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PubMed2004

On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites.

Viroids and most viral satellites have small, noncoding, and highly structured RNA genomes. How they cause disease symptoms without encoding proteins and why they have characteristic secondary structures are two longstanding questions. Recent studies have shown that both viroids and satellites are capable of inducing RNA silencing, suggesting a possible role of this mechanism in the pathology and evolution of these subviral RNAs. Here we show that preventing RNA silencing in tobacco, using a silencing suppressor, greatly reduces the symptoms caused by the Y satellite of cucumber mosaic virus. Furthermore, tomato plants expressing hairpin RNA, derived from potato spindle tuber viroid, developed symptoms similar to those of potato spindle tuber viroid infection. These results provide evidence suggesting that viroids and satellites cause disease symptoms by directing RNA silencing against physiologically important host genes. We also show that viroid and satellite RNAs are significantly resistant to RNA silencing-mediated degradation, suggesting that RNA silencing is an important selection pressure shaping the evolution of the secondary structures of these pathogens.

Base Sequence↗
PubMed1989

Secondary structure analysis of adenovirus tripartite leader.

RNA secondary structure analysis was performed to understand the translation function of the adenovirus tripartite leader, a 200-nucleotide 5' noncoding region found on all late viral mRNAs. The tripartite leader facilitates the translation of viral mRNAs at late but not early times after infection and eliminates the normal requirement for the eukaryotic initiation factor 4F or cap binding protein complex. Secondary structures were determined by probing 5' or 3' end-labeled tripartite leader RNAs under nondenaturing conditions with various single strand-specific nucleases, and the information was used to generate a potential model structure. The resulting structure is attractive since it may explain the unusual translation behavior conferred by the tripartite leader. We demonstrate that the first leader segment is predominantly single-stranded, a property consistent with the ability to enhance translation and provide independence from cap binding protein complex. In contrast, the remaining two leader segments form a moderately stable base-paired structure, except for a large hairpin loop. To confirm these findings, the secondary structure of the tripartite leader was also probed when it was attached to a large segment of a messenger RNA and was found to be very similar to that of the individual leader RNA. These findings suggest several possible mechanisms to account for the translation activity of the tripartite leader.

Adenoviridae↗
PubMed1997

The 5'-terminal 32 basepairs conserved between genome segments A and B contain a major promoter element of infectious bursal disease virus.

The regions of the infectious bursal disease virus (IBDV) genome with regulatory function are not known. In the present study, progressively deleted lengths of the 5' noncoding region of segment A were constructed in pGL3 vectors having SV40 enhancer or promoter, and a luciferase (LUC) reporter gene. Transient transfections of the constructs made in a promoter-less pGL3-Enhancer vector when transfected in Vero cells and the lysates assayed for LUC expression, allowed the localization of maximal activity to the 32-nucleotide stretch (precursor polyprotein ORF positions -131 to -100), which is highly conserved at the 5' end of both genome segments. This fragment, when evaluated in parallel in an enhancer-less pGL3-Promoter vector demonstrated no activity. To determine if this region is recognized by IBDV replicative proteins, we engineered modifications in an enhancer-less pGL3-Promoter vector where the terminal 32-bp fragment, the full-length noncoding region, or the noncoding region with the 32-bp fragment deleted was positioned in either the plus-sense or the minus-sense orientation immediately downstream of the SV40 promoter and upstream of the LUC gene. Transfections of these constructs in IBDV-infected and uninfected Vero cells resulted in the endogenous generation of recombinant viral-LUC RNAs containing the 5' terminal viral RNA sequences in either the plus-sense or the minus-sense orientation. LUC assays of the infected cell lysates showed up-regulated expression of LUC only with constructs containing the 32-bp fragment in the minus-sense orientation. Deletion of this 32-bp fragment abolished such LUC expression. We therefore conclude that the 5'-terminal 32 base pairs of genomic segment A contain a major promoter element in IBDV. In addition, our results show that IBDV replicative proteins recognize and transcribe single-stranded RNA in vivo.

Animals↗
PubMed1983

Nonallelic members of the cytochrome c multigene family of the rat may arise through different messenger RNAs.

