Compositional turnover and variation in Eemian pollen sequences in Europe

Vegetation History and Archaeobotany - The Eemian interglacial represents a natural experiment on how past vegetation with negligible human impact responded to amplified temperature changes...     

RNA Modified Uridines VI: Conformations of 3-[3-(S)-Amino-3-Carboxypropyl]Uridine (acp3U) from tRNA and 1-Methyl-3-[3-(S)-Amino-3-Carboxypropyl]Pseudouridine (m1acp3Ψ) from rRNA

Abstract

The conformation of chemically synthesized acp³U is 60& 3′-endo, gauche⁺, whereas that of m¹acp³Ψ is 60& 2′-endo, gauche⁺. We conclude that the difference in conformation probably imparts important local structures to their respective tRNA and rRNA.     

Specificity of Phage Display Selected Peptides for Modified Anticodon Stem and Loop Domains of tRNA

Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNA^Lys3 having 2-thiouridine (s²U₃₄) and pseudouridine (Ψ₃₉), bound the modified human ASL^Lys3(s²U₃₄;Ψ₃₉) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASL^Lys3(s²U₃₄;Ψ₃₉), followed by the hypermodified ASL^Lys3 (mcm⁵s²U₃₄;ms²t⁶A₃₇) and the unmodified ASL^Lys3, but bound poorly to singly modified ASL^Lys3 constructs (Ψ₃₉, ms²t⁶A_37, s²U₃₄), ASL^Lys1,2 (t⁶A₃₇) and Escherichia coli ASL^Glu (s²U₃₄). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides.     

Modified nucleotides in tRNA(Lys) and tRNA(Val) are important for translocation

Ribosomes translate genetic information encoded by messenger RNAs (mRNAs) into proteins. Accurate decoding by the ribosome depends on the proper interaction between the mRNA codon and the anticodon of transfer RNA (tRNA). tRNAs from all kingdoms of life are enzymatically modified at distinct sites, particularly in and near the anticodon. Yet, the role of these naturally occurring tRNA modifications in translation is not fully understood. Here we show that modified nucleosides at the first, or wobble, position of the anticodon and 3'-adjacent to the anticodon are important for translocation of tRNA from the ribosome's aminoacyl site (A site) to the peptidyl site (P site). Thus, naturally occurring modifications in tRNA contribute functional groups and conformational dynamics that are critical for accurate decoding of mRNA and for translocation to the P site during protein synthesis.     

Antibody-bound amyloid precursor protein upregulates ornithine decarboxylase expression

Alzheimer's disease is a neurodegenerative disorder characterised by extracellular accumulation of the Abeta peptide, derived from the amyloid precursor protein (APP). The function of APP as a cell surface receptor was examined by ligand-mimicking using an antibody against the APP extracellular domain. Alterations in gene expression evoked by antibody-bound APP were analysed using human pathway-finder gene arrays and the largest change in expression levels was found for ornithine decarboxylase (ODC). These results were confirmed by Western blotting which showed even higher upregulation on the protein level. APP knockdown by RNAi verified that upregulation of ODC was APP-mediated. This APP signalling event did not require gamma-secretase cleavage, as it was independent of the presence of presenilin-1 or -2. The induced ODC expression was rapid and biphasic, resembling growth-factor stimulated signalling events. This study shows that antibody-bound APP leads to altered gene expression that may be relevant to AD.     

A distinctive RNA fold: the solution structure of an analogue of the yeast tRNAPhe T Psi C domain.

The structure of an analogue of the yeast tRNAPhe T Psi C stem-loop has been determined by NMR spectroscopy and restrained molecular dynamics. The molecule contained the highly conserved modification ribothymidine at its naturally occurring position. The ribothymidine-modified T Psi C stem-loop is the product of the m5U54-tRNA methyltransferase, but is not a substrate for the m1A58-tRNA methyltransferase. Site-specific substitutions and 15N labels were used to confirm the assignment of NOESY cross-peaks critical in defining the global fold of the molecule. The structure is unusual in that the loop folds far over into the major groove of the curved stem. This conformation is stabilized by both stacking interactions and hydrogen bond formation. Furthermore, this conformation appears to be unique among RNA hairpins of similar size. There is, however, a considerable resemblance to the analogous domain in the crystal structure of the full-length yeast tRNAPhe. We believe, therefore, that the structure we have determined may represent an intermediate in the folding pathway during the maturation of tRNA.     

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNALeu(UUR) and tRNALys

5-Taurinomethyluridine (τm⁵U) and 5-taurinomethyl-2-thiouridine (τm⁵s²U) are located at the wobble position of human mitochondrial (hmt) tRNA^Leu(UUR) and tRNA^Lys, respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm⁵U and τm⁵s²U into 17-mers related to the sequence of the anticodon arms hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys, respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF₃) are compatible with the blockage of the canonical monomeric units. The synthesis of τm⁵s²U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.     

mt-tRNA Components: Synthesis of (2-Thio)Uridines Modified with Blocked Glycine/Taurine Moieties at C-5,1

In this paper, we discuss the usefulness of reductive amination of 5-formyl-2′,3′-O-isopropylidene(-2-thio)uridine with glycine or taurine esters in the presence of sodium triacetoxyborohydride (NaBH(OAc)₃) for the synthesis of the native mitochondrial (mt) tRNA components 5-carboxymethylaminomethyl(-2-thio)uridine (cmnm⁵(s²)U) and 5-taurinomethyl(-2-thio)uridine (τm⁵(s²)U) with a blocked amino acid function. 2-(Trimethylsilyl)ethyl and 2-(p-nitrophenyl)ethyl esters of glycine and 2-(2,4,5-trifluorophenyl)ethyl ester of taurine were selected as protection of carboxylic and sulfonic acid residues, respectively. The first synthesis of 5-formyl-2′,3′-O-isopropylidene-2-thiouridine is also reported.     

Presence and coding properties of 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um) in the wobble position of the anticodon of tRNA(Leu) (U*AA) from brewer's yeast.

The unknown modified nucleoside U* has been isolated by enzymatic and HPLC protocols from tRNA(Leu) (U*AA) recently discovered in brewer's yeast. The pure U* nucleoside has been characterized by electron impact mass spectroscopy, and comparison of its chromatographic and UV-absorption properties with those of appropriate synthetic compounds. The structure of U* was established as 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um). The yeast tRNA(Leu) (U*AA) is the only tRNA so far sequenced which has been shown to contain ncm5Um. The location of such a modified uridine at the first position of the anticodon restricts the decoding property to A of the leucine UUA codon.