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PubMed2025

Identification and Analysis of Small Nucleolar RNAs by Real-Time Quantitative PCR.

One of the greatest scientific achievements of the twenty-first century is the completion of The Human Genome Project (HGP). Thereafter, we came to know that the human genome codes nearly 2% for making proteins and thus named as coding genes, suggesting the rest of the genome as noncoding or junk. However, research in the past two decades has shown and established that noncoding RNAs are major contributors of regulating and modulating the various function of cells as well as tissues. Noncoding RNAs can be classified as basis of their sizes in two categories, long noncoding RNAs (>200&#xa0;nt) and small noncoding RNAs (<200&#xa0;nt). Small nucleolar RNAs (snoRNAs) are part of the small noncoding RNA family and primarily reside inside the nucleus of eukaryotes. Sno RNAs can be divided into two major categories based on their distinguished structure and function; these are C/D box and HACA box snoRNAs. They participate in the posttranscriptional modifications on ribosomal RNAs (r-RNAs), transfer RNAs (t-RNAs), messenger RNAs (m-RNAs), and small nuclear RNAs (snRNAs). Sno RNAs act as guide RNAs to modify other noncoding RNAs by pseudouridylation or 2'O ribomethylation. We discussed in this protocol about one of the widely used techniques for detection and analysis of snoRNAs, i.e., real-time quantitative PCR (RT-qPCR).

RNA, Small Nucleolar↗
PubMed1996

Specific interaction in vitro and in vivo of glyceraldehyde-3-phosphate dehydrogenase and LA protein with cis-acting RNAs of human parainfluenza virus type 3.

Human parainfluenza virus type 3 (HPIV3) genome RNA is transcribed and replicated by the virus-encoded RNA-dependent RNA polymerase, and specific cellular proteins play a regulatory role in these processes. To search for cellular proteins potentially interacting with HPIV3 cis-acting regulatory RNAs, a gel mobility shift assay was used. Two cellular proteins specifically interacted with the viral cis-acting RNAs containing the genomic 3'-noncoding region and the plus-sense leader sequence region. Surprisingly, by biochemical and immunological analyses, one of the cellular proteins was identified as the key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The other protein was characterized as the autoantigen, LA protein. Both GAPDH and LA protein also interacted with the same cis-acting RNA sequences in vivo and were found to be associated with the HPIV3 ribonucleoprotein complex in the infected cells. By double immunofluorescent labeling, GAPDH was found to be co-localized with viral ribonucleoprotein in the perinuclear region. These observations strongly suggest that cellular GAPDH and LA Protein participate in the regulation of HPIV3 gene expression.

Autoantigens↗
PubMed2003

Accumulation of mitochondrial DNA mutations in human immunodeficiency virus-infected patients treated with nucleoside-analogue reverse-transcriptase inhibitors.

Nucleoside reverse-transcriptase inhibitor (NRTI) therapy for human immunodeficiency virus (HIV) infection has been associated with mitochondrial DNA (mtDNA) polymerase-gamma inhibition and subsequent mtDNA depletion. Effects on mtDNA mutation, although suggested by critical involvement of polymerase-gamma in DNA-repair reactions, are unknown. In the present study, we assessed the nature and frequency of mitochondrial genome sequence differences in peripheral-blood samples taken prior to NRTI therapy and after 6-77 mo of treatment in 16 NRTI-treated patients. Samples from 10 HIV-infected, treatment-naive control individuals were taken at similar time intervals. Single-stranded conformation polymorphism (SSCP) and DNA-sequencing analysis techniques were used to detect mitochondrial genome sequence variants between paired longitudinal samples, and heteroplasmic populations were quantified after cloning and repeat SSCP/sequencing. Of 16 individuals treated with NRTIs, 5 exhibited altered SSCP profiles associated with the development of novel heteroplasmic DNA sequence changes, whereas no SSCP pattern change within these regions was observed in the control individuals. Heteroplasmic sequence changes were distributed across four regions of the genome: the noncoding region to 12S ribosomal RNA, reduced-nicotinamide-adenine-dinucleotide dehydrogenase 1, and cytochrome oxidase subunits I and III. Of the total of 26 patients who were examined in the present study, 4 of 5 patients with detectable mtDNA sequence changes since commencement of therapy developed evidence of peripheral fat wasting (lipoatrophy) between sample intervals (P=.031). One patient, without detectable sequence changes on NRTI therapy, also developed lipoatrophy. Levels of mtDNA copies/cell in blood samples were determined by quantitative PCR for 11 of the 16 NRTI-exposed patients; 7 of these 11 patients showed reduced levels of mtDNA in blood after therapy, including all 3 patients tested with evidence of mtDNA sequence changes on therapy. These data indicate that NRTI therapy provides conditions permissive for the development of peripheral-blood mtDNA mutations in vivo.