We determined the nucleotide sequences of three nonallelic cytochrome c genes (from recombinant clones Ch4A-RC5, 6 and 8) isolated from the rat cytochrome c gene family. In contrast with a fourth gene (from Ch4A-RC4), which has an intron and correctly encodes rat cytochrome c, these three appear to be pseudogenes and resemble mRNA molecules in two respects: they are all missing the intron of clone 4, and sequence homology with clone 4 in their 3' noncoding regions abruptly ends at two different A-rich tracts reminiscent of poly(A) tails. We also detect three cytochrome c mRNAs of sizes 1400, 1100 and 700 nucleotides in several tissues of the adult rat. The size differences among the mRNAs can be accounted for by length heterogeneity in their 3' noncoding regions. Two of the 3' ends map to the two points where the mRNA-like genes diverge from clone 4 at poly(A) tracts. Furthermore, short direct repeats flank the genes of clones 5, 6 and 8 at the positions where their sequences diverge. The observations suggest that these members of the cytochrome c multigene family may arise through insertion into the genome of DNA copies of cytochrome c mRNAs.

Animals↗
PubMed1995

Detection of enteroviruses and rhinoviruses in clinical specimens by PCR and liquid-phase hybridization.

A sensitive method based on PCR followed by liquid-phase hybridization for detection of enterovirus and rhinovirus RNAs in clinical specimens and cell culture supernatants is described. RNA was extracted from stool samples, throat swabs, nasopharyngeal aspirates, cerebrospinal fluid, urine, and plasma with a commercial phenol-guanidinium-chloroform reagent and purified on a polysulfone membrane, on which the reverse transcriptase reaction was also done. Two sets of oligonucleotide primers from the 5' noncoding region of picornaviruses were selected for DNA amplification of 153-bp (enterovirus) and 120-bp (rhinovirus) regions. Double-stranded amplicons were digested into single strands with T7 gene 6 exonuclease and quantitated by an assay using a europium-labeled probe, streptavidin- and biotinylated probe-coated microtitration wells, and time-resolved fluorometry. The sensitivity of the assay was about one template molecule when purified coxsackievirus A9 RNA was used. All enterovirus prototype strains, except echoviruses 22 and 23, and clinical isolates grown in cell culture or suckling mice were strongly positive by the enterovirus PCR-hybridization, as were selected prototype strains and untyped isolates of rhinoviruses by the rhinovirus PCR-hybridization. In a series of 100 clinical specimens tested, the results for 92 agreed with virus culture results. The detection method described will be useful in etiopathogenic studies on enteroviruses and rhinoviruses.

Animals↗
PubMed2001

Transcription and RNA replication of tacaribe virus genome and antigenome analogs require N and L proteins: Z protein is an inhibitor of these processes.

Tacaribe virus (TV), the prototype of the New World group of arenaviruses, comprises a single phylogenetic lineage together with four South American pathogenic producers of hemorrhagic disease. The TV genome consists of two single-stranded RNA segments called S and L. A reconstituted transcription-replication system based on plasmid-supplied TV-like RNAs and TV proteins was established. Plasmid expression was driven by T7 RNA polymerase supplied by a recombinant vaccinia virus. Plasmids were constructed to produce TV S segment analogs containing the negative-sense copy of chloramphenicol acetyltransferase (CAT) flanked at the 5' and 3' termini by sequences corresponding to those of the 5' and 3' noncoding regions of the S genome (minigenome) or the S antigenome (miniantigenome). In cells expressing N and L proteins, input minigenome or miniantigenome produced, respectively, encapsidated miniantigenome or minigenome which in turn produced progeny minigenome or progeny miniantigenome. Both minigenome and miniantigenome in the presence of N and L mediated transcription, which was analyzed as CAT expression. Coexpression of the small RING finger Z (p11) protein was highly inhibitory to both transcription and replication mediated by the minigenome or the miniantigenome. The effect depended on synthesis of Z protein rather than on plasmid or the RNA and was not ascribed to decreased amounts of plasmid-supplied template or proteins (N or L). N and L proteins were sufficient to support full-cycle RNA replication of a plasmid-supplied S genome analog in which CAT replaced the N gene. Replication of this RNA was also inhibited by Z expression.

Arenaviruses, New World↗
PubMed2006

Prediction of CsrA-regulating small RNAs in bacteria and their experimental verification in Vibrio fischeri.

The role of small RNAs as critical components of global regulatory networks has been highlighted by several recent studies. An important class of such small RNAs is represented by CsrB and CsrC of Escherichia coli, which control the activity of the global regulator CsrA. Given the critical role played by CsrA in several bacterial species, an important problem is the identification of CsrA-regulating small RNAs. In this paper, we develop a computer program (CSRNA_FIND) designed to locate potential CsrA-regulating small RNAs in bacteria. Using CSRNA_FIND to search the genomes of bacteria having homologs of CsrA, we identify all the experimentally known CsrA-regulating small RNAs and also make predictions for several novel small RNAs. We have verified experimentally our predictions for two CsrA-regulating small RNAs in Vibrio fischeri. As more genomes are sequenced, CSRNA_FIND can be used to locate the corresponding small RNAs that regulate CsrA homologs. This work thus opens up several avenues of research in understanding the mode of CsrA regulation through small RNAs in bacteria.

