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PubMed2005

Structural implications of novel diversity in eucaryal RNase P RNA.

Previous eucaryotic RNase P RNA secondary structural models have been based on limited diversity, representing only two of the approximately 30 phylogenetic kingdoms of the domain Eucarya. To elucidate a more generally applicable structure, we used biochemical, bioinformatic, and molecular approaches to obtain RNase P RNA sequences from diverse organisms including representatives of six additional kingdoms of eucaryotes. Novel sequences were from acanthamoeba (Acathamoeba castellanii, Balamuthia mandrillaris, Filamoeba nolandi), animals (Caenorhabditis elegans, Drosophila melanogaster), alveolates (Theileria annulata, Babesia bovis), conosids (Dictyostelium discoideum, Physarum polycephalum), trichomonads (Trichomonas vaginalis), microsporidia (Encephalitozoon cuniculi), and diplomonads (Giardia intestinalis). An improved alignment of eucaryal RNase P RNA sequences was assembled and used for statistical and comparative structural analysis. The analysis identifies a conserved core structure of eucaryal RNase P RNA that has been maintained throughout evolution and indicates that covariation in size occurs between some structural elements of the RNA. Eucaryal RNase P RNA contains regions of highly variable length and structure reminiscent of expansion segments found in rRNA. The eucaryal RNA has been remodeled through evolution as a simplified version of the structure found in bacterial and archaeal RNase P RNAs.

Acanthamoeba castellanii↗
PubMed1988

Localization of vasopressin mRNA in the rat brain using in situ hybridization with short cRNA probes made from synthetic DNA templates.

Short antisense and sense RNA sequences were transcribed from annealed double stranded oligodeoxynucleotide templates containing the T7 RNA polymerase promotor sequence and its complementary sequence, together with 5' overhanging sequence corresponding to either the sense or antisense sequences of part of the rat vasopressin gene to produce after transcription 35S-labelled sense or antisense RNA for use in situ hybridization histochemistry. Labelled antisense RNA coding for a vasopressin sequence visualized sites of vasopressin mRNA expression (as did the 35S-labelled antisense oligodeoxynucleotide sequence), whereas sense RNA sequences revealed no specific sites of hybridization. This method represents an accessible, convenient and general method for the generation of cRNA probes suitable for use in situ hybridization.

Animals↗
PubMed2002

Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition.

Previous studies have shown that human dihydrofolate reductase (DHFR) acts as an RNA-binding protein, in which it binds to its own mRNA and, in so doing, results in translational repression. In this study, we used RNA gel mobility shift and nitrocellulose filter-binding assays to further investigate the specificity of the interaction between human DHFR protein and human DHFR mRNA. Site-directed mutagenesis was used to identify the critical amino acid residues on DHFR protein required for RNA recognition. Human His-Tag DHFR protein specifically binds to human DHFR mRNA, while unrelated proteins including thymidylate synthase, p53 and glutathione-S-transferase were unable to form a ribonucleoprotein complex with DHFR mRNA. The Cys6 residue is essential for RNA recognition, as mutation at this amino acid with either an alanine (C6A) or serine (C6S) residue almost completely abrogated RNA-binding activity. Neither one of the cysteine mutant proteins was able to repress the in vitro translation of human DHFR mRNA. Mutations at amino acids Ile7, Arg28 and Phe34, significantly reduced RNA-binding activity. An RNA footprinting analysis identified three different RNA sequences, bound to DHFR protein, ranging in size from 16 to 45 nt, while a UV cross-linking analysis isolated an approximately 16 nt RNA sequence bound to DHFR. These studies begin to identify the critical amino acid residues on human DHFR that mediate RNA binding either through forming direct contact points with RNA or through maintaining the protein in an optimal structure that allows for the critical RNA-binding domain to be accessible.

Amino Acids↗
PubMed2002

Automatic RNA secondary structure prediction with a comparative approach.