Base Sequence↗
PubMed1998

Conserved subfamilies of the Drosophila HeT-A telomere-specific retrotransposon.

HeT-A, a major component of Drosophila telomeres, is the first retrotransposon proposed to have a vital cellular function. Unlike most retrotransposons, more than half of its genome is noncoding. The 3' end contains > 2.5 kb of noncoding sequence. Copies of HeT-A differ by insertions or deletions and multiple nucleotide changes, which initially led us to conclude that HeT-A noncoding sequences are very fluid. However, we can now report, on the basis of new sequences and further analyses, that most of these differences are due to the existence of a small number of conserved sequence subfamilies, not to extensive sequence change during each transposition event. The high level of sequence conservation within subfamilies suggests that they arise from a small number of replicatively active elements. All HeT-A subfamilies show preservation of two intriguing features. First, segments of extremely A-rich sequence form a distinctive pattern within the 3' noncoding region. Second, there is a strong strand bias of nucleotide composition: The DNA strand running 5' to 3' toward the middle of the chromosome is unusually rich in adenine and unusually poor in guanine. Although not faced with the constraints of coding sequences, the HeT-A 3' noncoding sequence appears to be under other evolutionary constraints, possibly reflecting its roles in the telomeres.

Animals↗
PubMed2005

Sequence turnover and tandem repeats in cis-regulatory modules in drosophila.

The path by which regulatory sequence can change, yet preserve function, is an important open question for both evolution and bioinformatics. The recent sequencing of two additional species of Drosophila plus the wealth of data on gene regulation in the fruit fly provides new means for addressing this question. For regulatory sequences, indels account for more base pairs (bp) of change than substitutions (between Drosophila melanogaster and Drosophila yakuba), though they are fewer in number. Using Drosophila pseudoobscura as an out-group, we can distinguish insertions from deletions (with maximum parsimony criteria), and find a ratio between 1 and 5 (insertions to deletions) that is species dependent and much larger than the ratio of 1/8 for neutral sequences (Petrov and Hartl 1998). Because neutral sequence is rapidly cleared from the genome, most noncoding regions which preserve their length between D. melanogaster-D. pseudoobscura and have an excess of insertions over deletions should be functional. A fraction of 15%-18% (i.e., more than 20 standard deviations from random expectation) of the regulatory sequence is covered by low copy number tandem repeats whose repeating unit has an average length of 5-10 bp and which occur preferentially (25%-45% coverage) in indels. All indels may be due to tandem repeats if we extrapolate the detection efficiency of the repeat-finding algorithms using the observed point mutation rate between the species we compare. Sequence creation by local duplication accords with the tendency for multiple copies of transcription factor-binding sites to occur in regulatory modules. Thus, indel events and tandem repeats in particular need to be incorporated into models of regulatory evolution because they can alter the rate at which beneficial variants arise and should also influence bioinformatic algorithms that parse regulatory sequences into binding sites.

Animals↗
PubMed1991

Hair pigmentation in transgenic mice.

A mouse tyrosinase minigene, mg-Tyrs-J, in which genomic 5' noncoding flanking sequence was fused to a mouse tyrosinase cDNA, was introduced into fertilized eggs of BALB/c albino mice. Six transformed mice that exhibited brown agouti hair with some variations in the intensity of pigmentation were obtained. By crossing the founder mice with BALB/c albino mice, transgenic lines and sublines were established. Each subline expressed a characteristic phenotype with their respective band patterns in Southern blot analysis. This indicates that the character is expressed by transgenes integrated at a single location of the chromosomes. The difference in phenotypes among sublines is probably due to the position effect of the chromatin where the transgene is integrated, although a possibility that modification in the nucleotide sequence of the transgene is responsible for the difference cannot be excluded at present.

Animals↗
PubMed2002

Cell-free replication of the hepatitis C virus subgenomic replicon.

