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PubMed1995

Structurally complex and highly active RNA ligases derived from random RNA sequences.

Seven families of RNA ligases, previously isolated from random RNA sequences, fall into three classes on the basis of secondary structure and regiospecificity of ligation. Two of the three classes of ribozymes have been engineered to act as true enzymes, catalyzing the multiple-turnover transformation of substrates into products. The most complex of these ribozymes has a minimal catalytic domain of 93 nucleotides. An optimized version of this ribozyme has a kcat exceeding one per second, a value far greater than that of most natural RNA catalysts and approaching that of comparable protein enzymes. The fact that such a large and complex ligase emerged from a very limited sampling of sequence space implies the existence of a large number of distinct RNA structures of equivalent complexity and activity.

Base Sequence↗
PubMed1981

Frequency and diversity of RNA sequences in different cell types of the fungus Schizophyllum commune.

In the basidiomycete Schizophyllum commune an RNA complexity of 13,500 average-sized (1100 nucleotides) RNA sequences was found by means of RNA excess hybridization to complementary DNA. 600 abundant poly(A)-containing RNA sequences were present in about 100 copies per cell, whereas the rare 13,000 poly(A)-containing RNA sequences were present in about 5 copies per cell on average. Homologous and heterologous cDNA hybridizations to poly(A)-rich RNAs revealed some quantitative, but no significant qualitative differences between the monokaryotic and the dikaryotic cell type. Heterologous hybridization and subsequent hybridizations of remaining single-stranded cDNA to poly(A)-rich RNAs from both cell types showed qualitative differences amounting to 2-3% of the rare sequences in the two cell types. However, from the results of similar hybridizations with total RNAs, i.e. including non-polyadenylated RNA, it could be calculated that the RNA sequences in monokaryons and dikaryons are at least 99% the same. Identity of RNA sequences in monokaryons and dikaryons was also inferred by comparison of the patterns of polypeptides synthesized by translation of total RNA in cell-free systems.

Agaricales↗
PubMed2026

Characterization of METTL3/14-mediated m6A modification in human transcriptome using Nanopore direct RNA sequencing.

Post-transcriptional RNA modifications modulate diverse aspects of RNA metabolism. N6-methyladenosine (m6A), one of the most abundant internal RNA modifications, is deposited by the core methyltransferase complex, METTL3 and METTL14. Oxford Nanopore Technologies (ONT) platform permits direct, single RNA molecule sequencing while preserving native modifications. However, without rigorous benchmarking, the accuracy and reproducibility of modification detection remain uncertain. Here, we leveraged ONT to comprehensively profile bona fide m6A modifications in cellular RNAs at single-nucleotide resolution by integrating two direct RNA sequencing chemistries (RNA002 and RNA004) with the m6Anet and Dorado modification-detection models. We independently depleted METTL3 and METTL14 in human cells and rigorously validated modification calls through several assays and independent orthogonal methods (GLORI and miCLIP). We find that Dorado detected a higher number of m6A events and enabled simultaneous detection of other RNA modifications (5-methylcytosine, pseudouridine, and inosine). Pairing Dorado with an in vitro transcribed, unmodified control under stringent filtering, we provide compelling evidence supporting a global reduction in m6A sites and stoichiometry within coding sequences and across genes, particularly in highly modified genes and sites, and at consensus DRACH motifs. We report a differential and complex regulation of modified transcripts, accompanied by a global reduction in poly(A) tail length. Notably, METTL3 and METTL14 depletion produced distinct transcript-specific effects, supporting non-redundant roles within the m6A writer complex. Together, our study illustrates a notable advancement of ONT capabilities and establishes a robust transcriptome-wide framework for RNA modification detection, thereby laying the groundwork for exploring the contribution of METTL3/METTL14 to cellular functions and disease.

Humans↗
PubMed2006

Consensus folding of unaligned RNA sequences revisited.

As one of the earliest problems in computational biology, RNA secondary structure prediction (sometimes referred to as "RNA folding") problem has attracted attention again, thanks to the recent discoveries of many novel non-coding RNA molecules. The two common approaches to this problem are de novo prediction of RNA secondary structure based on energy minimization and the consensus folding approach (computing the common secondary structure for a set of unaligned RNA sequences). Consensus folding algorithms work well when the correct seed alignment is part of the input to the problem. However, seed alignment itself is a challenging problem for diverged RNA families. In this paper, we propose a novel framework to predict the common secondary structure for unaligned RNA sequences. By matching putative stacks in RNA sequences, we make use of both primary sequence information and thermodynamic stability for prediction at the same time. We show that our method can predict the correct common RNA secondary structures even when we are given only a limited number of unaligned RNA sequences, and it outperforms current algorithms in sensitivity and accuracy.

