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PubMed1995

An APL-programmed genetic algorithm for the prediction of RNA secondary structure.

The possibilities of using a genetic algorithm for the prediction of RNA secondary structure were investigated. The algorithm, using the procedure of stepwise selection of the most fit structures (similarly to natural evolution), allows different models of fitness or driving forces determining RNA structure to be easily introduced. This can be used for simulation of the RNA folding process and for the investigation of possible folding pathways. Such an algorithm needs several modifications before it can predict RNA secondary structures. After modification, a fair number of correct stems are predicted, even when using computationally quick, but very crude, fitness criteria such as stem length and stacking energy, including elements of tertiary structure (pseudoknots). The fact that genetic algorithm simulation includes both stem formations and stem disruption allows one to observe intermediate structures that may be used in combination with phylogenetic or experimental research.

Algorithms↗

PubMed1999

Molecular diversity of K+ channels.

K+ channel principal subunits are by far the largest and most diverse of the ion channels. This diversity originates partly from the large number of genes coding for K+ channel principal subunits, but also from other processes such as alternative splicing, generating multiple mRNA transcripts from a single gene, heteromeric assembly of different principal subunits, as well as possible RNA editing and posttranslational modifications. In this chapter, we attempt to give an overview (mostly in tabular format) of the different genes coding for K+ channel principal and accessory subunits and their genealogical relationships. We discuss the possible correlation of different principal subunits with native K+ channels, the biophysical and pharmacological properties of channels formed when principal subunits are expressed in heterologous expression systems, and their patterns of tissue expression. In addition, we devote a section to describing how diversity of K+ channels can be conferred by heteromultimer formation, accessory subunits, alternative splicing, RNA editing and posttranslational modifications. We trust that this collection of facts will be of use to those attempting to compare the properties of new subunits to the properties of others already known or to those interested in a comparison between native channels and cloned candidates.

Alternative Splicing↗

PubMedDate not supplied

[Regulatory region of the MS2 phage RNA replicase cistron. Accesibility of MS2 RNA specific fragment to T1 RNAse digestion and chemical modification with kethoxal].

Partial digestion with T1 RNAase and chemical modification with kethoxal were used to study stability of two hairpin in the proposed secondary structure of the functionally active MS2 RNA fragment MS2 R(--53 leads to 6), containing the regulatory region of the phage replicase cistron. Analysis of the products obtained after the above treatments showed that T1 RNAase and kethoxal attacked predominantly the guanosine residues in the hairpin b of the MS2 R(--53 leads to 6). This implies that in contrast to the structurally stable hairpin a of the polynucleotide, hairpin b appears to be more labile and may exist under the present experimental conditions in equilibrium with its open form. The data of the competition experiments demonstrated that the kethoxal modified MS2 R(-53 leads to 6) and shorter polynucleotide MS2 R(-53 leads to-11) obtained from MS2 R(-53 leads to 6) after T1 RNAase digestion failed to bind with MS2 coat protein. The relatively unstable hairpin b region in the polynucleotide MS2 R(-53 leads to 6) is suggested to play essential role in the complex formation.

Aldehydes↗

PubMed1995

Posttranscriptional modification of the central loop of domain V in Escherichia coli 23 S ribosomal RNA.

Knowledge of the sites, structures, and functional roles of posttranscriptional modification in rRNAs is limited, despite steadily accumulating evidence that rRNA plays a direct role in the peptidyl transferase reaction and that modified nucleotides are concentrated at the functional center of the ribosome. Using methods based on mass spectrometry, modifications have been mapped in Escherichia coli 23 S rRNA in the central loop of domain V, a region of established interaction between 23 S RNA and tRNA. Two segments of RNA were isolated following protection with oligodeoxynucleotides and nuclease digestion: residues 2423-2473 (51-mer) and 2481-2519 (39-mer). Dihydrouridine was located at position 2449, within the RNase T1 hydrolysis product 2448-ADAACAGp-2454, as evidenced by a molecular mass 2 daltons higher than the gene sequence-predicted mass. This nucleoside, which is nearly ubiquitous in tRNA (where it is involved in maintenance of loop structure), is two bases from A-2551, a previously determined site of interaction between 23 S RNA and the CCA-aminoacyl terminus of tRNA at the ribosomal P-site. The oligonucleotide 2496-CACmCUCGp-2502 was isolated and accurately mass measured, and its nucleoside constituents were characterized by high performance liquid chromatography-mass spectrometry; there was no evidence of modification at position 2501 as implied by earlier work. Using similar techniques, the modified adenosine at position 2503 was unambiguously determined to be 2-methyladenosine in the fragment 2503-m2A psi Gp-2505.

