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PubMed1991

Eukaryotic origins: string analysis of 5S ribosomal RNA sequences from some relevant organisms.

Using the PHYLOGEN tree-forming programs, we evaluate the published 5S rRNA sequences in certain of the files in the Berlin DataBank in an attempt to identify the connection between archaebacteria and the eukaryotic protists. These programs are based on methods of string analysis developed by Sankoff and others. Their discriminatory power is derived from their continuous realignment of sequences through repeated assessment of insertions and deletions as well as substitutions. The programs demonstrate that even these small molecules (ca. 120 bases) retain substantial records of evolutionary events that occurred over a billion years ago. The eukaryotes seem to have been derived from ancestors near the common origins of the halobacterial and Methanococcales groups. Identifying what might have been a primordial eukaryote is more difficult because several of the species considered as early derivatives from the common root are isolated species with large genetic differences from each other and from all other extant forms that have been sequenced. The ameboid, flagellated, and ciliated protists seem to have emerged nearly simultaneously from an ancient cluster, but the sarcodinid protozoa have preference as the group of most ancient origin. The euglenozoa and the ciliates are of later derivation. Our ability to tease plausible trees from such small molecules suggests that the mode of analysis rather than the size of the molecule is often a major limitation in the reconstruction of acceptable ancient phylogenics.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2026

Integrated single-cell RNA sequencing and mendelian randomization analysis identifies causal immune-related driver genes in the heart failure inflammatory microenvironment.

BACKGROUND: Heart failure (HF) is a major global cause of cardiovascular death and disability. Chronic inflammation and immune dysregulation are critical in its development. The cardiac immune microenvironment, especially macrophages, drives HF progression, yet its molecular mechanisms and prognostic impact are not fully clear. This study aimed to identify causal immune-related driver genes in the HF inflammatory microenvironment. METHODS: We combined single-cell RNA sequencing (scRNA-seq) and Mendelian randomization (MR) to study how the inflammatory immune microenvironment affects HF risk. Using two public scRNA-seq datasets, we identified differentially expressed genes (DEGs) in HF heart tissues and selected 489 candidate genes. Causal relationships between these genes and HF were tested using expression quantitative trait loci (eQTL) data and HF genome-wide association study (GWAS) summary statistics. RESULTS: MR analysis showed that 65 genes were causally linked to HF risk. These genes were enriched in pathways related to cardiomyopathy, leukocyte migration, natural killer (NK) cell cytotoxicity, neutrophil extracellular traps, and NF-κB signaling. HF hearts displayed increased levels of macrophages, T cells, B cells, lymphoid cells, and mast cells, while neutrophils were reduced. CONCLUSIONS: Our integrated analysis reveals the central role of the cardiac inflammatory immune microenvironment in HF and identifies 65 key genes causally associated with HF susceptibility. These genes influence specific immune pathways and cell infiltration, shaping HF progression, and provide a basis for developing new biomarkers and immune-targeted therapies.

Heart failure (HF)↗

PubMed2004

The lymphocyte HIV reservoir in patients on long-term HAART is a memory of virus evolution.

OBJECTIVE: To investigate the dynamics of the lymphocyte HIV reservoir in patients on prolonged and effective highly active antiretroviral therapy (HAART). DESIGN: Nine HAART-treated patients were selected on the basis of long-term infection and long-term undetectable plasma viral RNA. Five patients had received antiretroviral therapy before HAART. We compared a polymorphic region of the env gene (C2V4), and the part of the pol gene encoding the reverse transcriptase in pre-HAART plasma and in the reservoir lymphocytes during HAART; the first plasma sample taken after structured treatment interruption was also studied in three patients. METHODS: Both regions of interest were amplified from plasma HIV RNA and cellular proviral DNA, then cloned, sequenced and subjected to phylogenetic analysis. RESULTS: Diversity of the lymphocyte reservoir was found in six of nine patients. Archiving of pre-HAART plasma clones was observed in six of nine patients. 'Wild-type' and zidovudine-resistant strains co-existed in reservoir T cells of two pre-HAART treated patients. In three patients, no resistant virus was found in the T-cell reservoir despite the detection of resistant virus in pre-HAART plasmas. However, virus archiving was documented in two of these three patients on the basis of C2V4 analysis. Latently infected T cells only partly accounted for the plasma viral load rebound after structured treatment interruption. CONCLUSIONS: The HIV lymphocyte reservoir is dynamic. Its diversity results mainly from successive archiving of circulating plasma viruses during the course of HIV infection. Archiving of resistant virus must be taken into account in therapeutic decisions.