Aliivibrio fischeri↗
PubMed2000

Demonstration of functional requirement of polypyrimidine tract-binding protein by SELEX RNA during hepatitis C virus internal ribosome entry site-mediated translation initiation.

Polypyrimidine tract-binding protein (PTB) has been previously shown to physically interact with the hepatitis C virus (HCV) RNA genome at its 5'- and 3'-noncoding regions. Using high affinity SELEX RNA molecules, we present evidence for the functional requirement of PTB during HCV internal ribosome entry site (IRES)-controlled translation initiation. This study was carried out in rabbit reticulocyte translation lysates in which the HCV IRES-driven reporter RNA was introduced along with the PTB-specific SELEX RNA molecules. The SELEX RNAs specifically inhibited the HCV IRES function in the context of mono- and dicistronic mRNAs. The cap-dependent translation of a reporter (chloramphenicol acetyltransferase) RNA or naturally capped brome mosaic virus RNA, however, was not affected by the presence of SELEX during in vitro translation assays. The SELEX-mediated inhibition of the HCV IRES is shown to be relieved by the addition of recombinant human PTB in an add-back experiment. The in vivo requirement of PTB was further confirmed by cotransfection of Huh7 cells with reporter RNA and PTB-specific SELEX RNA. The HCV IRES activity was inhibited by the SELEX RNA in these cells, but not by an unrelated control RNA. Together, these results demonstrate the functional requirement of cellular PTB in HCV translation and further support the feasible use of SELEX RNA strategy in demonstrating the functional relevance of cellular protein(s) in complex biological processes.

Base Sequence↗
PubMed1993

Profilaggrin is a major epidermal calcium-binding protein.

Profilaggrin is a major highly phosphorylated protein component of the keratohyalin granules of mammalian epidermis. It contains 10 to 12 tandemly repeated filaggrin units and is processed into the intermediate filament-associated protein filaggrin by specific dephosphorylation and proteolysis during terminal differentiation of the epidermal cells. Later, filaggrin itself is degraded to free amino acids that participate in maintenance of epidermal flexibility. The present paper describes the structural organization of the 5' region of the human profilaggrin gene as well as the amino terminus of the profilaggrin protein. The primary profilaggrin transcript consists of three exons and two introns. The first exon (exon I) is only 54 bp and is untranslated. The coding sequences are distributed between exon II (159 bp) and exon III, which contains the information for 10 to 12 filaggrin repeats (972 bp each) and the 3' noncoding sequences. A very large intron separates exons I and II. The combination of a very short exon I with an unusually long intron 1 makes the structure of the profilaggrin gene unique among the epidermally expressed genes investigated so far. Comparison of the expression patterns revealed by primer extension and RNase protection analysis of foreskin epidermal and cultured keratinocyte RNAs suggests that alternately spliced messages, which are different from profilaggrin mRNA, are transcribed from the profilaggrin gene system at earlier stages of epidermal differentiation. The amino terminus of profilaggrin exhibits a significant homology to the small calcium-binding S100-like proteins. It contains two alpha-helical regions, termed EF-hands, that bind calcium in vitro. This is the first example of functional calcium-binding domains fused to a structural protein. We suggest that in addition to its role in filament aggregation and the maintenance of epidermal flexibility, profilaggrin may play an important role in the differentiation of the epidermis by autoregulating its own processing in a calcium-dependent manner or by participating in the transduction of calcium signal in epidermal cells.

Amino Acid Sequence↗
PubMed2005

Natural antisense transcripts: sound or silence?

Antisense RNA was a rather uncommon term in a physiology environment until short interfering RNAs emerged as the tool of choice to knock down the expression of specific genes. As a consequence, the concept of RNA having regulatory potential became widely accepted. Yet, there is more to come. Computational studies suggest that between 15 and 25% of mammalian genes overlap, giving rise to pairs of sense and antisense RNAs. The resulting transcripts potentially interfere with each other's processing, thus representing examples of RNA-mediated gene regulation by endogenous, naturally occurring antisense transcripts. Concerns that the large-scale antisense transcription may represent transcriptional noise rather than a gene regulatory mechanism are strongly opposed by recent reports. A relatively small, well-defined group of antisense or noncoding transcripts is linked to monoallelic gene expression as observed in genomic imprinting, X chromosome inactivation, and clonal expression of B and T leukocytes. For the remaining, much larger group of bidirectionally transcribed genes, however, the physiological consequences of antisense transcription as well as the cellular mechanism(s) involved remain largely speculative.