Base Composition↗

PubMed1985

Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing.

N6-methyladenosine (m6A) residues are present as internal base modifications in most higher eucaryotic mRNAs; however, the biological function of this modification is not known. We describe a method for localizing and quantitating m6A within a large RNA molecule, the genomic RNA of Rous sarcoma virus. Specific fragments of 32P-labeled Rous sarcoma virus RNA were isolated by hybridization with complementary DNA restriction fragments spanning nucleotides 6185 to 8050. RNA was digested with RNase and finger-printed, and individual oligonucleotides were analyzed for the presence of m6A by paper electrophoresis and thin-layer chromatography. With this technique, seven sites of methylation in this region of the Rous sarcoma virus genome were localized at nucleotides 6394, 6447, 6507, 6718, 7414, 7424, and 8014. Further, m6A was observed at two additional sites whose nucleotide assignments remain ambiguous. A clustering of two or more m6A residues was seen at three positions within the RNA analyzed. Modification at certain sites was found to be heterogeneous, in that different molecules of RNA appeared to be methylated differently. Previous studies have determined that methylation occurs only in the sequences Gm6AC and Am6AC. We observed a high frequency of methylation at PuGm6ACU sequences. The possible involvement of m6A in RNA splicing events is discussed.

Adenosine↗

PubMed2002

The expanding snoRNA world.

In eukaryotes, the site-specific formation of the two prevalent types of rRNA modified nucleotides, 2'-O-methylated nucleotides and pseudouridines, is directed by two large families of snoRNAs. These are termed box C/D and H/ACA snoRNAs, respectively, and exert their function through the formation of a canonical guide RNA duplex at the modification site. In each family, one snoRNA acts as a guide for one, or at most two modifications, through a single, or a pair of appropriate antisense elements. The two guide families now appear much larger than anticipated and their role not restricted to ribosome synthesis only. This is reflected by the recent detection of guides that can target other cellular RNAs, including snRNAs, tRNAs and possibly even mRNAs, and by the identification of scores of tissue-specific specimens in mammals. Recent characterization of homologs of eukaryotic modification guide snoRNAs in Archaea reveals the ancient origin of these non-coding RNA families and offers new perspectives as to their range of function.

Animals↗

PubMed1998

Recent advances in mammalian RNA editing.

RNA editing is a posttranscriptional modification that results in the generation of nucleotides within an RNA transcript that do not match the bases present within the genome. Mammalian RNA editing events, often represented by cytidine-to-uridine and adenosine-to-inosine conversions, are predominantly mediated by base deamination. In the past decade, important advances have been made in the understanding of editing mechanisms, the identification of RNA sequences and structures necessary for editing regulation, and the cloning and characterization of editing enzymes. It has also recently been appreciated that RNA editing within mammalian substrates can have profound functional consequences in protein function, implicating this posttranscriptional modification as important in the production of molecular diversity.

Adenosine Deaminase↗

PubMed1975

Preparation and characteristics of the structure of methoxyamine-modified f2 RNA.

Treatment of phage f2 RNA with [14-C]methoxyamine under non-denaturing conditions resulted in modification of exposed cytosines only. On methoxyamine treatment in the presence of 6 M-guanidine, all cytosines were modified. Under the conditions applied, no modification of adenine base in RNA chain occurred. The structure of modified f2 RNA preparations was studied by melting and sedimentation analysis. The ratio between the modification products (N-4-methoxycytosine and N-4-methoxy-6-methoxyamino-5,6-dihydrocytosine) was determined in RNA preparations modified under non-denaturing and denaturing conditions.

