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PubMed1989

Organization and expression of 5S rRNA genes in the parasitic nematode, Brugia malayi.

DNA sequence analysis of genes encoding 5S rRNA in the human parasitic nematode Brugia malayi (B. malayi) indicates a surprising degree of heterogeneity. This variation in coding sequence is not accompanied by corresponding heterogeneity in flanking regions which are highly conserved. Six out of eight potential 5S coding regions differed; of these sequence variants, two were abundant in the B. malayi genome. Direct RNA sequence analysis indicated that one of these abundant variants accounts for most if not all of expressed 5S RNA at two stages of development.

Animals↗

PubMed1994

The 16S/23S ribosomal spacer region of Coxiella burnetti.

The 16S/23S spacer region of Coxiella burnetti isolate Nine Nile, phase 1, was sequenced. Sequence analysis revealed two tRNA coding regions for tRNA(Ile) and tRNA(Ala). DNA sequence alignment demonstrated significant homology with tRNA species from Pseudomonas aeruginosa and Rhodobacter sphaeroides, respectively. The non-coding tRNA spacer region was unique to Coxiella burnetti, based on database alignment.

Cloning, Molecular↗

PubMed1988

A sequence-specific conformational epitope on U1 RNA is recognized by a unique autoantibody.

An autoantibody from a patient with lupus-overlap syndrome was found to bind a specific region of U1 RNA. By using RNA sequence analysis, immunoprecipitation, and competition experiments with in vitro synthesized fragments of U1 RNA, a region of 40 nucleotides representing a stem-loop secondary structure was found to be an immunoreactive domain. This antibody recognized a conformational epitope because neither the RNA stem nor the RNA loop alone was immunoprecipitable. Antisense U1 RNA, U1 DNA, and other small RNAs were not reactive with the antibody. While the origins of nucleic acid-binding antibodies are unknown, this RNA-specific autoantibody probably originated by direct presentation to the immune system or as an anti-idiotype against a more common U1 small nuclear ribonucleoprotein-specific autoantibody. Thus, these findings have implications for the mechanisms of autoimmune recognition and provide an immunological approach to probing RNA structure and protein-RNA interactions.

Autoantibodies↗

PubMed1994

Matrix assisted laser desorption/ionization mass spectrometry of enzymatically synthesized RNA up to 150 kDa.

Enzymatically synthesized RNA samples (in vitro transcripts) were analysed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Spectra of RNA up to 150 kDA (461 nucleotides) are shown. Polymerase generated sample heterogeneity and its contribution to mass resolution are discussed. A time course exonuclease digest of a 55 nt in vitro transcript was analyzed to investigate the performance of MALDI-MS on complex mixtures. Based on these data, the analysis by MALDI-MS of DNA sequencing reactions, produced by the action of an RNA polymerase, is discussed.

Base Sequence↗

PubMed1995

The primary structure of Harpalus rufipes 5S ribosomal RNA: a contribution for understanding insect evolution.

The nucleotide sequence of 5S ribosomal RNA from the beetle Harpalus rufipes was determined and compared with primary structures of other insect 5S rRNAs. Sequence differences between two beetle 5S rRNAs may represent phylogenetic markers specific for two groups of Coleoptera - Adephaga and Polyphaga. Analysis of all insect sequences using parsimony allowed us to infer a phylogenetic tree of insects, which is consistent with morphological and paleobiological data.

Animals↗

PubMed2000

Molecular characterization of HERV-H variants associated with multiple sclerosis.

Our objective was to characterize retroviral sequences by RT-PCR with gag and env primers on RNA from RT-positive retroviral particles produced by multiple sclerosis (MS) derived B-lymphoblastoid cell lines. Sequence variants with high homology to the potentially functional subgroup RGH of the human endogenous retrovirus RTVL-H/HERV-H family were found. The same sequences were also specifically found in the particulate fraction of a series of MS patient plasma samples and were absent in controls. South-Western blots demonstrate the presence of a nucleic acid binding protein, corresponding in size and function to the nucleocapsid protein, Gag NC, of other retroviruses. We also present indications for transmission of the retrovirus to PHA-stimulated lymphocytes from healthy individuals.

B-Lymphocytes↗

PubMed2006

RegRNA: an integrated web server for identifying regulatory RNA motifs and elements.