This paper presents an algorithm, DCFold, that automatically predicts the common secondary structure of a set of aligned homologous RNA sequences. It is based on the comparative approach. Helices are searched in one of the sequences, called the 'target sequence', and compared to the helices in the other sequences, called the 'test sequences'. Our algorithm searches in the target sequence for palindromes that have a high probability to define helices that are conserved in the test sequences. This selection of significant palindromes is based on criteria that take into account their length and their mutation rate. A recursive search of helices, starting from these likely ones, is implemented using the 'divide and conquer' approach. Indeed, as pseudo-knots are not searched by DCFold, a selected palindrome (p, p') makes possible to divide the initial sequence into two sequences, the internal one and the one resulting from the concatenation of the two external ones. New palindromes can be searched independently in these subsequences. This algorithm was run on ribosomal RNA sequences and recovered very efficiently their common secondary structures.

Algorithms↗
PubMed2004

Identification of an hnRNP A1-dependent splicing silencer in the human papillomavirus type 16 L1 coding region that prevents premature expression of the late L1 gene.

We have previously identified cis-acting RNA sequences in the human papillomavirus type 16 (HPV-16) L1 coding region which inhibit expression of L1 from eukaryotic expression plasmids. Here we have determined the function of one of these RNA elements, and we provide evidence that this RNA element is a splicing silencer which suppresses the use of the 3' splice site located immediately upstream of the L1 AUG. We also show that this splice site is inefficiently utilized as a result of a suboptimal polypyrimidine tract. Introduction of point mutations in the L1 coding region that altered the RNA sequence without affecting the L1 protein sequence resulted in the inactivation of the splicing silencer and induced splicing to the L1 3' splice site. These mutations also prevented the interaction of the RNA silencer with a 35-kDa cellular protein identified here as hnRNP A1. The splicing silencer in L1 inhibits splicing in vitro, and splicing can be restored by the addition of RNAs containing an hnRNP A1 binding site to the reaction, demonstrating that hnRNP A1 inhibits splicing of the late HPV-16 mRNAs through the splicing silencer sequence. While we show that one role of the splicing silencer is to determine the ratio between partially spliced L2/L1 mRNAs and spliced L1 mRNAs, we also demonstrate that it inhibits splicing from the major 5' splice site in the early region to the L1 3' splice site, thereby playing an essential role in preventing late gene expression at an early stage of the viral life cycle. We speculate that the activity of the splicing silencer and possibly the concentration of hnRNP A1 in the HPV-16-infected cell determines the ability of the virus to establish a persistent infection which remains undetected by the host immune surveillance.

3' Untranslated Regions↗
PubMed1974

Influence of protein and ribonucleic acid synthesis on the replication of the bacteriocinogenic factor Clo DF13 in Escherichia coli cells and minicells.

The influence of ribonucleic acid (RNA) and protein synthesis on the replication of the cloacinogenic factor Clo DF13 was studied in Escherichia coli cells and minicells. In chromosomeless minicells harboring the Clo DF13 factor, Clo DF13 deoxyribonucleic acid (DNA) synthesis is slightly stimulated after inhibition of protein synthesis by chloramphenicol or puromycin and continues for more than 8 h. When minicells were treated with rifampin, a specific inhibitor of DNA-dependent RNA polymerase, Clo DF13 RNA and DNA synthesis appeared to stop abruptly. In cells, the Clo DF13 factor continues to replicate during treatment with chloramphenicol long after chromosomal DNA synthesis ceases. When rifampin was included during chloramphenicol treatment of cells, synthesis of Clo DF13 plasmid DNA was blocked completely. Isolated, supercoiled Clo DF13 DNA, synthesized in cells or minicells in the presence of chloramphenicol, appeared to be sensitive to ribonuclease and alkali treatment. These treatments convert a relatively large portion of the covalently closed Clo DF13 DNA to the open circular form, whereas supercoiled Clo DF13 DNA, isolated from non-chloramphenicol-treated cells or minicells, is not significantly affected by these treatments. These results indicate that RNA synthesis and specifically Clo DF13 RNA synthesis are involved in Clo DF13 DNA replication and that the covalently closed Clo DF13 DNA, synthesized in the presence of chloramphenicol, contains one or more RNA sequences. De novo synthesis of chromosomal and Clo DF13-specific proteins is not required for the replication of the Clo DF13 factor. Supercoiled Clo DF13 DNA, isolated from a polA107 (Clo DF13) strain which lacks the 5' --> 3' exonucleolytic activity of DNA polymerase I, is insensitive to ribonuclease or alkali treatment, indicating that in this mutant the RNA sequences are still removed from the RNA-DNA hybrid.