Binding Sites↗

PubMed1999

The splicing factor-associated protein, p32, regulates RNA splicing by inhibiting ASF/SF2 RNA binding and phosphorylation.

The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.

Carrier Proteins↗

PubMed2025

Defining the networks that connect RNase III and RNase J-mediated regulation of primary and specialized metabolism in Streptomyces venezuelae.

UNLABELLED: RNA metabolism involves coordinating RNA synthesis with RNA processing and degradation. Ribonucleases play fundamental roles within the cell, contributing to the cleavage, modification, and degradation of RNA molecules, with these actions ensuring appropriate gene regulation and cellular homeostasis. Here, we employed RNA sequencing to explore the impact of RNase III and RNase J on the transcriptome of Streptomyces venezuelae. Differential expression analysis comparing wild-type and RNase mutant strains at distinct developmental stages revealed significant changes in transcript abundance, particularly in pathways related to multicellular development, nutrient acquisition, and specialized metabolism. Both RNase mutants exhibited dysregulation of the BldD regulon, including altered expression of many cyclic-di-GMP-associated enzymes. We also observed precocious chloramphenicol production in these RNase mutants and found that in the RNase III mutant, this was associated with PhoP-mediated regulation. We further found that RNase III directly targeted members of the PhoP regulon, suggesting a link between RNA metabolism and a regulator that bridges primary and specialized metabolism. We connected RNase J function with translation through the observation that RNase J directly targets multiple ribosomal protein transcripts for degradation. These findings establish distinct but complementary roles for RNase III and RNase J in coordinating the gene expression dynamics critical for S. venezuelae development and specialized metabolism. IMPORTANCE: RNA processing and metabolism are mediated by ribonucleases and are fundamental processes in all cells. In the morphologically complex and metabolically sophisticated Streptomyces bacteria, RNase III and RNase J influence both development and metabolism through poorly understood mechanisms. Here, we show that both ribonucleases are required for the proper expression of the BldD developmental pathway and contribute to the control of chloramphenicol production, with an interesting connection to phosphate regulation for RNase III. Additionally, we show that both RNases have the potential to impact translation through distinct mechanisms and can function cooperatively in degrading specific transcripts. This study advances our understanding of RNases in Streptomyces biology by providing insight into distinct contributions made by these enzymes and the intriguing interplay between them.

Streptomyces↗

PubMed2005

Monomers for preparation of amide-linked RNA: asymmetric synthesis of all four nucleoside 5'-azido 3'-carboxylic acids.

[reaction: see text] Recent discovery of RNA interference as an efficient and naturally occurring mechanism of gene regulation has reinvigorated the interest in chemically modified RNA. For potential in-vivo applications small interfering RNAs require chemical modifications to fine-tune the thermal stability and increase the cellular delivery and potency and in vivo half-life of the RNA duplexes. From this perspective, amides as neutral and hydrophobic internucleoside linkages in RNA are highly interesting modifications for RNA interference. Amides are remarkably good mimics of the phosphodiester backbone of RNA and can be prepared using a relatively straightforward peptide coupling chemistry. However, the progress in the field has been hampered by the shortage of efficient methods to synthesize the monomeric building blocks for such couplings, the nucleoside amino acid equivalents. Herein, we report enantioselective synthesis of 5'-azido 3'-carboxylic acid derivatives of all four natural ribonucleosides. The key transformations in our synthesis are a double asymmetric ene reaction and a stereoselective iodolactonization that form the basic carbon skeleton of the modified ribose. Standard nucleoside synthesis is followed by a short and highly efficient protecting group manipulation to give the enantiomerically pure (>98%) title compounds in 9-10 steps and 15-19% overall yields starting from small achiral molecules. The present results are a significant improvement over our first-generation racemic synthesis and compare favorably with the previously reported synthesis from nucleoside and carbohydrate precursors.

Amides↗

PubMed1983