Alleles↗
PubMed2000

Localization of gurken RNA in Drosophila oogenesis requires elements in the 5' and 3' regions of the transcript.

During Drosophila oogenesis, signaling between the germline and the soma leads to the establishment of polarity in the egg and embryo. This process involves the interaction of gurken (grk), a TGFalpha-like protein, with torpedo (top), the Drosophila EGF receptor (Egfr). In early stage egg chambers, grk RNA is present predominantly along the posterior cortex of the oocyte, and in mid stage egg chambers, the grk transcript becomes tightly localized to the future dorsal anterior corner of the oocyte. This localization of grk RNA restricts the distribution of Gurken protein and is critical in defining both the anterior-posterior and dorsal-ventral axes of the egg. We have determined the genomic sequence of the grk gene. By testing the requirement of various fragments of grk RNA in the localization process, we find localization signals present in both the 5' and 3' regions of the gene. Sequences in the 5' noncoding region allow for accumulation of the transcript within the oocyte in early stage egg chambers, while signals in the coding region and the 3'UTR are necessary for localization in mid to late stage egg chambers. Active translation is not required for localization of the grk RNA. The mechanism of gurken RNA localization, therefore, differs from that of other localized RNAs studied to date.

3' Untranslated Regions↗
PubMed1981

Near identity of 3- RNA secondary structure in bromoviruses and cucumber mosaic virus.

The 3- terminal sequences of RNAs 1, 2, 3 and 4 from each of the three bromoviruses (brome mosaic, cowpea chlorotic mottle and broad bean mottle viruses) and also from cucumber mosaic virus display interviral sequence similarity in addition to strong intraviral homology. Interviral similarity is much more evident when RNA secondary, rather than primary, structures are compared. The last 190 bases of the various RNAs can fold into strikingly similar, extensively base-paired secondary structures whose common features are supported by RNA structure mapping. The extreme 3' end of each viral RNA can base-pair in two distinct configurations. Bromovirus RNA 3s each contain an unusually accessible internal oligo(A) sequence which, in brome mosaic virus at least, is located in the intercistronic noncoding region. Functional implications of these structural features are discussed.

Base Sequence↗
PubMed2006

Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae.

Mutations in RRP6 result in the accumulation of aberrant polyadenylated transcripts from small nucleolar RNA genes. We exploited this observation to search for novel noncoding RNA genes in the yeast genome. When RNA from rrp6Delta yeast is compared with wild-type on whole-genome microarrays, numerous intergenic loci exhibit an increased mutant/wild type signal ratio. Among these loci, we found one encoding a new C/D box small nucleolar RNA, as well as a surprising number that gave rise to heterogeneous Trf4p-polyadenylated RNAs with lengths of approximately 250-500 nt. This class of RNAs is not easily detected in wild-type cells and appears associated with promoters. Fine mapping of several such transcripts shows they originate near known promoter elements but do not usually extend far enough to act as mRNAs, and may regulate the transcription of downstream mRNAs. Rather than being uninformative transcriptional "noise," we hypothesize that these transcripts reflect important features of RNA polymerase activity at the promoter. This activity is normally undetectable in wild-type cells because the transcripts are somehow distinguished from true mRNAs and are degraded in an Rrp6p-dependent fashion in the nucleus.

2-Isopropylmalate Synthase↗
PubMed1991

Splice site requirement for the efficient accumulation of polyoma virus late mRNAs.

Polyoma virus late nuclear primary transcripts are giant and heterogeneous, containing tandem repeats of the late strand of the circular viral genome. Late pre-mRNA processing involves the splicing of noncoding 'leader' exons to each other (removing genome-length introns), with the joining of the last leader to a coding 'body' exon. We have constructed a number of mutants blocked only in leader-leader splicing, or blocked in both leader-leader and leader-body splicing. We examined the accumulation of both nuclear and cytoplasmic late-strand RNAs in NIH3T3 cells. Consistent with our previous results, mutants lacking the 3' splice site of the late leader (leader-leader splicing blocked) showed a 10-20 fold defect in late RNA accumulation. Mutants which lacked the leader 5' splice site (leader-body splicing blocked) had a more profound defect, exhibiting virtually no late-strand cytoplasmic or nuclear RNA. This result was unexpected as a substantial proportion of wild type late cytoplasmic messages are unspliced. A mutant with no intron, but having functional 3' and 5' splice sites bordering the leader exon, is capable of producing large amounts of unspliced late mRNA. This demonstrates that an excisable intron is not a requirement for late mRNA accumulation. The accumulation of polyoma late mRNAs requires the presence of leader exons bordered by functional 3' and 5' splice sites, whether or not these sites are used during pre-mRNA processing.

