Importance of a 5' stem-loop for longevity of papA mRNA in Escherichia coli.
High-level expression of the major pilus subunit (PapA) of uropathogenic strains of Escherichia coli results in part from the unusually long lifetime of the mRNA that encodes this protein. Here we report that the longevity of papA mRNA derives in large measure from the protection afforded by its 5' untranslated region. This papA RNA segment can prolong the lifetime of an otherwise short-lived mRNA to which it is fused. In vivo alkylation studies indicate that, in its natural milieu, the papA message begins with a stem-loop structure. This stem-loop is important for the stabilizing effect of the papA 5' untranslated region, as evidenced by the significant acceleration in papA mRNA decay that results from its removal. (+info
Neuronal BC1 RNA structure: evolutionary conversion of a tRNA(Ala) domain into an extended stem-loop structure.
By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5' domain of BC1 RNA, derived from tRNA(Ala), does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNA(Ala) domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3' end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA. (+info
Identification of the mass-silent post-transcriptionally modified nucleoside pseudouridine in RNA by matrix-assisted laser desorption/ionization mass spectrometry.
A new method using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry for the direct analysis of the mass-silent post-transcriptionally modified nucleoside pseudouridine in nucleic acids has been developed. This method utilizes 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide to derivatize pseudouridine residues. After chemical derivatization all pseudouridine residues will contain a 252 Da 'mass tag' that allows the presence of pseudouridine to be identified using mass spectrometry. Pseudouridine residues can be identified in intact nucleic acids by obtaining a mass spectrum of the nucleic acid before and after derivatization. The mass difference (in units of 252 Da) will denote the number of pseudouridine residues present. To determine the sequence location of pseudouridine, a combination of enzymatic hydrolysis and mass spectrometric steps are used. Here, MALDI analysis of RNase T1 digestion products before and after modification are used to narrow the sequence location of pseudouridine to specific T1 fragments in the gene sequence. Further mass spectrometric monitoring of exonuclease digestion products from isolated T1 fragments is then used for exact sequence placement. This approach to pseudouridine identification is demonstrated using Escherichia coli tRNAS: This new method allows for the direct determination of pseudouridine in nucleic acids, can be used to identify modified pseudouridine residues and can be used with general modification mapping approaches to completely characterize the post-transcriptional modifications present in RNAs. (+info
Possible interaction sites of mRNA, tRNA, translation factors and the nascent peptide in 5S, 5.8S and 28S rRNA in in vivo assembled eukaryotic ribosomal complexes.
We have investigated possible interaction sites for mRNA, tRNA, translation factors and the nascent peptide on 5S, 5.8S and 28S rRNA in in vivo assembled translational active mouse ribosomes by comparing the chemical footprinting patterns derived from native polysomes, salt-washed polysomes (mainly lacking translational factors) and salt-washed runoff ribosomes (lacking mRNA, tRNA and translational factors). Several ligand-induced footprints were observed in 28S rRNA while no reactivity changes were seen in 5S and 5.8S rRNA. Footprints derived from mRNA, tRNA and/or the nascent peptide chain were observed in domain I of 28S rRNA (hairpin 23), in domain II (helix 37/38 and helices 42 and 43 and in the eukaryotic expansion segment 15), in domain IV (helices 67 and 74) and in domain V (helices 94 and 96 and in the peptidyl transferase ring). Some of the protected sites were homologous to sites previously suggested to be involved in mRNA, tRNA and/or peptide binding in in vitro assembled prokaryotic complexes. Additional footprints were located in regions that have not previously been found involved in ligand binding. Part of these sites could derive from the nascent peptide in the exit channel of the ribosome. (+info
The brome mosaic virus RNA3 intergenic replication enhancer folds to mimic a tRNA TpsiC-stem loop and is modified in vivo.