Numerous regulatory structural motifs have been identified as playing essential roles in transcriptional and post-transcriptional regulation of gene expression. RegRNA is an integrated web server for identifying the homologs of regulatory RNA motifs and elements against an input mRNA sequence. Both sequence homologs and structural homologs of regulatory RNA motifs can be recognized. The regulatory RNA motifs supported in RegRNA are categorized into several classes: (i) motifs in mRNA 5'-untranslated region (5'-UTR) and 3'-UTR; (ii) motifs involved in mRNA splicing; (iii) motifs involved in transcriptional regulation; (iv) riboswitches; (v) splicing donor/acceptor sites; (vi) inverted repeats; and (vii) miRNA target sites. The experimentally validated regulatory RNA motifs are extracted from literature survey and several regulatory RNA motif databases, such as UTRdb, TRANSFAC, alternative splicing database (ASD) and miRBase. A variety of computational programs are integrated for identifying the homologs of the regulatory RNA motifs. An intuitive user interface is designed to facilitate the comprehensive annotation of user-submitted mRNA sequences. The RegRNA web server is now available at http://RegRNA.mbc.NCTU.edu.tw/.

Animals↗

PubMed2006

In silico analysis of 16S ribosomal RNA gene sequencing-based methods for identification of medically important anaerobic bacteria.

This study is the first study that provides useful guidelines to clinical microbiologists and technicians on the usefulness of full 16S rRNA sequencing, 5'-end 527-bp 16S rRNA sequencing and the existing MicroSeq full and 500 16S rDNA bacterial identification system (MicroSeq, Perkin-Elmer Applied Biosystems Division, Foster City, California, USA) databases for the identification of all existing medically important anaerobic bacteria. Full and 527-bp 16S rRNA sequencing are able to identify 52-63% of 130 Gram-positive anaerobic rods, 72-73% of 86 Gram-negative anaerobic rods and 78% of 23 anaerobic cocci. The existing MicroSeq databases are able to identify only 19-25% of 130 Gram-positive anaerobic rods, 38% of 86 Gram-negative anaerobic rods and 39% of 23 anaerobic cocci. These represent only 45-46% of those that should be confidently identified by full and 527-bp 16S rRNA sequencing. To improve the usefulness of MicroSeq, bacterial species that should be confidently identified by full and/or 527-bp 16S rRNA sequencing but not included in the existing MicroSeq databases should be included.

Bacteria, Anaerobic↗

PubMedDate not supplied

The development of a genetic profile of placental gene expression during the first trimester of pregnancy: a potential tool for identifying novel secreted markers.

OBJECTIVES: Many of the maternal serum markers used in prenatal screening have been developed or evolved based on serendipity. This study determines the feasibility of developing a genetic profile of placental gene expression during early pregnancy; such a gene repertoire may serve as a tool for identifying novel secreted markers. METHODS: RNA fingerprinting was used to produce a differential expression map in normal aborted placentae of weeks 9 and 13. RESULTS: Out of 212 gene expression differences, 115 were up-regulated at week 9 and the remaining 97 up-regulated at week 13. Ninety-four were found to be previously characterised genes and 118 were expressed sequence tags or novel genes. Seven of the known genes were found to be secreted proteins and included well-characterised pregnancy markers. mRNA levels of these secreted proteins were found to correlate with levels found in maternal serum. CONCLUSION: This approach enabled the identification of known secreted markers leaving open the possibility of finding new markers. With the human genome project nearing completion, it will be vital to use a systematic approach to understand the actual pattern of gene expression during pregnancy. This will also allow a more ordered and precise identification of novel prenatal diagnostic markers.

Amino Acid Sequence↗

PubMed2005

Computing expectation values for RNA motifs using discrete convolutions.

BACKGROUND: Computational biologists use Expectation values (E-values) to estimate the number of solutions that can be expected by chance during a database scan. Here we focus on computing Expectation values for RNA motifs defined by single-strand and helix lod-score profiles with variable helix spans. Such E-values cannot be computed assuming a normal score distribution and their estimation previously required lengthy simulations. RESULTS: We introduce discrete convolutions as an accurate and fast mean to estimate score distributions of lod-score profiles. This method provides excellent score estimations for all single-strand or helical elements tested and also applies to the combination of elements into larger, complex, motifs. Further, the estimated distributions remain accurate even when pseudocounts are introduced into the lod-score profiles. Estimated score distributions are then easily converted into E-values. CONCLUSION: A good agreement was observed between computed E-values and simulations for a number of complete RNA motifs. This method is now implemented into the ERPIN software, but it can be applied as well to any search procedure based on ungapped profiles with statistically independent columns.

Algorithms↗

PubMed2005

A method for aligning RNA secondary structures and its application to RNA motif detection.

