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PubMed1988

Structure of the rat insulin-like growth factor II transcriptional unit: heterogeneous transcripts are generated from two promoters by use of multiple polyadenylation sites and differential ribonucleic acid splicing.

The rat insulin-like growth factor II (rIGF-II) gene, which exists as a single copy in the genome, is expressed as a multitranscript family of mRNA molecules ranging in size from 4.6 to 1 kilobases. Part of this heterogeneity can be ascribed to the presence of two different promoters, each transcribing alternative 5'-noncoding regions which are spliced to common coding exons. In the present study we use a combination of DNA sequence analysis of the gene, mapping of the mRNA molecules by Northern analysis and ribonuclease protection experiments, and DNA sequence analysis of cDNA clones complementary to different regions of the genome to establish the structure of several rIGF-II mRNA species. These results indicate that RNA heterogeneity also arises from the use of different polyadenylation sites. In addition, a variant 2 kilobases RNA was observed that was colinear with the distal 1700 base pairs of the 3147 base pair long exon 3, and may arise by alternative RNA splicing. These posttranscriptional modifications of RNAs arising from the rIGF-II transcription unit may generate molecules with different functional potential.

Animals↗
PubMed2005

Kaposi's sarcoma-associated herpesvirus expresses an array of viral microRNAs in latently infected cells.

MicroRNAs (miRNAs) are an endogenously encoded class of small RNAs that have been proposed to function as key posttranscriptional regulators of gene expression in a range of eukaryotic species, including humans. The small size of miRNA precursors makes them potentially ideal for use by viruses as inhibitors of host cell defense pathways. Here, we demonstrate that the pathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) encodes an array of 11 distinct miRNAs, all of which are expressed at readily detectable levels in latently KSHV infected cells. Individual KSHV miRNAs were expressed at up to 2,200 copies per cell. The KSHV miRNAs are expressed from what appears to be a single genetic locus that largely coincides with an approximately 4-kb noncoding sequence located between the KSHV v-cyclin and K12/Kaposin genes, both of which are also expressed in latently infected cells. Computer analysis of potential mRNA targets for these viral miRNAs identified a number of interesting candidate genes, including several mRNAs previously shown to be down-regulated in KSHV-infected cells. We hypothesize that these viral miRNAs play a critical role in the establishment and/or maintenance of KSHV latent infection in vivo and, hence, in KSHV-induced oncogenesis.

Base Sequence↗
PubMed1993

A cytoplasmic 57-kDa protein that is required for translation of picornavirus RNA by internal ribosomal entry is identical to the nuclear pyrimidine tract-binding protein.

Initiation of translation of the RNA genomes of picornaviruses such as poliovirus and encephalomyocarditis virus is cap-independent and results from interaction of ribosomes with a segment of the 5' noncoding region of these mRNAs termed the internal ribosomal entry site. Genetic and biochemical studies have previously shown that a 57-kDa cytoplasmic RNA-binding protein (p57) plays an essential role in this translation mechanism. We have now found that p57 shares physical, biochemical, and antigenic properties with the pyrimidine tract-binding protein (PTB), a nuclear protein that has been implicated in various processes involving pre-mRNA. These data indicate that p57 and PTB are the same protein. Purified recombinant PTB bound specifically to a bulged hairpin within the internal ribosomal entry site of encephalomyocarditis virus and had a much lower affinity for a mutated derivative of this hairpin and for unrelated RNAs. Immunodepletion of p57/PTB from a HeLa cell-free lysate inhibited translation of poliovirus and encephalomyocarditis virus mRNAs but had no effect on translation of beta-globin mRNA, confirming the essential role of p57 in translation by internal ribosomal entry.

Base Sequence↗
PubMed2002

Functional analysis of yeast snoRNA and snRNA 3'-end formation mediated by uncoupling of cleavage and polyadenylation.

Many nuclear and nucleolar small RNAs are accumulated as nonpolyadenylated species and require 3'-end processing for maturation. Here, we show that several genes coding for box C/D and H/ACA snoRNAs and for the U5 and U2 snRNAs contain sequences in their 3' portions which direct cleavage of primary transcripts without being polyadenylated. Genetic analysis of yeasts with mutations in different components of the pre-mRNA cleavage and polyadenylation machinery suggests that this mechanism of 3"-end formation requires cleavage factor IA (CF IA) but not cleavage and polyadenylation factor activity. However, in vitro results indicate that other factors participate in the reaction besides CF IA. Sequence analysis of snoRNA genes indicated that they contain conserved motifs in their 3" noncoding regions, and mutational studies demonstrated their essential role in 3"-end formation. We propose a model in which CF IA functions in cleavage and polyadenylation of pre-mRNAs and, in combination with a different set of factors, in 3"-end formation of nonpolyadenylated polymerase II transcripts.