Algorithms↗
PubMed1993

[Heterogeneity of delta virus RNA sequences in Japan].

Hepatitis delta virus RNA sequences were determined in isolates from two Japanese patients, M and S, by polymerase chain reaction and direct nucleotide sequencing and compared with three isolates from Italy, USA and Taiwan. The sequence obtained for hepatitis delta virus RNA from patient M was 92-96% identical to the sequences obtained for three other strains of hepatitis delta virus, whereas the sequence of hepatitis delta virus RNA obtained from patient S was approximately 80% identical to the other sequenced strains. This suggests that the delta agent in Japan has a heterogeneous origin and the delta virus RNA sequence from Japanese patient S is the most divergent delta virus isolate yet analyzed.

Adult↗
PubMed2025

Enzymes in high-throughput RNA sequencing: Applications and challenges.

High-throughput RNA sequencing provides genome-wide information on the dynamics of RNA in each cell and how the dynamics responds to environmental changes. Next-generation sequencing by the Illumina platform currently provides the highest information output as compared to other platforms. A key component of next generation sequencing of each RNA is the successful end-to-end reverse-transcription into a cDNA strand. This can be highly challenging given the propensity of each RNA to adopt ordered structures and to contain post-transcriptional modifications. While many reverse transcriptase (RT) enzymes have been developed over the years to maximize read-through of an RNA, their processivity and efficiency varies, raising the question of how to select the RT for the experiment at hand. Here, we use tRNA as a model for genome-wide sequencing, as tRNA has a stable secondary and tertiary structure and has a high density and wide variety of post-transcriptional modifications, presenting one of the most challenging problems of sequencing RNA. We compare the efficiency of end-to-end cDNA synthesis of tRNA among several recent RT enzymes and provide a general sequencing workflow that is applicable to most of these enzymes.

High-Throughput Nucleotide Sequencing↗
PubMed1978

The precursor of mouse beta-globin messenger RNA contains two intervening RNA sequences.

We have investigated the locations of the poly(A), the mRNA-specific sequences and the RNA sequences that are eventually cleaved from the 1860 nucleotide precursor of mouse beta-globin mRNA. Biochemical and electron microscopic data demonstrate that there are two intervening RNA moieties in the precursor which separate the beta-globin mRNA sequences into three portions containing 480, 205 and 155 nucleotides. One of the two intervening RNA moieties contains 780 nucleotides. The size of the smaller intervening RNA has not been determined precisely, but it is 125 nucleotides or less. The largest mRNA-specific fragment is derived from the 3' terminus of the precursor, and contains the 3' terminal poly (A) and 330 mRNA-specific transcribed nucleotides. At least one, and probably both, intervening RNAs occur within the coding portion of the mRNA sequences. The larger of the intervening RNAs is located next to the 480 nucleotide mRNA-specific fragment, and the smaller intervening RNA is located between the 205 and 155 nucleotide mRNA-specific fragments. These experiments are consistent with the notion that the intervening sequences in the DNA of mouse beta-globin genes are transcribed into the mRNA precursor and are excised from the RNA by post-transcriptional events.

Animals↗
PubMed1992

Compilation of small RNA sequences.

This is an update containing small RNA sequences published during 1991. Approximately two hundred small RNA sequences are available in this and earlier compilations. The hard copy print out of this set will be available directly from us (inquiries should be addressed to R. Reddy). These files are also available on GenBank computer. Sequences from various sources covered in earlier compilations (see Reddy, R. Nucl. Acids Res. 16:r71; Reddy, R. and Gupta, S. Nucl Acids Res. 1990 Supplement, 18:2231 and 1991 Supplement, 19:2073) are not included in this update but are listed below.

Animals↗
PubMed1979

In vitro DNA dependent synthesis of globin RNA sequences from erythroleukemic cell chromatin.