Alkalies↗
PubMed1991

Sequence-independent RNA cleavages generate the primers for plus strand DNA synthesis in hepatitis B viruses: implications for other reverse transcribing elements.

Reverse transcription of RNA into duplex DNA requires accurate initiation of both minus and plus strand DNA synthesis; this in turn requires the generation of specific primer molecules. We have examined plus strand primer generation in the hepatitis B viruses, small DNA viruses that replicate via reverse transcription. The plus strand primer in these viruses is a short capped RNA derived from the 5' end of the RNA template by cleavage at a specific set of sites. To elucidate the cleavage mechanism we constructed a series of viral mutants bearing alterations in and around the cleavage sites. Our results reveal that the cleavage reaction is sequence-independent and indicate that the cleavage sites are positioned by measurement of the distance from the 5' end of the RNA. Comparison of these findings with what is known about RNase H-mediated primer generation in retroviruses and other retroid elements suggests that, despite many divergent features, some common molecular features are preserved.

Base Sequence↗
PubMed1993

Secondary structure of RNase MRP RNA as predicted by phylogenetic comparison.

RNase MRP is a ribonucleoprotein endoribonuclease that has been shown to cleave mitochondrial primer RNA sequences from a variety of sources. The bulk of RNase MRP activity is found in the nucleus where its function remains unknown. Two different approaches have resulted in predictions of distinct secondary structures for RNase MRP RNA. In order to analyze more definitively the higher-order structure of RNase MRP RNA, we have conducted a phylogenetic comparison of the available RNase MRP RNA sequences from human, mouse, rat, cow, toad, and yeast. The resulting secondary structure shares features in common with previously described structures for prokaryotic and eukaryotic RNase P RNAs (1) and RNase MRP RNAs (2, 3). In addition, the phylogenetic structure is consistent with available chemical modification data on RNase MRP RNA and with the detailed analysis of the To antigen binding domain located near the 5' end of the RNase MRP RNA. The structure is not limited to RNase MRP RNAs, but can be expanded to cover both eukaryotic RNase P RNAs and RNase P/MRP RNAs from plants.

Animals↗
PubMed2026

Uncovering host transcriptional responses to tilapia lake virus (TiLV) through De novo RNA-seq assembly in Nile tilapia, Oreochromis niloticus.

Tilapia lake virus (TiLV) has emerged as an important pathogen that negatively impacts tilapia farming globally. Using RNA sequencing technology, this study investigated the liver transcriptomic profile of apparently healthy and TiLV-infected Oreochromis niloticus from wild. RNA sequence libraries generated 3,356 differentially expressed genes (DEGs), with 1,726 genes that were upregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 680 different pathways with differential regulation of the metabolic and immune-related pathways indicating that TiLV may interfere in host metabolism and replicate to establish the infection. This study provides transcriptomic insights into the liver responses of naturally TiLV-infected wild O. niloticus and highlights key immune and metabolic pathways associated with viral infection.

Animals↗
PubMed2006

Phylogenetic comparison of protein-coding versus ribosomal RNA-coding sequence data: a case study of the Lecanoromycetes (Ascomycota).

