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PubMed1995

Mutations within noncoding terminal sequences of model RNAs of Sendai virus: influence on reporter gene expression.

A reverse-genetics system employing the chloramphenicol acetyltransferase (CAT) reporter gene has been established previously for Sendai virus. We utilized PCR-directed mutagenesis to introduce nucleotide additions, deletions, and/or substitutions within terminal Sendai virus RNA sequences. The influence of these mutations on replication-transcription of these model Sendai-CAT RNAs was analyzed by assaying CAT activity. Results from these experiments indicate that (i) Sendai-CAT RNAs expressing wild-type levels of CAT activity conform to the Sendai virus rule of six, (ii) apparent exceptions to the rule of six exist in that the 5' terminus of the Sendai-CAT RNA is more tolerant than the 3' terminus of nucleotide additions or deletions, and (iii) the 3' leader region of Sendai-CAT RNA appears to be sensitive not only to mutagenesis (single-nucleotide addition or deletion) but also to changes in its total nucleotide length.

Base Sequence↗

PubMed2005

Large-scale transcriptome analyses reveal new genetic marker candidates of head, neck, and thyroid cancer.

A detailed genome mapping analysis of 213,636 expressed sequence tags (EST) derived from nontumor and tumor tissues of the oral cavity, larynx, pharynx, and thyroid was done. Transcripts matching known human genes were identified; potential new splice variants were flagged and subjected to manual curation, pointing to 788 putatively new alternative splicing isoforms, the majority (75%) being insertion events. A subset of 34 new splicing isoforms (5% of 788 events) was selected and 23 (68%) were confirmed by reverse transcription-PCR and DNA sequencing. Putative new genes were revealed, including six transcripts mapped to well-studied chromosomes such as 22, as well as transcripts that mapped to 253 intergenic regions. In addition, 2,251 noncoding intronic RNAs, eventually involved in transcriptional regulation, were found. A set of 250 candidate markers for loss of heterozygosis or gene amplification was selected by identifying transcripts that mapped to genomic regions previously known to be frequently amplified or deleted in head, neck, and thyroid tumors. Three of these markers were evaluated by quantitative reverse transcription-PCR in an independent set of individual samples. Along with detailed clinical data about tumor origin, the information reported here is now publicly available on a dedicated Web site as a resource for further biological investigation. This first in silico reconstruction of the head, neck, and thyroid transcriptomes points to a wealth of new candidate markers that can be used for future studies on the molecular basis of these tumors. Similar analysis is warranted for a number of other tumors for which large EST data sets are available.

Alternative Splicing↗

PubMed2006

A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA).

MicroRNAs represent a class of short (approximately 22 nt), noncoding regulatory RNAs involved in development, differentiation, and metabolism. We describe a novel microarray platform for genome-wide profiling of mature miRNAs (miChip) using locked nucleic acid (LNA)-modified capture probes. The biophysical properties of LNA were exploited to design probe sets for uniform, high-affinity hybridizations yielding highly accurate signals able to discriminate between single nucleotide differences and, hence, between closely related miRNA family members. The superior detection sensitivity eliminates the need for RNA size selection and/or amplification. MiChip will greatly simplify miRNA expression profiling of biological and clinical samples.

Animals↗

PubMed2005

RNA sequence and secondary structure participate in high-affinity CsrA-RNA interaction.

The global Csr regulatory system controls bacterial gene expression post-transcriptionally. CsrA of Escherichia coli is an RNA binding protein that plays a central role in repressing several stationary phase processes and activating certain exponential phase functions. CsrA regulates translation initiation of several genes by binding to the mRNA leaders and blocking ribosome binding. CsrB and CsrC are noncoding regulatory RNAs that are capable of sequestering CsrA and antagonizing its activity. Each of the known target transcripts contains multiple CsrA binding sites, although considerable sequence variation exists among these RNA targets, with GGA being the most highly conserved element. High-affinity RNA ligands containing single CsrA binding sites were identified from a combinatorial library using systematic evolution of ligands by exponential enrichment (SELEX). The SELEX-derived consensus was determined as RUACARGGAUGU, with the ACA and GGA motifs being 100% conserved and the GU sequence being present in all but one ligand. The majority (51/55) of the RNAs contained GGA in the loop of a hairpin within the most stable predicted structure, an arrangement similar to several natural CsrA binding sites. Strikingly, the identity of several nucleotides that were predicted to form base pairs in each stem were 100% conserved, suggesting that primary sequence information was embedded within the base-paired region. The affinity of CsrA for several selected ligands was measured using quantitative gel mobility shift assays. A mutational analysis of one selected ligand confirmed that the conserved ACA, GGA, and GU residues were critical for CsrA binding and that RNA secondary structure participates in CsrA-RNA recognition.