Base Sequence↗
PubMed1991

Host-derived 5' ends and overlapping complementary 3' ends of the two mRNAs transcribed from the ambisense S segment of Uukuniemi virus.

Two mRNAs, coding for the N and NSS proteins, are transcribed from the small (S) Uukuniemi virus RNA segment by an ambisense strategy (J. F. Simons, U. Hellman, and R. F. Pettersson, J. Virol. 64:247-255, 1990). In this report, we describe the analysis of the 5' and 3' ends of the two mRNAs. Primer extension as well as cloning and sequencing of individual mRNAs showed that the 5' ends of both mRNAs contained nonviral sequences ranging from 7 to 25 residues in length (mean, 12 residues), indicating a cap-snatching mechanism similar to the one originally described for priming of influenza virus mRNA synthesis. In 35% of the cases, the first virion-specified nucleotide (an A residue) was substituted with a G residue. Between the translation termination codons of N and NSS, there is a 74-residue-long noncoding intergenic region (Simons et al., J. Virol. 64:247-255, 1990). Nuclease protection assays using both RNA and DNA hybridization probes showed that the 3' ends of the N and NSS mRNAs overlap each other by about 100 nucleotides. The 3' end of the NSS mRNA extends into the coding sequence of the N mRNA, whereas the N mRNA is terminated just prior to the stop codon of NSS. To our knowledge, this is the first example of overlapping complementary mRNAs in viruses with an ambisense coding strategy. No obvious transcription termination sequence was identified. However, because of a short palindromic sequence in the intergenic region, the 3' ends of both mRNAs (and consequently also the template RNAs) can be folded into an A/U-rich hairpin structure. It remains to be determined whether this structure plays any role in transcription termination.

Amino Acid Sequence↗
PubMed1997

Host range phenotype induced by mutations in the internal ribosomal entry site of poliovirus RNA.

Most poliovirus strains infect only primates. The host range (HR) of poliovirus is thought to be primarily determined by a cell surface molecule that functions as poliovirus receptor (PVR), since it has been shown that transgenic mice are made poliovirus sensitive by introducing the human PVR gene into the genome. The relative levels of neurovirulence of polioviruses tested in these transgenic mice were shown to correlate well with the levels tested in monkeys (H. Horie et al., J. Virol. 68:681-688, 1994). Mutants of the virulent Mahoney strain of poliovirus have been generated by disruption of nucleotides 128 to 134, at stem-loop II within the 5' noncoding region, and four of these mutants multiplicated well in human HeLa cells but poorly in mouse TgSVA cells that had been established from the kidney of the poliovirus-sensitive transgenic mouse. Neurovirulence tests using the two animal models revealed that these mutants were strongly attenuated only in tests with the mouse model and were therefore HR mutants. The virus infection cycle in TgSVA cells was restricted by an internal ribosomal entry site (IRES)-dependent initiation process of translation. Viral protein synthesis and the associated block of cellular protein synthesis were not observed in TgSVA cells infected with three of four HR mutants and was evident at only a low level in the remaining mutant. The mutant RNAs were functional in a cell-free protein synthesis system from HeLa cells but not in those from TgSVA and mouse neuroblastoma NS20Y cells. These results suggest that host factor(s) affecting IRES-dependent translation of poliovirus differ between human and mouse cells and that the mutant IRES constructs detect species differences in such host factor(s). The IRES could potentially be a host range determinant for poliovirus infection.

Animals↗
PubMed2004

Systematic identification of abundant A-to-I editing sites in the human transcriptome.

RNA editing by members of the ADAR (adenosine deaminases acting on RNA) family leads to site-specific conversion of adenosine to inosine (A-to-I) in precursor messenger RNAs. Editing by ADARs is believed to occur in all metazoa, and is essential for mammalian development. Currently, only a limited number of human ADAR substrates are known, whereas indirect evidence suggests a substantial fraction of all pre-mRNAs being affected. Here we describe a computational search for ADAR editing sites in the human transcriptome, using millions of available expressed sequences. We mapped 12,723 A-to-I editing sites in 1,637 different genes, with an estimated accuracy of 95%, raising the number of known editing sites by two orders of magnitude. We experimentally validated our method by verifying the occurrence of editing in 26 novel substrates. A-to-I editing in humans primarily occurs in noncoding regions of the RNA, typically in Alu repeats. Analysis of the large set of editing sites indicates the role of editing in controlling dsRNA stability.