The resolving power and statistical support provided by two protein-coding (RPB1 and RPB2) and three ribosomal RNA-coding (nucSSU, nucLSU, and mitSSU) genes individually and in various combinations were investigated based on maximum likelihood bootstrap analyses on lichen-forming fungi from the class Lecanoromycetes (Ascomycota). Our results indicate that the optimal loci (single and combined) to use for molecular systematics of lichen-forming Ascomycota are protein-coding genes (RPB1 and RPB2). RPB1 and RPB2 genes individually were phylogenetically more efficient than all two- and three-locus combinations of ribosomal loci. The 3rd codon position of each of these two loci provided the most characters in support of phylogenetic relationships within the Lecanoromycetes. Of the three ribosomal loci we used in this study, mitSSU contributed the most to phylogenetic analyses when combined with RPB1 and RPB2. Except for the mitSSU, ribosomal genes were the most difficult to recover because they often contain many introns, resulting in PCR bias toward numerous and intronless co-extracted contaminant fungi (mainly Dothideomycetes, Chaetothyriomycetes, and Sordariomycetes in the Ascomycota, and members of the Basidiomycota), which inhabit lichen thalli. Maximum likelihood analysis on the combined five-locus data set for 82 members of the Lecanoromycetes provided a well resolved and well supported tree compared to existing phylogenies. We confirmed the monophyly of three recognized subclasses in the Lecanoromycetes, the Acarosporomycetidae, Ostropomycetidae, and Lecanoromycetideae; the latter delimited as monophyletic for the first time, with the exclusion of the family Umbilicariaceae and Hypocenomyce scalaris. The genus Candelariella (formerly in the Candelariaceae, currently a member of the Lecanoraceae) represents the first evolutionary split within the Lecanoromycetes, before the divergence of the Acarosporomycetidae. This study provides a foundation necessary to guide the selection of loci for future multilocus phylogenetic studies on lichen-forming and allied ascomycetes.

Ascomycota↗
PubMed1983

The 5'-terminal ribonucleotide of an in vitro ColE1 transcript is not complementary to the DNA template.

A set of overlapping promoters in ColE1 DNA is identified using DNA and RNA sequencing methods. These promoters initiate a divergent pattern of in vitro transcription from a region near the colicin E1 promoter. A comparison of the 5'-terminal RNA sequences of the in vitro transcripts with the DNA sequence of the template indicates that one transcript initiates with a ribonucleotide (pppG) which is not complementary to the DNA template (a thymidylate residue is present on the coding strand at the designated transcription start point). A structural and functional counterpart for this phenomenon is suggested to exist near the opposite end of the colicin E1 gene.

Bacteriocin Plasmids↗
PubMed2004

Evolutionary rate variation and RNA secondary structure prediction.

Predicting RNA secondary structure using evolutionary history can be carried out by using an alignment of related RNA sequences with conserved structure. Accurately determining evolutionary substitution rates for base pairs and single stranded nucleotides is a concern for methods based on this type of approach. Determining these rates can be hard to do reliably without a large and accurate initial alignment, which ideally also has structural annotation. Hence, one must often apply rates extracted from other RNA families with trusted alignments and structures. Here, we investigate this problem by applying rates derived from tRNA and rRNA to the prediction of the much more rapidly evolving 5'-region of HIV-1. We find that the HIV-1 prediction is in agreement with experimental data, even though the relative evolutionary rate between A and G is significantly increased, both in stem and loop regions. In addition we obtained an alignment of the 5' HIV-1 region that is more consistent with the structure than that currently in the database. We added randomized noise to the original values of the rates to investigate the stability of predictions to rate matrix deviations. We find that changes within a fairly large range still produce reliable predictions and conclude that using rates from a limited set of RNA sequences is valid over a broader range of sequences.

Algorithms↗
PubMed2025

Single-Cell Splicing Isoform Atlas of the Adult Human Heart and Heart Failure.