Murine erythroleukemic cells in culture accumulate cytoplasmic globin mRNA during differentiation induced by dimethyl sulfoxide (DMSO)1. Chromatin was prepared from DMSO induced erythroleukemic cells that were transcribing globin RNA in order to determine whether in vitro synthesis of globin RNA sequences was possible from chromatin. RNA was synthesized in vitro using 5-mercuriuridine triphosphate and exogenous Escheria coli RNA polymerase. Newly synthesized mercurated RNA was purified from endogenous chromatin associated RNA by affinity chromatography on a sepharose sulfhydryl column, and the globin RNA sequence content of the mercurated RNA was assayed by hybridization to cDNA globin. The synthesis of globin RNA sequences was shown to occur and to be sensitive to actinomycin and rifampicin and insensitive to alpha-amanitin. In contrast, synthesis of globin RNA sequence synthesis was not detected in significant amounts from chromatin prepared from uninduced erythroleukemic cells, nor from uninduced cell chromatin to which globin RNA was added prior to transcription. Isolated RNA:cDNA globin hybrids were shown to contain mercurated RNA by affinity chromatography. These results indicated that synthesis of globin RNA sequences from chromatin can be performed by E. coli RNA polymerase.

Cell Line↗
PubMed1979

Direct chemical method for sequencing RNA.

Four different base-specific chemical reactions generate a means of directly sequencing RNA terminally labeled with 32P. After a partial, specific modification of each kind of RNA base, an amine-catalyzed strand scission generates labeled fragments whose lengths determine the position of each nucleotide in the sequence. Dimethyl sulfate modifies guanosine. Diethyl pyrocarbonate attacks primarily adenosine. Hydrazine attacks uridine and cytidine, but salt suppresses the reaction with uridine. In all cases, aniline induces a subsequent strand scission. The electrophoretic fractionation of the labeled fragments on a polyacrylamide gel, followed by autoradiography, determines the RNA sequence. RNA labeled at the 3' end yields clean cleavage patterns for each purine and pyrimidine and allows a determination of the entire RNA sequence out to 100-200 bases from the labeled terminus.

Base Sequence↗
PubMed1994

Compilation of small RNA sequences.

This is an update containing small RNA sequences deposited in GenBank recently. Over four hundred small RNA sequences are available in this and earlier complications.

Animals↗
PubMed2000

In vitro selection of an RNA sequence that interacts with high affinity with thymidylate synthase.

Previous studies have shown that the repressive effect of thymidylate synthase (TS) mRNA translation is mediated by direct binding of TS itself to two cis-acting elements on its cognate mRNA. To identify the optimal RNA nucleotides that interact with TS, we in vitro synthesized a completely degenerate, linear RNA pool of 25 nt and employed in vitro selection to isolate high affinity RNA ligands that bind human TS protein. After 10 rounds of selection and amplification, a single RNA molecule was selected that bound TS protein with nearly 20-fold greater affinity than native, wild-type TS RNA sequences. Secondary structure analysis of this RNA sequence predicted it to possess a stem-loop structure. Deletion and/or modification of the UGU loop element within the RNA sequence decreased binding to TS by up to 1000-fold. In vivo transfection experiments revealed that the presence of the selected RNA sequence resulted in a significant increase in the expression of a heterologous luciferase reporter construct in human colon cancer H630 and TS-overexpressing HCT-C:His-TS+ cells, but not in HCT-C18 cells expressing a functionally inactive TS. In addition, the presence of this element in H630 cells leads to induced expression of TS protein. An immunoprecipitation method using RT-PCR confirmed a direct interaction between human TS protein and the selected RNA sequence in transfected human cancer H630 cells. This study identified a novel RNA sequence from a degenerate RNA library that specifically interacts with TS.

Base Sequence↗
PubMed1980

Frequency distribution of pre-messenger RNA sequences in polyadenylated and non-polyadenylated nuclear RNA from Friend cells.

Hybridisation of cDNA probes for abundant and rare polysomal polyadenylated RNAs with polyadenylated and non-polyadenylated nuclear RNA from Friend cells indicated that the abundant polysomal polyadenylated RNA sequences were present at a higher concentration in the nucleus than rare polysomal sequences, but at a reduced range of concentrations. The ratio of the concentrations of abundant and rare sequences was about 3 in non-polyadenylated nuclear RNA, 9 in polyadenylated nuclear RNA and 13 in polysomal polyadenylated RNA. This suggests that polyadenylation may play a role in the quantitative selection of sequences for transport to the cytoplasm. Polyadenylation cannot be the only signal for transport, since a highly complex population of nucleus-confined polyadenylated molecules exists, each of which is present on average at less than one copy per cell.

Animals↗
PubMedDate not supplied

Synthesis of uridine phosphoramidite analogs: reagents for site-specific incorporation of photoreactive sites into RNA sequences.