BACKGROUND: Alignment of RNA secondary structures is important in studying functional RNA motifs. In recent years, much progress has been made in RNA motif finding and structure alignment. However, existing tools either require a large number of prealigned structures or suffer from high time complexities. This makes it difficult for the tools to process RNAs whose prealigned structures are unavailable or process very large RNA structure databases. RESULTS: We present here an efficient tool called RSmatch for aligning RNA secondary structures and for motif detection. Motivated by widely used algorithms for RNA folding, we decompose an RNA secondary structure into a set of atomic structure components that are further organized by a tree model to capture the structural particularities. RSmatch can find the optimal global or local alignment between two RNA secondary structures using two scoring matrices, one for single-stranded regions and the other for double-stranded regions. The time complexity of RSmatch is O(mn) where m is the size of the query structure and n that of the subject structure. When applied to searching a structure database, RSmatch can find similar RNA substructures, and is capable of conducting multiple structure alignment and iterative database search. Therefore it can be used to identify functional RNA motifs. The accuracy of RSmatch is tested by experiments using a number of known RNA structures, including simple stem-loops and complex structures containing junctions. CONCLUSION: With respect to computing efficiency and accuracy, RSmatch compares favorably with other tools for RNA structure alignment and motif detection. This tool shall be useful to researchers interested in comparing RNA structures obtained from wet lab experiments or RNA folding programs, particularly when the size of the structure dataset is large.

Algorithms↗

PubMed2019

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis.

Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

Cell Line↗

PubMed1996

Clustering of Argentinean tospoviruses with existing species in the genus by sequence analysis of a 450-nucleotide RNA region of the N gene.

The genomic diversity of Argentine Tospoviruses from different geographical areas, and from several distinct crops was analysed here. For each isolate, RT-PCR, cloning and sequencing of a 450 nt fragment of the N gene were performed. Comparisons of RNA and predicted amino acid sequence identity and similarity were made. A partial sequence of the N gene was able to classify our local isolates within three Tospovirus species previously described (Tomato spotted wilt virus, TSWV; tomato chlorotic spot virus, TCSV and groundnut ringspot virus, GRSV). With the sequence data currently available, we performed a cladistic phylogenetic analysis which gave a possible genealogy among members of the Tospovirus genus.

Amino Acid Sequence↗

PubMed1998

cis-acting RNA elements required for replication of bovine viral diarrhea virus-hepatitis C virus 5' nontranslated region chimeras.

Pestiviruses, such as bovine viral diarrhea virus (BVDV), share many similarities with hepatitis C virus (HCV) yet are more amenable to virologic and genetic analysis. For both BVDV and HCV, translation is initiated via an internal ribosome entry site (IRES). Besides IRES function, the viral 5' nontranslated regions (NTRs) may also contain cis-acting RNA elements important for viral replication. A series of chimeric RNAs were used to examine the function of the BVDV 5' NTR. Our results show that: (1) the HCV and the encephalomyocarditis virus (EMCV) IRES element can functionally replace that of BVDV; (2) two 5' terminal hairpins in BVDV genomic RNA are important for efficient replication; (3) replacement of the entire BVDV 5' NTR with those of HCV or EMCV leads to severely impaired replication; (4) such replacement chimeras are unstable and efficiently replicating pseudorevertants arise; (5) pseudorevertant mutations involve deletion of 5' sequences and/or acquisition of novel 5' sequences such that the 5' terminal 3-4 bases of BVDV genome RNA are restored. Besides providing new insight into functional elements in the BVDV 5' NTR, these chimeras may prove useful as pestivirus vaccines and for screening and evaluation of anti-HCV IRES antivirals.

5' Untranslated Regions↗

PubMed2026

Identification of biallelic loss-of-function PREP variants in three individuals with syndromic intellectual disability.

BACKGROUND: Neurodevelopmental disorders are one of the most prevalent reasons for genetic testing in childhood. Despite the identification of over 1950 associated genes, many proposed candidate genes lack convincing gene-disease validity. The gene PREP encodes the broadly expressed prolyl endopeptidase whose exact function remains largely unknown. A homozygous PREP variant has been reported once as a candidate gene in two siblings with intellectual disability but no functional studies were conducted. METHODS: Exome and trio genome sequencing were performed in two unrelated families as part of larger cohorts. Segregation analysis, RNA sequencing and immunoblots were performed to further examine the pathogenicity of detected PREP variants. RESULTS: We report three individuals from two unrelated families who presented with intellectual disability, behavioural abnormalities, strabismus, generalised muscular hypotonia, dysmorphic facial features and epilepsy. Exome and genome sequencing identified two different homozygous rare PREP variants: c.1570_1573dup, p.(Asn525Thrfs*5) and c.1839-2A>G, p.?. RNA sequencing confirmed the detected intronic variant to result in two aberrant mRNA isoforms. In patient-derived cells immunoblots showed absence of PREP protein. CONCLUSION: Our data suggest PREP deficiency as the underlying cause of a syndromic neurodevelopmental disorder.