BACKGROUND: Alternative splicing plays crucial roles in normal heart development and cardiac disease by influencing protein-coding sequences, functional domains, and molecular networks. However, a detailed characterization of the human heart isoform landscape remains incomplete. METHODS: Leveraging long-read single-nucleus RNA sequencing and computational analysis, we dissected full-length isoform heterogeneities, expression patterns, and usage shifts across cell types, cell states, and cardiac conditions of the adult left ventricle. We applied in silico approaches to assess the functional relevance of identified isoforms; validated isoform compositions of representative cardiac genes using reverse transcription quantitative polymerase chain reaction and targeted amplicon sequencing; and developed a web server for interactive navigation of our results. RESULTS: The data revealed that isoform heterogeneity is widespread in the cardiac cellular system, serving as a posttranscriptional buffer mechanism that calibrates the molecule reservoirs in human hearts. In healthy left ventricles, ≈30% of cell type-specific genes were polyform, using multiple isoforms tailored to cell type-specific programs. Among ubiquitously expressed genes, >300 showed differential isoform usage with cell type specificity in normal hearts. Comparisons of cardiomyocytes across conditions uncovered 379 genes with marked isoform usage shifts, most of which are predicted to change protein coding outcomes through direct changes in protein coding sequences and switches between intron retention and non-protein-coding biotypes. In contrast, cell state-specific programs tend to operate on monoform genes associated with changes among cell states. In addition, our data revealed heart failure-associated differential isoform usage events in stromal and immune cell types in the cardiac microenvironment. CONCLUSIONS: We present a comprehensive atlas of splicing isoforms in the normal adult heart and heart failure through long-read single-nucleus RNA sequencing and computational analyses. The results suggest crucial roles of isoforms in buffering core cellular programs and contributing to disease-associated cell states. The full-length details of these cell-specific isoforms serve as an important reference for downstream translational and mechanistic studies and are available on our online data portal at https://github.com/gaolabtools/heart-isoform-atlas.

Humans↗
PubMed1978

Histone gene transcripts in the cleavage and mesenchyme blastula embryo of the sea urchin, S. purpuratus.

Two distinct populations of histone gene transcripts have been identified in the sea urchin embryo. Both late cleavage and mesenchyme blastula stages contain histone transcripts which hybridize to a full-length histone repeat recombinant DNA, pCO1. The histone RNAs of the two stages, however, are dissimilar in sequence. While the transcripts of the cleavage embryo form well matched hybrids with the plasmid DNA which are relatively resistant to RNAase, the hybrids containing the mesenchyme blastula transcripts melt some 10 degrees C lower and are twice as sensitive to RNAase. Hybridization of the two RNA samples to the Hha I fragments of the histone DNA, or to segments of the histone repeat subcloned in other plasmids, shows that many regions scattered along the repeat are complementary to widely diverged transcripts in the mesenchyme blastula RNA. The two RNA populations consist predominantly of polysomal RNA sequences and are most probably mRNAs for the five histones. The mesenchyme blastula RNA sequences in both S. purpuratus and L. pictus form hybrids with pCO1 DNA that are less stable than those containing L. pictus cleavage RNA, indicating the wide divergence of the two histone RNA populations. The bulk of the histone genes in S. purpuratus appear to be of the type coding for the early mRNAs. Only a small percentage of the several hundred gene copies are candidates for the type coding for the late mRNAs. The melting characteristics of the hybrids and the sensitivity of RNAase provide an assay for the late embryonic histone genes. Of the total RNA labeled during a 10 min pulse in the cleavage embryo, histone transcripts represent approximately 9.7 and 6.5% of the radioactivity in S. purpuratus and L. pictus, respectively. These values fall to 0.57 and 1.4%, respectively, at the mesenchyme blastula stage. Although histone genes are transcribed during these two periods, the type of gene which is active is switched at some point prior to the mesenchyme blastula stage.

Animals↗
PubMed1991

Analysis of genomic variability of hepatitis C virus.