Bacterial Proteins↗

PubMed2004

Identification and analysis of the minimal promoter activity of a novel noncoding nuclear RNA gene, AncR-1, from the honeybee (Apis mellifera L.).

Previously, we identified a gene for a noncoding nuclear RNA, termed Ks-1, that is expressed preferentially in a restricted set of neurons in the honeybee brain. In the present study, we identified another novel gene, termed AncR-1, whose transcripts were localized to nuclei in the whole cortex region of the honeybee brain, as a candidate novel noncoding nuclear RNA gene. RNA fluorescent in situ hybridization revealed that AncR-1 and Ks-1 transcripts were located in a distinct portion of a single neural nucleus, suggesting that they have distinct functions in brain neurons. cDNA cloning revealed that the AncR-1 transcripts were up to 7 kb in size, had mRNA-like structures, and were alternatively spliced. The reporter assay using Drosophila SL-2 cells demonstrated that a TATA box-like sequence located -30 bp upstream of the 5' end of AncR-1 cDNA had promoter activity. None of the alternatively spliced AncR-1 cDNA variants contained significant open reading frames, strongly suggesting that AncR-1 transcripts function as novel noncoding nuclear RNAs. Furthermore, in situ hybridization revealed that AncR-1 was expressed not only in the brain but also in the sex organs in the queen and drones and in the hypopharyngeal glands and oenocytes of the worker bees, suggesting that AncR-1 is involved in diverse organ functions. Some of the AncR-1 transcripts enriched in the nuclei of the hypopharyngeal glands were polyadenylated, indicating the presence of mRNA-like AncR-1 transcripts in the nuclei.

Animals↗

PubMed2004

Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs.

The factors regulating the expression of microRNAs (miRNAs), a ubiquitous family of approximately 22-nt noncoding regulatory RNAs, remain undefined. However, it is known that miRNAs are first transcribed as a largely unstructured precursor, termed a primary miRNA (pri-miRNA), which is sequentially processed in the nucleus, to give the approximately 65-nt pre-miRNA hairpin intermediate, and then in the cytoplasm, to give the mature miRNA. Here we have sought to identify the RNA polymerase responsible for miRNA transcription and to define the structure of a full-length human miRNA. We show that the pri-miRNA precursors for nine human miRNAs are both capped and polyadenylated and report the sequence of the full-length, approximately 3433-nt pri-miR-21 RNA. This pri-miR-21 gene sequence is flanked 5' by a promoter element able to transcribe heterologous mRNAs and 3' by a consensus polyadenylation sequence. Nuclear processing of pri-miRNAs was found to be efficient, thus largely preventing the nuclear export of full-length pri-miRNAs. Nevertheless, an intact miRNA stem-loop precursor located in the 3' UTR of a protein coding gene only moderately inhibited expression of the linked open reading frame, probably because the 3' truncated mRNA could still be exported and expressed. Together, these data show that human pri-miRNAs are not only structurally similar to mRNAs but can, in fact, function both as pri-miRNAs and mRNAs.

Base Sequence↗

PubMed2004

Drosophila male-specific lethal 2 protein controls sex-specific expression of the roX genes.

The MSL complex of Drosophila upregulates transcription of the male X chromosome, equalizing male and female X-linked gene expression. Five male-specific lethal proteins and at least one of the two noncoding roX RNAs are essential for this process. The roX RNAs are required for the localization of MSL complexes to the X chromosome. Although the mechanisms directing targeting remain speculative, the ratio of MSL protein to roX RNA influences localization of the complex. We examine the transcriptional regulation of the roX genes and show that MSL2 controls male-specific roX expression in the absence of any other MSL protein. We propose that this mechanism maintains a stable MSL/roX ratio that is favorable for localization of the complex to the X chromosome.

Animals↗

PubMed2004

A small modulatory dsRNA specifies the fate of adult neural stem cells.

Discovering the molecular mechanisms that regulate neuron-specific gene expression remains a central challenge for CNS research. Here, we report that small, noncoding double-stranded (ds) RNAs play a critical role in mediating neuronal differentiation. The sequence defined by this dsRNA is NRSE/RE1, which is recognized by NRSF/REST, known primarily as a negative transcriptional regulator that restricts neuronal gene expression to neurons. The NRSE dsRNA can trigger gene expression of neuron-specific genes through interaction with NRSF/REST transcriptional machinery, resulting in the transition from neural stem cells with neuron-specific genes silenced by NRSF/REST into cells with neuronal identity that can express neuronal genes. The mechanism of action appears to be mediated through a dsRNA/protein interaction, rather than through siRNA or miRNA. The discovery of small modulatory dsRNAs (smRNAs) extends the important contribution of noncoding RNAs as key regulators of cell behavior at both transcriptional and posttranscriptional levels.

Animals↗

PubMed2005

Fewer genes, more noncoding RNA.