Base Sequence↗
PubMed2003

Dosage compensation: an intertwined world of RNA and chromatin remodelling.

Dosage compensation mechanisms in flies and mammals provide an exquisite example of chromatin associated RNAs in chromosome-wide transcription regulation. Recent progress shows that chromatin modifications are also closely linked to these processes. Concerted action of the RNA/chromatin-modifying enzymes may play a crucial role in determining transcriptional output. Furthermore, non-coding RNAs appear to play a dual role, being targeting modules as well as encoding for target sites for complex recognition.

Animals↗
PubMed1999

Protein 2A of grapevine fanleaf nepovirus is implicated in RNA2 replication and colocalizes to the replication site.

RNA2 of grapevine fanleaf virus is replicated in trans by the RNA1-encoded replication machinery. Full processing of the RNA2-encoded polyprotein P2 yields protein 2A of unknown function, the movement protein 2B(MP), and the coat protein 2C(CP). Analysis of a set of deletion mutants in the P2-coding sequence revealed that protein 2A is necessary but not sufficient for RNA2 replication. In addition to the 5' and 3' noncoding sequences and the 2A-coding sequence, an additional sequence coding for 2B(MP) and/or 2C(CP) or the green fluorescent protein (GFP) is necessary for RNA2 replication. When 2A fused to GFP (2AGFP) was transiently expressed in uninfected T-BY2 protoplasts, 2AGFP appeared as punctate structures evenly distributed in the cytoplasm. However, in cells cotransfected with grapevine fanleaf virus RNAs and the 2AGFP construct, 2AGFP was predominantly found in a juxtanuclear location along with 1D(pro) and 1C(VPg), two RNA1-encoded proteins involved in RNA replication. Viral RNA replication as traced by 5-bromouridine 5' triphosphate (BrUTP) incorporation into newly synthesized RNA occurred at the same location. This colocalization is consistent with the hypothesis that 2A enables RNA2 replication through its association with the replication complex assembled from RNA1-encoded proteins.

Base Sequence↗
PubMed2005

Homologous crossovers among molecules of brome mosaic bromovirus RNA1 or RNA2 segments in vivo.

Previously we demonstrated frequent homologous crossovers among molecules of the RNA3 segment in the tripartite brome mosaic bromovirus (BMV) RNA genome (A. Bruyere, M. Wantroba, S. Flasinski, A. Dzianott, and J. J. Bujarski, J. Virol. 74:4214-4219, 2000). To further our knowledge about mechanisms of viral RNA genome variability, in this paper we have studied homologous recombination in BMV RNA1 and RNA2 components during infection. We have found that basal RNA-RNA crossovers could occur within coding regions of both RNAs, although recombination frequencies slightly varied at different RNA sections. In all cases, the frequencies were much lower than the rate observed for the intercistronic recombination hot spot in BMV RNA3. Probability calculations accounted for at least one homologous crossover per RNA molecule per replication cycle. In addition, we have demonstrated an efficient repair of mutations within the conserved 3' and 5' noncoding regions, most likely due to error-prone BMV RNA replication. Overall, our data verify that homologous crossovers are common events a during virus life cycle, and we discuss their importance for viral RNA genetics.

Bromovirus↗
PubMed1989

Regulation of gene expression during adipocyte differentiation: a review.

The differentiation of adipose precursor cells is accompanied by the acquisition of adipocyte-specific messenger (m) RNAs allowing characteristic changes in protein composition. The development of methods for cloning and characterizing individual genes has provided the opportunity to study selective gene expression by adipocytes at the molecular level. In this review, the information obtained to date regarding transcriptional and post-transcriptional regulatory mechanisms utilized by adipocytes is summarized. Included are descriptions of conserved DNA sequences found in noncoding regions of adipose genes and of how protein-DNA interactions at these regions are thought to regulate the initiation of transcription. Among the transcription factors implemented in regulation of adipocyte-specific gene expression are the protein product of the proto-oncogene c-fos, the triiodothyronine receptor and steroid hormone receptors. Data also are discussed that indicate that RNA editing and determinants of mRNA stability contribute to the adipocyte phenotype. It is hoped that a review of the regulatory aspects of gene expression during adipocyte differentiation will identify areas deserving further study by researchers interested in understanding adipose development.

Adipose Tissue↗