s. 632-643
  • Journal article (peer-reviewed)abstract
    • There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNAAla-based body (tRNAAlaB) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNAPheB body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNAAlaB body than from the tRNAPheB body. At ∼1 µM EF-Tu, tRNAAlaB conferred considerably faster incorporation kinetics than tRNAPheB, especially in the case of the bulky bK. In contrast, the swap to the tRNAAlaB body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNAAlaB and tRNAPheB bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.
  •  
42.
  • Imbert, Arthur, et al. (author)
  • FISH-quant v2 : a scalable and modular tool for smFISH image analysis
  • 2022
  • In: RNA. - : COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. - 1355-8382 .- 1469-9001. ; 28:6, s. 786-795
  • Journal article (peer-reviewed)abstract
    • Regulation of RNA abundance and localization is a key step in gene expression control. Single-molecule RNA fluorescence in situ hybridization (smFISH) is a widely used single-cell-single-molecule imaging technique enabling quantitative studies of gene expression and its regulatory mechanisms. Today, these methods are applicable at a large scale, which in turn come with a need for adequate tools for data analysis and exploration. Here, we present FISH-quant v2, a highly modular tool accessible for both experts and non-experts. Our user-friendly package allows the user to segment nuclei and cells, detect isolated RNAs, decompose dense RNA clusters, quantify RNA localization patterns and visualize these results both at the single-cell level and variations within the cell population. This tool was validated and applied on large-scale smFISH image data sets, revealing diverse subcellular RNA localization patterns and a surprisingly high degree of cell-to-cell heterogeneity.
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43.
  • Innocenti, Nicolas, 1986-, et al. (author)
  • Whole-genome mapping of 5′ RNA ends in bacteria by tagged sequencing: a comprehensive view in Enterococcus faecalis
  • 2015
  • In: RNA. - : RNA Society. - 1355-8382 .- 1469-9001.
  • Journal article (peer-reviewed)abstract
    • Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first snapshot of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5' RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites (TSSs) and 352 processing sites (PSSs) in E. faecalis. A blind motif search retrieved canonical features of SigA-and SigN-dependent promoters preceding transcription start sites mapped. We discovered 85 novel putative regulatory RNAs, small-and antisense RNAs, and 72 transcriptional antisense organizations. Presented data constitute a significant insight into bacterial RNA landscapes and a step toward the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner.
  •  
44.
  • Ivanova, Natalia, et al. (author)
  • Structure probing of tmRNA in distinct stages of trans-translation
  • 2007
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 13:5, s. 713-722
  • Journal article (peer-reviewed)abstract
    • Ribosomes stalled on problematic mRNAs in bacterial cells can be rescued by transfer-messenger RNA (tmRNA), its helperprotein (small protein B, SmpB), and elongation factor Tu (EF-Tu) through a mechanism called trans-translation. In this work weused lead(II) footprinting to probe the interactions of tmRNA with SmpB and other components of the translation machinery atdifferent steps of the trans-translation cycle. Ribosomes with a short nascent peptide stalled on a truncated mRNA were reactedwith Ala-tmRNA EF-Tu GTP, SmpB, and other translation components to initiate and execute trans-translation. Free tmRNA was                  d      dprobed with lead(II) acetate with and without SmpB, and ribosome bound tmRNA was probed in one of four different trans-translation states stabilized by antibiotic addition or selective exclusion of translation components. For comparison, we alsoanalyzed lead(II) cleavage patterns of tmRNA in vivo in a wild-type as well as in an SmpB-deficient Escherichia coli strain. Weobserved some specific cleavages/protections in tmRNA for the individual steps of trans-translation, but the overall tmRNAconformation appeared to be similar in the stages analyzed. Our findings suggest that, in vivo, a dominant fraction of tmRNA isin complex with SmpB and that, in vitro, SmpB remains tmRNA bound at the initial steps of trans-translation.
  •  
45.
  • Johansson, Marcus J O, et al. (author)
  • Dual function of the tRNA(m(5)U54)methyltransferase in tRNA maturation
  • 2002
  • In: RNA. - 1355-8382 .- 1469-9001. ; 8:3, s. 324-335
  • Journal article (peer-reviewed)abstract
    • A 5-methyluridine (m(5)U) residue at position 54 is a conserved feature of bacterial and eukaryotic tRNAs. The methylation of U54 is catalyzed by the tRNA(m5U54)methyltransferase, which in Saccharomyces cerevisiae is encoded by the nonessential TRM2 gene. In this study, we identified four different strains with mutant forms of tRNA(Ser)CGA. The absence of the TRM2 gene in these strains decreased the stability of tRNA(Ser)CGA and induced lethality. Two alleles of TRM2 encoding catalytically inactive tRNA(m5U54)methyltransferases were able to stabilize tRNA(Ser)CGA in one of the mutants, revealing a role for the Trm2 protein per se in tRNA maturation. Other tRNA modification enzymes interacting with tRNA(Ser)CGA in the maturation process, such as Pus4p, Trm1 p, and Trm3p were essential or important for growth of the tRNA(Ser)CGA mutants. Moreover, Lhp1p, a protein binding RNA polymerase III transcripts, was required to stabilize the mutant tRNAs. Based on our results, we suggest that tRNA modification enzymes might have a role in tRNA maturation not necessarily linked to their known catalytic activity.
  •  
46.