Recent studies showing that most "messenger" RNAs do not encode proteins finally explain the long-standing discrepancy between the small number of protein-coding genes found in vertebrate genomes and the much larger and ever-increasing number of polyadenylated transcripts identified by tag-sampling or microarray-based methods. Exploring the role and diversity of these numerous noncoding RNAs now constitutes a main challenge in transcription research.

Animals↗

PubMed2006

Alternative splicing of the first intron of the steroid receptor RNA activator (SRA) participates in the generation of coding and noncoding RNA isoforms in breast cancer cell lines.

The Steroid Receptor RNA Activator 1 (SRA1) has originally been described as a noncoding RNA specifically activating steroid receptor transcriptional activity. We have, however, identified, in human breast tissue, exon- 1 extended SRA1 isoforms containing two initiating AUG codons and encoding a protein we called SRAP. We recently reported a decreased estrogen receptor activity in breast cancer cells overexpressing SRAP, suggesting antagonist roles played by SRA1 RNA and SRAP. SRA1 appears to be the first example of a molecule active both at the RNA and at the protein level. No data are currently available regarding the mechanisms possibly involved in the generation of coding and noncoding functional SRA1 RNAs. Using 5'-Rapid Amplification of cDNA Extremities (5'-RACE), we have herein identified several putative transcription initiation sites surrounding the second methionine codon and used to generate coding SRA1 transcripts. In the process, we also identified an alternatively spliced noncoding SRA1 transcript still containing an intron-1 sequence. Using targeted RT-PCR approaches, we confirmed the presence in breast cancer cell lines of SRA1 RNAs containing a full as well as a partial intron-1 sequence and established that the relative proportion of these RNAs varied within breast cancer cell lines. Using a "minigene" strategy, we also showed that artificial RNAs containing the SRA1 intron-1 sequence are alternatively spliced in breast cancer cell lines. Interestingly, the splicing pattern of the minigene products parallels the one of the endogenous SRA1 transcripts. Altogether, our data suggest that the primary genomic sequence in and around intron-1 is sufficient to lead to a differential splicing of this intron. We propose that alternative splicing of intron-1 is one mechanism used by breast cancer cells to regulate the balance between coding and functional noncoding SRA1 RNAs.

Alternative Splicing↗

PubMed1999

Noncoding RNA genes.

Some genes produce RNAs that are functional instead of encoding proteins. Noncoding RNA genes are surprisingly numerous. Recently, active research areas include small nucleolar RNAs, antisense riboregulator RNAs, and RNAs involved in X-dosage compensation. Genome sequences and new algorithms have begun to make systematic computational screens for noncoding RNA genes possible.

Animals↗

PubMed2006

Identification of a large noncoding RNA in extremophilic eubacteria.

We have discovered a large and highly conserved RNA motif that typically resides in a noncoding section of a multigene messenger RNA in extremophilic Gram-positive eubacteria. RNAs of this class adopt an ornate secondary structure, are large compared with most other noncoding RNAs, and have been identified only in certain extremophilic bacteria. These ornate, large, extremophilic (OLE) RNAs have a length of approximately 610 nucleotides, and the 35 representatives examined exhibit extraordinary conservation of nucleotide sequence and base pairing. Structural probing of the OLE RNA from Bacillus halodurans corroborates a complex secondary structure model predicted from comparative sequence analysis. The patterns of structural conservation, and its unique phylogenetic distribution, suggest that OLE RNA carries out a complex and critical function only in certain extremophilic bacteria.

Bacteria↗

PubMed2007

A structured viroid RNA serves as a substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RNA-induced silencing complex-mediated degradation.

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.

Arabidopsis↗

PubMed1991

Direct introduction and transient expression of capped and non-capped RNA in Saccharomyces cerevisiae.

We report the introduction of functional RNA molecules into yeast spheroplasts. Plasmids containing the firefly luciferase coding region were transcribed to yield RNAs suitable for introduction into yeast cells and direct assay of their translation products. The 5' noncoding regions of the RNAs were derived either from the 5' noncoding regions of firefly luciferase, poliovirus, or yeast virus-like-particle (VLP) L-A or M1 RNAs. Capped and non-capped mRNAs were made by T7 RNA polymerase-directed transcription and introduced into yeast spheroplasts. The peak time of luciferase transient expression from introduced RNAs was 2-4 h after their introduction. In contrast, transient expression of luciferase from a non-replicative, luciferase-encoding plasmid introduced into the cells was maximal at 16 h. For capped mRNAs, luciferase activity increased linearly with transcript amount for both yeast and human (HeLa) cells. Although non-capped luciferase mRNAs were expressed more efficiently following introduction into yeast than into HeLa cells, the 5' noncoding sequences from yeast double-stranded (ds)RNA VLP RNAs conferred no greater apparent cap-independence than non-VLP RNA sequences in this transient expression assay. The RNA transient expression system will allow the study of translation of capped and non-capped RNAs in yeast cells and of the replicative cycle of yeast virus-like RNA genomes.