Antiretroviral Therapy, Highly Active↗

PubMed2025

Expanding and improving analyses of nucleotide recoding RNA-seq experiments with the EZbakR suite.

Nucleotide recoding RNA sequencing methods (NR-seq; TimeLapse-seq, SLAM-seq, TUC-seq, etc.) are powerful approaches for assaying transcript population dynamics. In addition, these methods have been extended to probe a host of regulated steps in the RNA life cycle. Current bioinformatic tools significantly constrain analyses of NR-seq data. To address this limitation, we developed EZbakR (https://github.com/isaacvock/EZbakR), an R package to facilitate a more comprehensive set of NR-seq analyses, and fastq2EZbakR (https://github.com/isaacvock/fastq2EZbakR), a Snakemake pipeline for flexible preprocessing of NR-seq datasets, collectively referred to as the EZbakR suite. Together, these tools generalize many aspects of the NR-seq analysis workflow. The fastq2EZbakR pipeline can assign reads to a diverse set of genomic features (e.g., genes, exons, splice junctions), and EZbakR can perform analyses on any combination of these features. EZbakR extends standard NR-seq mutational modeling to support multi-label analyses (e.g., s4U and s6G dual labeling), and implements an improved hierarchical model to better account for transcript-to-transcript variance in metabolic label incorporation. EZbakR also generalizes dynamical systems modeling of NR-seq data to support analyses of premature mRNA processing and flow between subcellular compartments. Finally, EZbakR implements flexible and well-powered comparative analyses of all estimated parameters via design matrix-specified generalized linear modeling. The EZbakR suite will thus allow researchers to make full, effective use of NR-seq data.

Software↗

PubMed1991

Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein.

In Saccharomyces cerevisiae, temperature-sensitive mutations in the genes RNA14 and RNA15 correlate with a reduction of mRNA stability and poly(A) tail length. Although mRNA transcription is not abolished in these mutants, the transcripts are rapidly deadenylated as in a strain carrying an RNA polymerase B(II) temperature-sensitive mutation. This suggests that the primary defect could be in the control of the poly(A) status of the mRNAs and that the fast decay rate may be due to the loss of this control. By complementation of their temperature-sensitive phenotype, we have cloned the wild-type genes. They are essential for cell viability and are unique in the haploid genome. The RNA14 gene, located on chromosome H, is transcribed as three mRNAs, one major and two minor, which are 2.2, 1.5, and 1.1 kb in length. The RNA15 gene gives rise to a single 1.2-kb transcript and maps to chromosome XVI. Sequence analysis indicates that RNA14 encodes a 636-amino-acid protein with a calculated molecular weight of 75,295. No homology was found between RNA14 and RNA15 or between RNA14 and other proteins contained in data banks. The RNA15 DNA sequence predicts a protein of 296 amino acids with a molecular weight of 32,770. Sequence comparison reveals an N-terminal putative RNA-binding domain in the RNA15-encoded protein, followed by a glutamine and asparagine stretch similar to the opa sequences. Both RNA14 and RNA15 wild-type genes, when cloned on a multicopy plasmid, are able to suppress the temperature-sensitive phenotype of strains bearing either the rna14 or the rna15 mutation, suggesting that the encoded proteins could interact with each other.

Amino Acid Sequence↗

PubMed1984

Expression of unspliced NS1 mRNA, spliced NS2 mRNA, and a spliced chimera mRNA from cloned influenza virus NS DNA in an SV40 vector.

Influenza virus gene 8 codes for two nonstructural proteins (NS1 and NS2) which are translated, respectively, from a colinear and an interrupted mRNA. To investigate the mechanism of transcription processing by splicing, cloned full-length NS DNA was inserted into the late region of an SV40 vector. In this manner, transcription of NS RNA from the recombinant initiates and terminates using SV40 control sequences. RNA mapping and sequence analysis showed that the RNA transcripts are processed to produce both the spliced and unspliced NS mRNAs in approximately equal abundance. The junction sequence of the spliced mRNA is identical to that of NS2 mRNA found in influenza virus-infected cells. In addition, another mRNA species was found to contain a chimera splice between SV40 and NS sequences. Both NS1 and NS2 polypeptides are produced in the recombinant-infected cells as predicted from sequence analysis. These studies establish that during influenza virus infections processing of the NS1 mRNA transcript undergoes a mechanism of splicing similar to that occurring with DNA-directed RNA transcription.