The hepatitis C virus (HCV) contains a plus-strand RNA genome. The 5' noncoding region (NCR) of the viral genome functions as an internal ribosome entry site, and its unique 3' NCR is required for the assembly of the replication complex during initiation of HCV RNA replication. Lohmann et al. (V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilman, and R. Batenschlager, Science 285:110-113, 1999) developed a subgenomic HCV replicon system, which represents an important tool in studying HCV replication in cultured cells. In this study, we describe a cell-free replication system that utilizes cytoplasmic lysates prepared from Huh-7 cells harboring the HCV subgenomic replicons. These lysates, which contain ribonucleoprotein complexes associated with cellular membranes, were capable of incorporating [alpha(32)P]CTP into newly synthesized RNA from subgenomic replicons in vitro. Replicative forms (RFs) and replicative intermediates (RIs) were synthesized from the endogenous HCV RNA templates. Consistent with previous observations, RFs were found to be resistant to RNase A digestion, whereas RIs were sensitive to RNase treatment. The radiolabeled HCV RF-RI complexes contained both minus and plus strands and were specific to the lysates derived from replicon-expressing cells. The availability of a cell-free replication system offers opportunities to probe the mechanism(s) of HCV replication. It also provides a novel assay for potential therapeutic agents.

Cell Line↗
PubMed2004

What can we learn from noncoding regions of similarity between genomes?

BACKGROUND: In addition to known protein-coding genes, large amounts of apparently non-coding sequence are conserved between the human and mouse genomes. It seems reasonable to assume that these conserved regions are more likely to contain functional elements than less-conserved portions of the genome. METHODS: Here we used a motif-oriented machine learning method based on the Relevance Vector Machine algorithm to extract the strongest signal from a set of non-coding conserved sequences. RESULTS: We successfully fitted models to reflect the non-coding sequences, and showed that the results were quite consistent for repeated training runs. Using the learned models to scan genomic sequence, we found that they often made predictions close to the start of annotated genes. We compared this method with other published promoter-prediction systems, and showed that the set of promoters which are detected by this method is substantially similar to that detected by existing methods. CONCLUSIONS: The results presented here indicate that the promoter signal is the strongest single motif-based signal in the non-coding functional fraction of the genome. They also lend support to the belief that there exists a substantial subset of promoter regions which share several common features including, but not restricted to, a relative abundance of CpG dinucleotides. This subset is detectable by a variety of distinct computational methods.

Animals↗
PubMed1997

[Functions of the segment-specific noncoding regions of influenza virus genome RNA].

The genome of influenza A viruses consists of eight negative-strand RNA segments. These segments contain the untranslated regions (UTRs), ranging from 20 to 61 nucleotides, at their 3' and 5' ends. The UTRs are composed of the highly conserved terminal nucleotides and the segment-specific nonconserved nucleotides located adjacent the open reading frame of the viral RNAs. Utilizing the virus-like model RNAs, whose nonconserved UTRs were mutated, deleted or replaced with those of other segments, the unique features of the nonconserved UTRs have been elucidated in the steps of transcription, translation, replication and RNA packaging into virus particles. Here I summarize current understanding of the functions of the segment-specific nonconserved UTRs of virus RNA.

Genome, Viral↗
PubMed1998

[Polymorphism of noncoding regions of the mitochondrial genome in the indigenous population of Southeastern Tuva Republic].

Deletion-insertion polymorphism of the V region and restriction polymorphism of D-loop for seven restriction enzymes--AspS9I (Cfr3I isoschizomere), BamHI, Bme18I (AvaII), EcoRV, HaeIII, KpnI, and Kzo9I (Sau3AI)--in mitochondrial DNA (mt DNA) were studied in the indigenous population of southeastern Tuva Republic. The results were compared with the data on Russians, Mongols, Buryats, and Altaians. Tuvinians significantly differed from Mongols in the frequency of the 9-bp deletion of the V region (1.89 and 8.07%, respectively). Additionally, significant differences between Tuvinians and samples of Mongols and Russians were obtained for frequencies of the AspS9I, HaeIII, and KpnI restriction sites. A comparison of the polymorphism of mitochondrial genome in individuals of different sexes and ages was performed in the studied sample.

Adult↗
PubMed2006

Genomic heterogeneity in the density of noncoding single-nucleotide and microsatellite polymorphisms in Plasmodium falciparum.