The genome of brome mosaic virus (BMV), a positive-strand RNA virus in the alphavirus-like superfamily, consists of three capped, messenger-sense RNAs. RNA1 and RNA2 encode viral replication proteins 1a and 2a, respectively. RNA3 encodes the 3a movement protein and the coat protein, which are essential for systemic infection in plants but dispensable for RNA3 replication in plants and yeast. A subset of the 250-base intergenic region (IGR), the replication enhancer (RE), contains all cis-acting signals necessary for a crucial, early template selection step, the 1a-dependent recruitment of RNA3 into replication. One of these signals is a motif matching the conserved box B sequence of RNA polymerase III transcripts. Using chemical modification with CMCT, kethoxal, DMS, DEPC, and lead, we probed the structure of the IGR in short, defined transcripts and in full-length RNA3 in vitro, in yeast extracts, and in whole yeast cells. Our results reveal a stable, unbranched secondary structure that is not dependent on the surrounding ORF sequences or on host factors within the cell. Functional 5' and 3' deletions that defined the minimal RE in earlier deletion studies map to the end of a common helical segment. The box B motif is presented as a hairpin loop of 7 nt closed by G:C base pairs in perfect analogy to the TpsiC-stem loop in tRNA(Asp). An adjacent U-rich internal loop, a short helix, and another pyrimidine-rich loop were significantly protected from base modifications. This same arrangement is conserved between BMV and cucumoviruses CMV, TAV, and PSV. In the BMV box B loop sequence, uridines corresponding to tRNA positions T54 and psi55 were found to be modified in yeast and plants to 5mU and pseudouridine. Together with the aminoacylated viral 3'-end, this is thus the second RNA replication signal within BMV where the virus has evolved a tRNA structural mimicry to a degree that renders it a substrate for classical tRNA modification reactions in vivo. (+info
Structural insights into group II intron catalysis and branch-site selection.
Group II self-splicing introns catalyze autoexcision from precursor RNA transcripts by a mechanism strikingly similar to that of the spliceosome, an RNA-protein assembly responsible for splicing together the protein-coding parts of most eukaryotic pre-mRNAs. Splicing in both cases initiates via nucleophilic attack at the 5' splice site by the 2' OH of a conserved intron adenosine residue, creating a branched (lariat) intermediate. Here, we describe the crystal structure at 3.0 A resolution of a 70-nucleotide RNA containing the catalytically essential domains 5 and 6 of the yeast ai5gamma group II self-splicing intron, revealing an unexpected two-nucleotide bulged structure around the branch-point adenosine in domain 6. (+info
Structure of the 5' nontranslated region of the coxsackievirus b3 genome: Chemical modification and comparative sequence analysis.
Coxsackievirus B3 (CVB3) is a picornavirus which causes myocarditis and pancreatitis and may play a role in type I diabetes. The viral genome is a single 7,400-nucleotide polyadenylated RNA encoding 11 proteins in a single open reading frame. The 5' end of the viral genome contains a highly structured nontranslated region (5'NTR) which folds to form an internal ribosome entry site (IRES) as well as structures responsible for genome replication, both of which are critical for virulence. A structural model of the CVB3 5'NTR, generated primarily by comparative sequence analysis and energy minimization, shows seven domains (I to VII). While this model provides a preliminary basis for structural analysis, the model lacks comprehensive experimental validation. Here we provide experimental evidence from chemical modification analysis to determine the structure of the CVB3 5'NTR. Chemical probing results show that the theoretical model for the CVB3 5'NTR is largely, but not completely, supported experimentally. In combination with our chemical probing data, we have used the RNASTRUCTURE algorithm and sequence comparison of 105 enterovirus sequences to provide evidence for novel secondary and tertiary interactions. A comprehensive examination of secondary structure is discussed, along with new evidence for tertiary interactions. These include a loop E motif in domain III and a long-range pairing interaction that links domain II to domain V. The results of our work provide mechanistic insight into key functional elements in the cloverleaf and IRES, thereby establishing a base of structural information from which to interpret experiments with CVB3 and other picornaviruses. (+info
Probing RNA structure with chemical reagents and enzymes.