| 1. |
- Alalam, Hanna, et al.
(författare)
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Global SLAM-Seq for accurate mRNA decay determination and identification of NMD targets.
- 2022
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 28:7
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Tidskriftsartikel (refereegranskat)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.
(författare)
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A possible tertiary rRNA interaction between expansion segments ES3 and ES6 in eukaryotic 40S ribosomal subunits
- 2003
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 9:1, s. 20-24
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Tidskriftsartikel (refereegranskat)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.
(författare)
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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
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 10:3, s. 403-411
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Loop-loop interaction in an adenine-sensing riboswitch: a molecular dynamics study
- 2013
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Ingår i: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 19:7, s. 916-926
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Nucleotide modifications and tRNA anticodon-mRNA codon interactions on the ribosome
- 2011
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Ingår i: RNA (New York, N.Y.). - : Cold Spring Harbor Laboratory. - 1469-9001 .- 1355-8382. ; 17:12, s. 2177-2188
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Tidskriftsartikel (refereegranskat)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.
(författare)
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The bacterial toxin ReIE induces specific mRNA cleavage in the A site of the eukaryote ribosome
- 2008
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Ingår i: RNA. - : RNA Society. - 1355-8382 .- 1469-9001. ; 14:2, s. 233-239
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Tidskriftsartikel (refereegranskat)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.
(författare)
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MicroRNAs in Amoebozoa : Deep sequencing of the small RNA population in the social amoeba Dictyostelium discoideum reveals developmentally regulated microRNAs
- 2012
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 18:10, s. 1771-1782
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Tidskriftsartikel (refereegranskat)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.
(författare)
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An S6 : S18 complex inhibits translation of E. coli rpsF.
- 2015
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 21:12, s. 2039-46
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Tidskriftsartikel (refereegranskat)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.
(författare)
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Fitness advantages conferred by the L20-interacting RNA cis-regulator of ribosomal protein synthesis in Bacillus subtilis
- 2018
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Ingår i: RNA. - : Cold Spring Harbor Laboratory. - 1355-8382 .- 1469-9001. ; 24:9, s. 1133-1143
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Tidskriftsartikel (refereegranskat)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.
(författare)
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RNA:RNA interaction in ternary complexes resolved by chemical probing
- 2022
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Ingår i: RNA. - : Cold Spring Harbor Laboratory Press (CSHL). - 1355-8382 .- 1469-9001. ; 29:3, s. 317-329
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Tidskriftsartikel (refereegranskat)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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