Amino Acid Sequence↗

PubMed2004

A novel gene homologous to teashirt is differentially expressed in neonatal mouse skin during development of hair follicles.

Neonatal mouse skin is useful for studying changes in gene expression during development of hair follicles, as the mitotic activity of skin cells changes shortly after birth. Using ribonucleic acid (RNA) differential display, a 261-nt message has been identified in the skin, specifically on d 3-5 but not on d 2 after birth. Confirmation of its expression by ribonuclease protection assay showed that stronger expression is seen on d 3-5 compared with d 1-2. Using RNA ligase-mediated rapid amplification of 5' complementary deoxyribonucleic acid ends, we have successfully isolated a 3046-bp gene, which has 93% sequence homology to a mouse teashirt1 gene. Amino acid analysis showed that it has 74% identity to the mouse teashirt1 protein and possesses zinc-finger motifs 1, 2, and 3. In situ hybridization data revealed that it is mainly expressed in the follicle bulb, including dermal papilla and matrix cells. As the proliferation of bulb cells is important to follicle development during this period, the finding of its strong expression on d 3-5 suggests that the identified gene is a potential candidate for follicle growth.

Amino Acid Sequence↗

PubMed2001

Molecular evidence of homosexual transmission of HIV type 2 in Spain.

Eight HIV-2-infected Caucasian men living in the same geographical area in Gipuzkoa (northern Spain) have been identified in the last 5 years. HIV-2 infection in this area is uncommon, and no other cases of HIV-2 infection have been found after extensive testing for HIV-1/2 antibodies. Epidemiological data suggested a possible link among the identified subjects, with homosexual contact being the most likely way of transmission. A genetic analysis of four of the subjects, from whom specimens were available, was conducted. Phylogenetic and signature pattern studies of the reverse transcriptase (RT) and env genes supported a single source of infection. Interindividual nucleotide variability ranged from 2.4 to 4.8% in the RT region and from 5.2 to 6.1% in the env gene, whereas the mean divergence between patient and control strains was 9.8 and 18.3%, respectively. The nucleotide and amino acid signature patterns were closely related in viruses from the four examined individuals. This is the first report of a cluster of HIV-2 infections with genetic sequence data support. The singularity of this cluster should alert clinicians on the possibility of HIV-2 outside endemic areas.

Adult↗

PubMed2002

Sequence analysis of Chinese and Japanese Curcuma drugs on the 18S rRNA gene and trnK gene and the application of amplification-refractory mutation system analysis for their authentication.

The botanical origins of Chinese and Japanese Curcuma drugs were determined to be Curcuma longa, C. phaeocaulis, the Japanese population of C. zedoaria, C. kwangsiensis, C. wenyujin, and C. aromatica based on a comparison of their 18S rRNA gene and trnK gene sequences with those of six Curcuma species reported previously. Moreover, to develop a more convenient identification method, amplification-refractory mutation system (ARMS) analysis of both gene regions was performed on plants. The ARMS method for the 18S rRNA gene was established using two types of forward primers designed based on the nucleotide difference at position 234. When DNAs of four Curcuma species were used as templates, PCR amplification with either of the two primers only generated a fragment of 912 base pairs (bp). However, when DNAs of the purple-cloud type of C. kwangsiensis and C. wenyujin were used, PCR amplifications with both primers unexpectedly generated the fragment, suggesting that these two were heterozygotes. The ARMS method for the trnK gene was also established using a mixture of four types of specific reverse primers designed on the basis of base substitutions and indels among six species, and common reverse and forward primers. C. phaeocaulis or the Chinese population of C. zedoaria, the Japanese population of C. zedoaria or the purple-cloud type of C. kwangsiensis, the pubescent type of C. kwangsiensis or C. wenyujin, and C. aromatica were found to show specific fragments of 730, 185, 527 or 528, and 641 or 642 bp, respectively. All species including C. longa also showed a common fragment of 897-904 bp. Using both ARMS methods, together with information on producing areas, the identification of Curcuma plants was achieved. Moreover, the ARMS method for the trnK gene was also useful for authentication of Curcuma drugs.

Curcuma↗

PubMed2006

Demonstration of two novel methods for predicting functional siRNA efficiency.