  • Kamel, Wael, 1986-, et al. (author)
  • An Ago2-associated capped transcriptional start site small RNA suppresses adenovirus DNA replication
  • 2017
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 23:11, s. 1700-1711
  • Journal article (peer-reviewed)abstract
    • Here we show that the adenovirus major late promoter produces a 31-nucleotide transcriptional start site small RNA (MLP-TSS-sRNA) that retains the 7-methylguanosine (m7G)-cap and is incorporated onto Ago2-containing RNA-induced silencing complexes (RISC) in human adenovirus-37 infected cells. RNA polymerase II CLIP (UV-cross linking immunoprecipitation) experiments suggest that the MLP-TSS-sRNA is produced by promoter proximal stalling/termination of RNA polymerase II transcription at the site of the small RNA 3' end. The MLP-TSS-sRNA is highly stable in cells and functionally active, down-regulating complementary targets in a sequence and dose-dependent manner. The MLP-TSS-sRNA is transcribed from the opposite strand to the adenoviral DNA polymerase and preterminal protein mRNAs, two essential viral replication proteins. We show that the MLP-TSS-sRNA act in trans to reduce DNA polymerase and preterminal protein mRNA expression. As a consequence of this, the MLP-TSS-sRNA has an inhibitory effect on the efficiency of viral DNA replication. Collectively, our results suggest that this novel sRNA may serve a regulatory function controlling viral genome replication during a lytic and/or persistent adenovirus infection in its natural host.
  •  
47.
  • Kang, Wenjing, et al. (author)
  • Survey of 800+data sets from human tissue and body fluid reveals xenomiRs are likely artifacts
  • 2017
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 23:4, s. 433-445
  • Journal article (peer-reviewed)abstract
    • miRNAs are small 22-nucleotide RNAs that can post-transcriptionally regulate gene expression. It has been proposed that dietary plant miRNAs can enter the human bloodstream and regulate host transcripts; however, these findings have been widely disputed. We here conduct the first comprehensive meta-study in the field, surveying the presence and abundances of cross-species miRNAs (xenomiRs) in 824 sequencing data sets from various human tissues and body fluids. We find that xenomiRs are commonly present in tissues (17%) and body fluids (69%); however, the abundances are low, comprising 0.001% of host human miRNA counts. Further, we do not detect a significant enrichment of xenomiRs in sequencing data originating from tissues and body fluids that are exposed to dietary intake (such as liver). Likewise, there is no significant depletion of xenomiRs in tissues and body fluids that are relatively separated from the main bloodstream (such as brain and cerebro-spinal fluids). Interestingly, the majority (81%) of body fluid xenomiRs stem from rodents, which are a rare human dietary contribution but common laboratory animals. Body fluid samples from the same studies tend to group together when clustered by xenomiR compositions, suggesting technical batch effects. Last, we performed carefully designed and controlled animal feeding studies, in which we detected no transfer of plant miRNAs into rat blood, or bovine milk sequences into piglet blood. In summary, our comprehensive computational and experimental results indicate that xenomiRs originate from technical artifacts rather than dietary intake.
  •  
48.
  • Karlsson, H, et al. (author)
  • A robust and versatile method for production and purification of large-scale RNA samples for structural biology
  • 2020
  • In: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 26:8, s. 1023-1037
  • Journal article (peer-reviewed)abstract
    • Recent findings in genome-wide transcriptomics revealed that RNAs are involved in almost every biological process, across all domains of life. The characterization of native RNAs of unknown function and structure is particularly challenging due to their typical low abundance in the cell and the inherent sensitivity toward ubiquitous RNA degrading enzymes. Therefore, robust in vitro synthesis and extensive work-up methods are often needed to obtain samples amenable for biochemical, biophysical, and structural studies. Here, we present a protocol that combines the most recent advances in T7 in vitro transcription methodology with reverse phase ion pairing and ion exchange HPLC purification of RNAs for the production of yield-optimized large-scale samples. The method is easy to follow, robust and suitable for users with little or no experience within the field of biochemistry or chromatography. The complete execution of this method, for example, for production of isotopically labeled NMR samples, can be performed in less than a week.
  •  
49.
  • Kirsebom, Leif A. (author)
  • Sidney Altman : A brief memoir
  • 2022
  • In: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 28:11, s. 1404-1405
  • Journal article (pop. science, debate, etc.)
  •  
50.
  • Klaue, Y, et al. (author)
  • Biochemical analysis and scanning force microscopy reveal productive and non-productive ADAR2 binding to RNA substrates
  • 2003
  • In: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 9:7, s. 839-846
  • Journal article (peer-reviewed)abstract
    • Scanning force microscopy (SFM) can be used to image biomolecules at high resolution. Here we demonstrate that single-molecule analysis by SFM complements biochemical data on RNA protein binding and can provide information that cannot be obtained by the usual biochemical methods. We have used this method to study the interaction between the RNA editing enzyme ADAR2 and RNA transcripts containing selective and nonselective editing sites. The natural selectively edited R/G site from glutamate receptor subunit B (GluR-B) was inserted into an RNA backbone molecule consisting of a completely double-stranded (ds) central part and incompletely paired ends derived from potato spindle tuber viroid (PSTVd). This molecule was efficiently edited at the R/G site, but promiscuous editing occurred at nonselective sites in the completely double-stranded region. The construct was also used to analyze binding of ADAR2 to wild-type and modified R/G editing sites in relation to binding at other nonselectively edited sites. Editing analysis together with SFM allow us to differentiate between binding and enzymatic activity. ADAR2 has                     been reported to have a general affinity to dsRNA. However, we show that there is a prominent bias for stable binding at sites selectively edited over other edited sites. On the other hand, promiscuous editing at nonselective sites apparently results from transient binding of the enzyme to the substrate. Furthermore, we find distinct sites with nonproductive binding of the enzyme.
  •  
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