Adenosine↗
PubMed1997

Ribonucleic acid expression of the clustered imprinted genes, p57KIP2, insulin-like growth factor II, and H19, in adrenal tumors and cultured adrenal cells.

The recently cloned cyclin-dependent kinase inhibitor gene p57KIP2 is genomically imprinted and located on human chromosome 11p15.5. This region contains two other imprinted genes, insulin-like growth factor II (IGF-II) and H19, both of which seem to be implicated in adrenal neoplasms. We analyzed the expression of the putative tumor suppressor p57KIP2 gene by Northern blotting in normal and hyperplastic adrenals, adrenocortical tumors, and pheochromocytomas. The expression of p57KIP2 messenger ribonucleic acid (mRNA) correlated positively with H19 and negatively with IGF-II RNA in adrenocortical tissues. p57KIP2 mRNA (and H19 RNA) was abundantly expressed in normal human adrenals, adrenocortical adenomas from patients with Cushing's or Conn's syndrome or without clinical evidence of hormone overproduction, hyperplastic adrenals, and tumor-adjacent adrenal tissues, in which IGF-II mRNA expression was low. In most adrenocortical carcinomas and virilizing adrenal adenomas, very low levels of both p57KIP2 and H19 RNAs were observed, whereas IGF-II was highly expressed. In pheochromocytomas, p57KIP2 and H19 RNA expression was highly variable, but on the average it was about 45% and 27%, respectively, of that in normal and tumor-adjacent adrenals. In cultured adrenocortical cells, ACTH and dibutyryl cAMP treatment slightly reduced the predominant 1.7-kilobase (kb) transcript of p57KIP2 gene, but induced a 2.5-kb transcript with a simultaneous increase in H19 RNA expression. The stimulatory effect of ACTH on the 2.5-kb p57KIP2 and H19 transcript accumulation was enhanced by exogenous IGF-II and IGF-I. Our data show that p57KIP2 and H19 RNAs are expressed usually in parallel in normal and pathological adrenocortical tissues. The decreased expression of both p57KIP2 and H19 RNAs in conjunction with elevated IGF-II mRNA expression in hormonally active adrenocortical carcinomas suggests that the loss of expression of the putative tumor suppressor genes p57KIP2 and H19 may be involved in the pathogenesis of these neoplasms.

Adrenal Gland Neoplasms↗
PubMed1985

Five pseudoknots are present at the 204 nucleotides long 3' noncoding region of tobacco mosaic virus RNA.

The 104 nucleotides long 3' terminal region of TMV RNA was shown previously to contain two pseudoknotted structures (Rietveld et al. (1984), EMBO J. 3, 2613-2619). We here present evidence for the occurrence, within the 204 nucleotides long 3' noncoding region, of another highly structured domain located immediately adjacent to the tRNA-like structure of 95 nucleotides (Joshi et al. (1985) Nucleic Acids Res. 13, 347-354). A model for the three-dimensional folding of this region, containing three more pseudoknots, is proposed on the basis of chemical modification and enzymatic digestion. The existence of these three consecutive pseudoknots was supported by sequence comparisons with the RNA from the related tobamoviruses TMV-L, CcTMV and CGMMV. Coaxial stacking of the six double helical segments involved gives rise to the formation of a 25 basepair long quasi-continuous double helix. The results show that the three-dimensional folding of the 3' non-translated region of tobamoviral RNAs is largely maintained by the formation of five pseudoknots. The organisation of this region in the RNA of the tobamovirus CcTMV suggests that recombinational events among aminoacylatable plant viral RNAs have to be considered.

Base Sequence↗
PubMed2003

Characterization of adapt33, a stress-inducible riboregulator.