BACKGROUND: siRNAs are small RNAs that serve as sequence determinants during the gene silencing process called RNA interference (RNAi). It is well know that siRNA efficiency is crucial in the RNAi pathway, and the siRNA efficiency for targeting different sites of a specific gene varies greatly. Therefore, there is high demand for reliable siRNAs prediction tools and for the design methods able to pick up high silencing potential siRNAs. RESULTS: In this paper, two systems have been established for the prediction of functional siRNAs: (1) a statistical model based on sequence information and (2) a machine learning model based on three features of siRNA sequences, namely binary description, thermodynamic profile and nucleotide composition. Both of the two methods show high performance on the two datasets we have constructed for training the model. CONCLUSION: Both of the two methods studied in this paper emphasize the importance of sequence information for the prediction of functional siRNAs. The way of denoting a bio-sequence by binary system in mathematical language might be helpful in other analysis work associated with fixed-length bio-sequence.

Algorithms↗

PubMed2024

Nanopore sequencing to detect A-to-I editing sites.

Adenosine-to-inosine (A-to-I) RNA editing, mediated by the ADAR family of enzymes, is pervasive in metazoans and functions as an important mechanism to diversify the proteome and control gene expression. Over the years, there have been multiple efforts to comprehensively map the editing landscape in different organisms and in different disease states. As inosine (I) is recognized largely as guanosine (G) by cellular machineries including the reverse transcriptase, editing sites can be detected as A-to-G changes during sequencing of complementary DNA (cDNA). However, such an approach is indirect and can be confounded by genomic single nucleotide polymorphisms (SNPs) and DNA mutations. Moreover, past studies rely primarily on the Illumina platform, which generates short sequencing reads that can be challenging to map. Recently, nanopore direct RNA sequencing has emerged as a powerful technology to address the issues. Here, we describe the use of the technology together with deep learning models that we have developed, named Dinopore (Detection of inosine with nanopore sequencing), to interrogate the A-to-I editome of any organism.

Inosine↗

PubMedDate not supplied

Tracking Cryptosporidium parvum by sequence analysis of small double-stranded RNA.

We sequenced a 173-nucleotide fragment of the small double-stranded viruslike RNA of Cryptosporidium parvum isolates from 23 calves and 38 humans. Sequence diversity was detected at 17 sites. Isolates from the same outbreak had identical double-stranded RNA sequences, suggesting that this technique may be useful for tracking Cryptosporidium infection sources.

Animals↗

PubMed1997

Chlamydomonas U2, U4 and U6 snRNAs. An evolutionary conserved putative third interaction between U4 and U6 snRNAs which has a counterpart in the U4atac-U6atac snRNA duplex.

The spliceosomal UsnRNAs U2, U4 and U6 from the green alga Chlamydomonas reinhardtii (Cre) were sequenced using a combination of RNA and cDNA sequencing methods and were compared to other sequenced UsnRNAs. The lengths of Cre U6 and Cre U2 RNAs are similar to those of their higher plant equivalents. Cre U4 RNA is shorter (139 nt) than its counterpart from higher plants (150-154 nt), and contains stem IV and loop D which are absent, with the exception of the Tetrahymena U4 RNA, from the U4 RNAs of other unicellular organisms studied to date. Base-pairing interactions between U6 and U4 RNAs and between U6 and U2 RNAs, identical to those described for mammalian and yeast systems, are structurally feasible in the Cre system. In addition, based on comparative analyses of the predicted U4/U6 RNA duplex from various species, an evolutionary conserved third putative U6-U4 interaction was found. Interestingly, it can also be formed with the recently discovered U6atac and U4atac RNAs. This is a strong support in favor of the possible biological significance of this third putative interaction. Based on comparative analysis, an extension of the earlier described U6-U2 interaction patterns is also proposed.

Alternative Splicing↗

PubMed2004

Structural analysis of hepatitis C RNA genome using DNA microarrays.

Many studies have tried to identify specific nucleotide sequences in the quasispecies of hepatitis C virus (HCV) that determine resistance or sensitivity to interferon (IFN) therapy, unfortunately without conclusive results. Although viral proteins represent the most evident phenotype of the virus, genomic RNA sequences determine secondary and tertiary structures which are also part of the viral phenotype and can be involved in important biological roles. In this work, a method of RNA structure analysis has been developed based on the hybridization of labelled HCV transcripts to microarrays of complementary DNA oligonucleotides. Hybridizations were carried out at non-denaturing conditions, using appropriate temperature and buffer composition to allow binding to the immobilized probes of the RNA transcript without disturbing its secondary/tertiary structural motifs. Oligonucleotides printed onto the microarray covered the entire 5' non-coding region (5'NCR), the first three-quarters of the core region, the E2-NS2 junction and the first 400 nt of the NS3 region. We document the use of this methodology to analyse the structural degree of a large region of HCV genomic RNA in two genotypes associated with different responses to IFN treatment. The results reported here show different structural degree along the genome regions analysed, and differential hybridization patterns for distinct genotypes in NS2 and NS3 HCV regions.