The anti-C100-enzyme-linked immunosorbent assay, the new four-antigen antibody recombinant immunoblot assay, and detection of viral RNA sequences by copy DNA-polymerase chain reaction were used to establish the course of hepatitis C virus (HCV) infection in recipients of HCV-infected blood products. Different patterns of infection were observed: (1) persistent HCV infection with and without chronic hepatitis, and with acute resolved hepatitis; and (2) acute resolved hepatitis with clearance of HCV. In order to determine whether different infection- and anti-HCV recognition patterns are correlated to differences in viral nucleotide sequences, we compared sequences in the NS3 region between isolates from recipient(s) and their infective donors. Based on these comparisons we conclude that in The Netherlands two types of molecular variants circulate; one resembling the Japanese prototype isolate JH1, and the other the HCV-1 isolate from the U.S.A. The difference in sequence homology between the two types is approximately 24%. Comparison of sequences of donors and involved recipients determined in isolates prepared from blood samples four years after transfusion revealed that viral RNA sequences are strongly conserved (greater than 96.8%) in the NS3 region. These data indicate that the observed differences in anti-HCV immune response patterns between recipients are more a reflection of their immune reactivity than of divergence of viral strains.

Cardiac Surgical Procedures↗
PubMed1997

Characterization of HIV-related periodontitis in AIDS patients: HIV-infected macrophage exudate in gingival crevicular fluid as a hallmark of distinctive etiology.

In an attempt to clarify the immunobiological events featuring periodontitis lesions of AIDS patients in the late stage of the disease, peripheral blood (PB) and gingival crevicular fluid (GCF) leucocytes from periodontitis lesions of 23 late-stage AIDS patients were analysed by three-colour flow cytometry for detection and identification of intracytoplasmic p24+ cell fractions. The cells were reacted with CD14 and CD68 for mononuclear phagocytes or with CD4 and CD14 for Th cells, then with anti-p24 MoAb. To detect HIV proviral sequences and intracellular p24 RNA sequences, genomic DNA and cellular RNA from leucocytes were extracted for semi-nested polymerase chain reaction (PCR) amplification. CD68+/p24+ and CD14+/CD68+/p24+ fractions were larger in GCF than in PB (P<0.0001; P < 0.003). CD14+/p24+ fraction was lower in GCF than in PB (P < 0.05). The fluorescence intensities (FI) for intracellular p24 in CD68+ and CD14+/CD68+ cells were higher in GCF than in PB (P < 0.003; P < 0.02), whereas those of CD14+ macrophages did not differ. The p24 FI of CD68+ macrophages in GCF correlated with CD4+ lymphocyte counts in PB (P < 0.005). p24 FI levels of CD14+ monocytes in GCF and PB significantly correlated (P < 0.02), whereas that of CD68+ macrophages did not. PCR and reverse transcriptase (RT)-PCR of cellular DNA and RNA yielded positive signals, demonstrating viral integration and production in GCF leucocytes. These results show that periodontitis lesions in AIDS patients can be characterized by a rapid macrophage turnover, and these HIV-infected macrophage exudates in GCF may be considered as a within-mouth source of virus.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

[Region of intermolecular complementarity in Escherichia coli 16S rRNA, mRNA, and tRNA molecules].

The results of computer analysis of complementarity regions in the sequences of E. coli 16S rRNA, mRNAs and tRNAs are reported in this article. The potential regions of intermolecular RNA-RNA hybridization, or clinger fragments, in 16S rRNA, which are complementary to the sites frequently occurring in mRNAs and tRNAs, were found. Major clinger fragments on 16S rRNA are universal for genes that belong to different functional groups. Our results show there are adaptations of the structural organization of the 16S rRNA molecule to messenger and transport RNA sequences. RNA interaction with clinger fragments may contribute to upregulation of translation process through increasing the local concentration of mRNAs and tRNAs in the vicinity of the ribosome and their proper positioning, as well as decrease the efficiency of translation through non-specific mRNA-16SrRNA interactions.

Escherichia coli↗