Base Sequence↗

PubMed1990

Factors affecting efficient infection of tobacco with in vitro RNA transcripts from cloned cDNAs of satellite tobacco mosaic virus.

Recombinant cDNA clones of the complete satellite tobacco mosaic virus (STMV) genome (1059 ribonucleotides) were constructed with unique Xbal and HindIII or Pstl restriction sites engineered at the 5' and 3' termini, respectively. The genome-length cDNAs were positioned downstream of T7 or SP6 phage promoters. Genome-sense RNAs transcribed in vitro from the T7 promoter were biologically active, while negative-sense RNAs transcribed in vitro from the SP6 promoter were not. Constructs that were identical to STMV and two other constructs in which there were two or six specific nucleotide differences in the 3' noncoding region yielded RNAs that were infectious. Sequence analysis of the progeny RNA derived from infections with transcripts containing nucleotide differences between nucleotides 682 and 753 revealed that these changes in sequence were maintained. In contrast, differences in the nucleotide sequence between nucleotides 989 and 1059 were not maintained in progeny RNA; one mutant reverted to the wild-type sequence, and the other generated a new sequence during infection.

Base Sequence↗

PubMed1997

Secondary structure of the 5'-noncoding region of border disease virus genome RNAs.

The computer predicted secondary structures from the 5'-NC genome region of four border disease virus (BDV) strains collected from sheep in England and Scotland over a period 1976-1986 were prepared. The FOLD program from GCG sequence analysis software package was used for the analysis of a 243 bp RNA fragment. Two typical shapes of secondary structures were observed which contained multiple imperfect stem-loop motifs. The shape of those structures exactly fit with the grouping of BDV strains to two phylogenetic groups. Secondary structures are typical only of BDV strains and they are different from the structures prepared for NADL (BVDV) and Alfort (CSFV) strains.

Animals↗

PubMed2002

Avian bic, a gene isolated from a common retroviral site in avian leukosis virus-induced lymphomas that encodes a noncoding RNA, cooperates with c-myc in lymphomagenesis and erythroleukemogenesis.

bic is a novel gene identified at a common retroviral integration site in avian leukosis virus-induced lymphomas and has been implicated as a collaborator with c-myc in B lymphomagenesis. It lacks an extensive open reading frame and is believed to function as an untranslated RNA (W. Tam, Gene 274:157-167, 2001; W. Tam, D. Ben-Yehuda, and W. S. Hayward, Mol. Cell. Biol. 17:1490-1502, 1997). The oncogenic potential of bic, particularly its ability to cooperate with c-myc in oncogenesis, was tested directly by expressing c-myc and bic, either singly or in pairwise combination, in cultured chicken embryo fibroblasts (CEFs) and in chickens using replication-competent retrovirus vectors. Coexpression of c-myc and bic in CEFs caused growth enhancement of cells. Most importantly, chick oncogenicity assays demonstrated that bic can cooperate with c-myc in lymphomagenesis and erythroleukemogenesis. The present study provides direct evidence for the involvement of untranslated RNAs in oncogenesis and provides further support for the role of noncoding RNAs as riboregulators.

Animals↗

PubMed2003

Inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid RNA.

Viroids are small noncoding and infectious RNAs that replicate autonomously and move systemically throughout an infected plant. The RNAs of the family Pospiviroidae contain a central conserved region (CCR) that has long been thought to be involved in replication. Here, we report that the CCR of Potato spindle tuber viroid (PSTVd) also plays a role in pathogenicity. A U257A change in the CCR converted the intermediate strain PSTVd(Int) to a lethal strain that caused severe growth stunting and premature death of infected plants. PSTVd with nucleotide U257 changed to C or G did not cause such symptoms. The pathogenic effect of the U257A substitution was abolished by a C259U substitution in the same RNA. Analyses of the pathogenic effects of the U257A substitution in three other PSTVd variants established A257 as a new pathogenicity determinant that functions independently and synergistically with the classic pathogenicity domain. The U257A substitution did not alter PSTVd secondary structure, replication levels, or tissue tropism. The stunted growth of PSTVd(Int)U257A-infected tomato plants resulted from restricted cell expansion but not cell division or differentiation. This was correlated positively with the downregulated expression of an expansin gene, LeExp2. Our results demonstrate that specific nucleotides in a noncoding, pathogenic RNA have a profound effect in altering distinct cellular responses, which then lead to well-defined alterations in plant growth and developmental patterns. The feasibility of correlating viroid RNA sequence/structure with the altered expression of specific host genes, cellular processes, and developmental patterns makes viroid infection a valuable system in which to investigate host factors for symptom expression and perhaps also to characterize the mechanisms of RNA regulation of gene expression in plants.

Base Sequence↗