Conserved Sequence↗

PubMed1998

Detection and sequence analysis of borna disease virus p24 RNA from peripheral blood mononuclear cells of patients with mood disorders or schizophrenia and of blood donors.

Borna disease virus (BDV) p24 RNA was detected in the peripheral blood mononuclear cells (PBMCs) of psychiatric patients and blood donors by nested reverse transcriptase PCR (RT-PCR). The prevalences of BDV p24 RNA in patients with mood disorders (4%) and schizophrenia (4%) were not significantly different from that in blood donors (2%). This finding was inconsistent with previous reports that showed either a high prevalence or absence of BDV p24 RNA in patients with psychiatric disorders. The differences in BDV p24 RNA prevalence in these studies may be due to differences in the criteria for positivity, the number of PBMCs used for RNA extraction, or the amount of RNA tested for nested RT-PCR or to laboratory contamination. Sequence analysis of BDV p24 RNA from the PBMCs of patients and blood donors showed a high nucleotide sequence conservation but definite nucleotide mutations compared with horse BDV p24 RNA sequences. In comparison with human BDV p24 RNA sequences previously reported from Japan and Germany, there were several positions with silent nucleotide mutations among these clones.

Adult↗

PubMed1998

Molecular evidence for mother-to-child transmission of multiple variants by analysis of RNA and DNA sequences of human immunodeficiency virus type 1.

We have examined the viral selection that may occur during transmission by studying the env gene sequences from four cases of mother-to-child transmission of human immunodeficiency virus type 1. The V3 region sequences were directly amplified from both plasma viral RNA and peripheral blood mononuclear cells containing proviral DNA from mothers at delivery and at the time of diagnosis for children. Transmission occurred perinatally in three cases. The similarity of the viral sequences in each infant sample contrasted with the heterogeneous viral populations in the mothers. Phylogenetic analysis indicated the transmission of one or a few closely related maternal minor virus variants. In contrast, the child virus population in the fourth case was as heterogeneous as that of his mother, and phylogenetic analysis strongly suggested the transmission of multiple maternal variants. This case of multiple transmission was confirmed by analyzing sequences obtained at three times after delivery. Strains with sequences corresponding to the syncytium-inducing phenotype were also transmitted in this fourth case, and this was associated with the rapid development of disease in the child. There was no evidence for transmission of particular viral variants from mother to infant. We have thus described a particular case of vertical human immunodeficiency virus type 1 transmission with the transmission of multiple maternal variants to the infant and a rapid, fatal outcome in the child.

Amino Acid Sequence↗

PubMed2004

Conserved RNA secondary structures in viral genomes: a survey.

SUMMARY: The genomes of RNA viruses often carry conserved RNA structures that perform vital functions during the life cycle of the virus. Such structures can be detected using a combination of structure prediction and co-variation analysis. Here we present results from pilot studies on a variety of viral families performed during bioinformatics computer lab courses in past years.

Algorithms↗

PubMed2003

A software tool-box for analysis of regulatory RNA elements.

We describe an integrated tool-box to identify regulatory RNA elements. The RNA analyzer collects general and specific information on any submitted RNA sequence or batch of sequences in FASTA format. It determines and rapidly scans the different regions of an RNA (including 5' UTR, CDS, 3' UTR in mRNA) and screens for specific RNA signals (in each of these regions, e.g. polyA-site, AU rich region etc. in 3' UTR). It runs a fast folding RNA routine to provide an overview of the RNA fold. Furthermore it analyzes structure content, fold energy and stem loops. In addition, consensus templates are used to determine whether there are any functional structures present for translational control (template: IRE), structured RNA (template: tRNA consensus) or catalytic RNA (template: trans-splicing RNA), giving indications as to how well the structures found match to these templates. The tool box has been implemented as a WWW server at http://wb2x01.biozentrum.uni-wuerzburg.de/.

Internet↗