Female↗

PubMed1999

Confirmation of Norwalk-like virus amplicons after RT-PCR by microplate hybridization and direct sequencing.

A large number of Norwalk-like viruses (NLVs) have been identified from stool samples by RT-PCR by amplifying part of the polymerase-coding gene. A set of probes were selected based on sequence analysis of the viruses circulating in Finland during the years 1996-97 for confirmation of the findings by hybridization. A microplate hybridization test, which provides a rapid semi-automatic detection for PCR products, was designed and compared with agarose gel electrophoresis. From the material of 210 stool samples, mainly from diarrheal outbreaks during years 1997-1998, three probes, one for NLV genogroup GGI and one for each of the two GGII subgroups (Toronto-like and Lordsdale-like), were sufficient to detect 87.8% (36/41) of GGI and 89.0% (49/55) of GGII samples positive by gel electrophoresis. Amplicon sequencing of the strains not detected by the above probes revealed genetic variability in the sequences. Biotin-streptavidin binding was used both for microplate hybridization assays and for direct sequencing to identify the amplicons. Based on the sequences three more probes for the hybridization panel were added so that all the different NLVs of this study could be recognized.

Base Sequence↗

PubMed2005

Isolation, cloning, and expression of three prepro-GnRH mRNAs in Atlantic croaker brain and pituitary.

Three prepro-gonadotropin-releasing hormones, seabream GnRH (sbGnRH), chicken GnRH-II (cGnRH-II), and salmon GnRH (sGnRH) were isolated by cDNA cloning from the brain of the Atlantic croaker, Micropogonias undulatus. The amino acid sequences of croaker GnRH precursors show greatest similarities to those of the gilthead and red sea breams and European sea bass. In situ hybridization of croaker brain sections revealed more abundant sbGnRH mRNA expression in the preoptic area (POA) than in other brain regions. sbGnRH mRNA expression was also observed in the olfactory bulb (OB; but not in the terminal nerve ganglion cells [TNgc]), ventral telencephalon (vTEL), and anterior hypothalamus. In addition, specific sbGnRH mRNA signals were detected in the pituitary. cGnRH-II mRNA expression was limited to the midbrain tegmentum. Neuronal elements expressing sGnRH mRNA were detected in the OB including the TNgc, vTEL, and POA, indicating an overlap of the sbGnRH and sGnRH systems in certain ventral forebrain areas. The results of quantitative reverse transcriptase-polymerase chain reaction of the three GnRH mRNAs in different brain areas and the pituitary are consistent with their localization by in situ hybridization. Interestingly, a few sbGnRH mRNA-expressing neuronal elements were observed arranged in a row in the anteroventral hypothalamus projecting toward the pituitary. The results provide a morphological basis for a putative role of sbGnRH as the gonadotropin-releasing hormone. Moreover, localization of sbGnRH mRNA in a teleost pituitary points to sbGnRH synthesis, and its potential role as a local regulator, within the pituitary, similar to the role of GnRH-I in mammals.

Animals↗

PubMed1995

Distant strains of the fish rhabdovirus VHSV maintain a sixth functional cistron which codes for a nonstructural protein of unknown function.

We used direct RNA sequencing to determine the genomic organization of the region downstream from the G gene of viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus. This region contains a gene coding for a protein, identified as nonvirion protein (NV), and the gene coding for the RNA polymerase (L). Thus, VHSV genome organization was confirmed to be 3'-N-P-M-G-NV-L-5'. In both a virulent European (07-71) and an avirulent North American (Makah) strain, the NV gene is transcribed into a small mRNA that codes for a protein of 122 amino acids. It has no significant sequence similarity with the infectious hematopoietic necrosis virus NV protein nor with any other known protein. We expressed the NV protein as a fusion protein with the glutathione S-transferase of Schistosoma japonicum and used the purified fusion protein to immunize rabbits. The rabbit antiserum precipitated from infected cell extracts--and not from noninfected cells or purified virions--a protein of 14 kDa, well in accordance with the expected NV gene product size. The prediction that the NV protein is a nonstructural protein is supported by its absence from mature virions although it is present in infected cells.

Amino Acid Sequence↗