The density and distribution of single-nucleotide polymorphisms (SNPs) across the genome has important implications for linkage disequilibrium mapping and association studies, and the level of simple-sequence microsatellite polymorphisms has important implications for the use of oligonucleotide hybridization methods to genotype SNPs. To assess the density of these types of polymorphisms in P. falciparum, we sampled introns and noncoding DNA upstream and downstream of coding regions among a variety of geographically diverse parasites. Across 36,229 base pairs of noncoding sequence representing 41 genetic loci, a total of 307 polymorphisms including 248 polymorphic microsatellites and 39 SNPs were identified. We found a significant excess of microsatellite polymorphisms having a repeat unit length of one or two, compared to those with longer repeat lengths, as well as a nonrandom distribution of SNP polymorphisms. Almost half of the SNPs localized to only three of the 41 genetic loci sampled. Furthermore, we find significant differences in the frequency of polymorphisms across the two chromosomes (2 and 3) examined most extensively, with an excess of SNPs and a surplus of polymorphic microsatellites on chromosome 3 as compared to chromosome 2 (P=0.0001). Furthermore, at some individual genetic loci we also find a nonrandom distribution of polymorphisms between coding and flanking noncoding sequences, where completely monomorphic regions may flank highly polymorphic genes. These data, combined with our previous findings of nonrandom distribution of SNPs across chromosome 2, suggest that the Plasmodium falciparum genome may be a mosaic with regard to genetic diversity, containing chromosomal regions that are highly polymorphic interspersed with regions that are much less polymorphic.

Animals↗
PubMed2001

Interaction of poly(rC)-binding protein 2 with the 5'-terminal stem loop of the hepatitis C-virus genome.

The 5' noncoding region (NCR) of hepatitis C virus (HCV) contains an internal ribosome entry site for translation initiation. Cellular proteins (e.g. La, polypyrimidine tract-binding protein, and p25) that interact with HCV 5' NCR have been implicated in facilitating efficient internal initiation. The 5' NCR may also contain RNA structures and specific RNA sequences that interact with cellular proteins to promote RNA replication. UV crosslinking experiments revealed a 43-kDa cellular protein (p43) also interacts with the HCV 5' NCR. Further UV crosslinking experiments with deletion mutants of HCV 5' NCR demonstrated that p43 bound specifically to the 5'-terminal stem-loop of the HCV 5' NCR. Achromobactor proteinase I digests, competition experiments, and immunoprecipitation confirmed that p43 was identical to human poly(rC)-binding protein 2 (PCBP2). We prepared a PCBP2-immunodepleted rabbit reticulocyte lysate with an anti-PCBP2 antibody. Translation activity promoted by the HCV internal ribosome-entry site was the same in PCBP2-depleted lysates as in mock-depleted lysates. In conclusion, PCBP2 specifically interacted with the 5' terminus of HCV genome but had no effect on HCV translation. We speculate that PCBP2's interaction with HCV 5' NCR may be involved in the replication-initiation complex of HCV.

5' Untranslated Regions↗
PubMed2006

A comparative genomics strategy for targeted discovery of single-nucleotide polymorphisms and conserved-noncoding sequences in orphan crops.

Completed genome sequences provide templates for the design of genome analysis tools in orphan species lacking sequence information. To demonstrate this principle, we designed 384 PCR primer pairs to conserved exonic regions flanking introns, using Sorghum/Pennisetum expressed sequence tag alignments to the Oryza genome. Conserved-intron scanning primers (CISPs) amplified single-copy loci at 37% to 80% success rates in taxa that sample much of the approximately 50-million years of Poaceae divergence. While the conserved nature of exons fostered cross-taxon amplification, the lesser evolutionary constraints on introns enhanced single-nucleotide polymorphism detection. For example, in eight rice (Oryza sativa) genotypes, polymorphism averaged 12.1 per kb in introns but only 3.6 per kb in exons. Curiously, among 124 CISPs evaluated across Oryza, Sorghum, Pennisetum, Cynodon, Eragrostis, Zea, Triticum, and Hordeum, 23 (18.5%) seemed to be subject to rigid intron size constraints that were independent of per-nucleotide DNA sequence variation. Furthermore, we identified 487 conserved-noncoding sequence motifs in 129 CISP loci. A large CISP set (6,062 primer pairs, amplifying introns from 1,676 genes) designed using an automated pipeline showed generally higher abundance in recombinogenic than in nonrecombinogenic regions of the rice genome, thus providing relatively even distribution along genetic maps. CISPs are an effective means to explore poorly characterized genomes for both DNA polymorphism and noncoding sequence conservation on a genome-wide or candidate gene basis, and also provide anchor points for comparative genomics across a diverse range of species.

Base Sequence↗
PubMed2005

The small introns of antisense genes are better explained by selection for rapid transcription than by "genomic design".