The synthesis of three new photoactive RNA phosphoramidites, 5-bromouridine, 5-iodouridine, and O4-triazolouridine, is reported. The 5' OH of bromouridine and iodouridine were protected as dimethoxytrityl ether using dimethoxytrityl chloride and pyridine. Selective protection of 2' OH was achieved as the corresponding tert-butyldimethylsilyl ether. Protected ribonucleosides were converted to phosphoramidites using 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. O4-Triazolouridine phosphoramidite monomer was prepared in one step from uridine phosphoramidite. These phosphoramidites were used to incorporate photoprobes at any chosen sites in the RNA sequences during chemical syntheses. The modified monomers were incorporated into RNA oligomers with coupling yields > 98%. After chemical synthesis, O4-triazolouridine was converted to 4-thiouridine by the addition of thiolacetic acid during standard deprotection methods. The extent of thiation and incorporation of modified nucleotides into RNA sequences were confirmed by nuclease digest, HPLC, and gel electrophoresis.

Alkaline Phosphatase↗
PubMed1975

Mapping of adenovirus 2 RNA sequences in lytically infected cells and transformed cell lines.

The strands of the six EcoRI fragments and the HpaI fragments E and C of Ad2 DNA were separated by electrophoresis in agarose gels. Using 32P-labeled fragment strands in solution hybridization experiments, the fraction of each strand complementary to RNA extracted from infected or transformed cells was assayed by chromatography on hydroxylapatite. In this manner, a tentative map of the cytoplasmic RNA sequences has been constructed for viral RNA extracted from cells both early and late during infection (see Fig. 16; in the map shown, the two strands of Ad2 are named the r and l strands following the bacteriophage convention). Since early cytoplasmic RNA anneals to four distinct regions of the genome, Ad2 probably codes for at least four early gene functions. Summation experiments have shown that all RNA sequences found in the cytoplasm of cells early during infection are also present in the cells' cytoplasm at late times. Viral RNA sequences in five independently isolated and cloned transformed rat cell lines were also mapped on the Ad2 genome. One class of Ad2-transformed rat cells contains RNA sequences complementary to only the segment of Ad2 DNA from 0.03-0.10 on the physical map, and this corresponds to one of the four regions of the genome expressed early during infection. If a viral gene product is necessary to maintain the transformed phenotype of the cell or codes for the virus-specific tumor (T) antigen, this genetic information must be at the left end of the genome (see Fig. 16). The two other classes of Ad2-transformed rat cells contain viral RNA sequences complementary to two or three of the regions of the genome transcribed into early cytoplasmic RNA. At both early and late times during the lytic cycle, the nucleus of the infected cell contains viral RNA sequences that are not transported to the cell's cytoplasm, suggesting that RNA processing and selection may play a role in the regulation of viral mRNA production.

Adenoviridae↗
PubMed2026

A modular class-aware workflow for small RNA sequencing analysis using mouse sperm as a case study.

BACKGROUND: Small RNA sequencing analysis is challenging because RNA classes differ in biogenesis, sequence redundancy, genomic organization, and annotation reliability. Integrated workflows accommodating these constraints remain limited, particularly for fragment-level and cluster-level analysis. METHODS: We present a reproducible, containerized, class-aware workflow for small RNA sequencing analysis, using mouse sperm as a case study. The workflow combines standardized preprocessing with complementary annotation and quantification strategies for microRNAs (miRNAs), transfer RNA-derived small RNAs (tsRNAs), ribosomal RNA-derived small RNAs (rsRNAs), and PIWI-interacting RNA (piRNA)-enriched genomic clusters. Using sperm small RNA data from offspring of lipopolysaccharide (LPS)-exposed male mice, we compared integrated-reference mapping, multi-class annotation, fragment-level tsRNA profiling, and genome-based piRNA cluster analysis, with custom modules for locus-aware harmonization and condition-specific cluster analysis. RESULTS: Integrated-reference mapping aligned 88.17% of reads and retained 690 features after filtering. It identified 11 differentially expressed miRNAs between LPS and controls, while other classes showed limited signal. Fragment-level profiling improved tsRNA resolution. piRNA cluster analysis identified 958 control and 940 LPS clusters, with 18 control-specific and no LPS-specific clusters. CONCLUSION: This workflow supports transparent, reproducible, class-aware interpretation of small RNA sequencing data while emphasizing cautious interpretation of piRNA-enriched signals from total small RNA sequencing.

Small non-coding RNA analysis↗