Several models have been proposed to explain why expression parameters of a gene might be related to the size of the gene's introns. These include the idea that an energetic cost of transcription should favor smaller introns in highly expressed genes (the "economy selection" argument) and that tissue-specific genes reside in genomic locations with complex chromatin level control requiring large amounts of noncoding DNA (the "genomic design" hypothesis). We recently proposed a modification of the economy model arguing that, for some genes, the time that expression takes is more important than the energetic cost, such that some weakly but rapidly expressed genes might also have small introns. We suggested that antisense genes might be such a class and showed that the data appear to be consistent with this. We now reexamine this model to ask (a) whether the effects described were owing solely to the fact that antisense genes are often noncoding RNA and (b) whether we can confidently reject the "genomic design" model as an explanation for the facts. We show that the effects are not specific to noncoding RNAs and that the predictions of the "genomic design" model for the most part are not upheld.

Antisense Elements (Genetics)↗
PubMed2006

[Noncoding RNA, a new focus of functional genomic study].

Approximately 97% of all transcriptional output of the human being is noncoding RNA (ncRNA), which has been found to play roles in a great variety of processes, and is becoming more and more attractive. This review is a general introduction to the concept, classification and functions of ncRNA.

Genomics↗
PubMed1997

Organization and diversity of the 3'-noncoding region of classical swine fever virus genome.

Specific PCR primers were selected to amplify a 359 bp DNA fragment flanking the 3'-part of the polymerase gene and the 3'-noncoding (3'-NC) region of the genome of classical swine fever virus (CSFV). In RT-PCR the selected fragment was amplified from the genomes of 27 viral strains collected from Europe, America and Asia over a period of a half century as well as from three vaccine strains of CSFV. Eight PCR products were sequenced using an automatic sequencing device. Nucleotide sequence analysis was performed by computer programs DNASTAR and PHYLIP. The comparative studies revealed that the 3'-NC region contains a variable region of nucleotides which is located immediately after the stop codon TGA or TAA. Furthermore, a strongly conserved constant region was identified near to the extreme 3'-terminus. Two imperfect repeated sequences were found both in the variable and in the constant regions. In addition, the variable region was characterized by the occurrence of a 50 bp long poly AT track. Phylogenetic analysis with different mathematical approaches (MegAlign, Neighbor-Joining method, Maximum Likelihood, Parsimony) revealed that the studied CSFV strains were clustered into two main phylogenetic groups. Group I was comprised of the reference strain Brescia, together with old American and European field isolates and a Brazilian vaccine strain. Group II included the reference strain Alfort (Tübingen) and recent European field strains. The Congenital Tremor strain formed a distinct lineage which, although being highly divergent, was more closely related to group I than to group II. In conclusion, the present phylogenetic grouping yielded very similar results as previous studies base on comparison of the E2 (gp55) region. The agreement of the phylogenetic analysis in the two distinct regions confirms the reliability of the genetic grouping of CSFV strains into two main genogroups.

Animals↗
PubMed1988

Sequence and secondary structure analysis of the 5'-terminal region of flavivirus genome RNA.

The 5'-terminal noncoding region sequences were determined for the genome RNAs of seven strains of St. Louis encephalitis virus (SLEV) and one strain of West Nile virus (WNV) using a single synthetic cDNA primer complementary to the 5'-terminus of the coding region of a strain of WNV RNA. The 5'-terminal sequences obtained for the SLEV and WNV RNAs were compared with published sequences for yellow fever virus (YFV), Murray Valley encephalitis virus (MVEV), and dengue virus. While only short regions within the 5'-noncoding sequence were conserved among different flavivirus RNAs, significant homology was observed in this region among members of the same flavivirus subgroup and almost complete conservation was observed between different strains of the same virus. For example, seven strains of SLE, isolated from different geographic locations over a 17-year period and differing in their neurovirulence phenotype, contained only two to four nucleotide changes in the 5'-noncoding region. Interestingly, each of three low-virulence strains shared the same unique base substitution at position 16. Secondary structures predicted to be formed by the 5'-termini of each of the different flavivirus genome RNAs were of similar size and shape, in each case consisting of a stem with a small top loop and a larger side loop. The prediction of a common structure among a number of different flaviviruses, despite the lack of extensive sequence homology, suggests that this secondary structure is functionally important. An additional stem and loop structure is predicted to be formed in the region spanning the translation initiation codon. This structure showed significantly less conservation of size and shape than the 5'-terminal secondary structure.

Base Sequence↗