We have identified adapt33 as a multiple stress-responsive gene that is induced under conditions of a cytoprotective "adaptive response." adapt33 RNA does not contain any appreciable open reading frame nor produce a protein product and is therefore classified as a stress-inducible riboregulator. Although a number of oxidant stress-modulated, protein-encoding genes have been reported and characterized, very few stress-inducible riboregulator RNAs are known. Here we extend previous studies toward understanding the underlying regulation of expression and function of this rare mammalian riboregulator. mRNA stability and transcription studies determined that adapt33 induction by hydrogen peroxide is at the mRNA stability level, and that adapt33 has a very short half-life. Surprisingly, adapt33 mRNA also exhibits altered electrophoretic migration in response to both hydrogen peroxide and cis-platinum treatment. Although no transcriptional modulation in response to hydrogen peroxide was observed, fusion promoter constructs revealed that adapt33 has an unusually strong promoter that is active in both hamster and human cells. Analysis of expression following the stimulation of apoptosis with hydrogen peroxide and staurosporine revealed a strong correlation with apoptosis, suggesting a possible novel, noncoding RNA component of the apoptotic mechanism. We conclude that adapt33 is a stress-inducible, apoptosis-associated RNA with unique structural and gene promoter characteristics.

Animals↗
PubMed2001

Nucleotides at the extremities of the viral RNA of influenza C virus are involved in type-specific interactions with the polymerase complex.

Influenza A and C viruses share common sequences in the terminal noncoding regions of the viral RNA segments. Differences at the 5'- and 3'-ends exist, however, that could contribute to the specificity with which the transcription/replication signals are recognized by the cognate polymerase complexes. Previously, by making use of a transient expression system for the transcription and replication of a reporter RNA template bearing either type A or type C extremities, it was shown that a type C RNA template is transcribed and replicated with equal efficiency by either the type A or the type C polymerase complex, whereas a type A RNA template is less efficiently transcribed and replicated by the type C polymerase complex than by the type A complex. To explore the contribution of the nucleotides at the extremities of the RNAs to this type-specificity, the effect of mutations introduced either alone or in combination at nucleotide 5 at the 3'-end and at nucleotides 3', 6' or 8' at the 5'-end of type A or C RNA templates were studied in the presence of either the type A or the type C polymerase complex. The results indicate that the nature of nucleotides 5 and 6' contribute to type-specificity. Moreover, these results underline the importance of the base pairing between nucleotide 3' and 8' at the 5'-end of the RNA. Thus, it could be suggested that the nature of the nucleotides as well as the stability of the secondary structure at the extremities of the viral RNA are important determinants of type-specificity.

DNA-Directed RNA Polymerases↗
PubMed2001

The non-coding RNAs as riboregulators.

The non-coding RNAs database (http://biobases.ibch.poznan.pl/ncRNA/) contains currently available data on RNAs, which do not have long open reading frames and act as riboregulators. Non-coding RNAs are involved in the specific recognition of cellular nucleic acid targets through complementary base pairing to control cell growth and differentiation. Some of them are connected with several well known developmental and neuro-behavioral disorders. We have divided them into four groups. This paper is a short introduction to the database and presents its latest, updated edition.

Animals↗
PubMed2005

Attenuation of bunyavirus replication by rearrangement of viral coding and noncoding sequences.

Bunyamwera virus (BUN) is the prototype virus of the family Bunyaviridae. BUN has a tripartite negative-sense RNA genome comprising small (S), medium (M), and large (L) segments. Partially complementary untranslated regions (UTRs) flank the coding region of each segment. The terminal 11 nucleotides of these UTRs are conserved between the three segments, while the internal regions are unique. The UTRs direct replication and transcription of viral RNA and are sufficient to allow encapsidation of viral RNA into ribonucleoprotein complexes. To investigate the segment-specific functions of the UTRs, we have used reverse genetics to recover a recombinant virus (called BUN MLM) in which the L segment open reading frame (ORF) is flanked by the M segment UTRs. Compared to wild-type virus, BUN MLM virus shows growth attenuation in cultured mammalian cells and a slower disease progression in mice, produces small plaques, expresses reduced levels of L mRNA and L (RNA polymerase) protein, synthesizes less L genomic and antigenomic RNA, and has an increased particle-to-PFU ratio. Our data suggest that the packaging of BUN RNAs is not segment specific. In addition, the phenotype of BUN MLM virus supports the finding that BUN UTRs differ in their regulation of RNA synthesis but suggests that the interplay between each segment UTR and its cognate ORF may contribute to that regulation. Since BUN MLM virus is attenuated due to an essentially irreversible mutation, the rearrangement of UTRs is a feasible strategy for vaccine design for the more pathogenic members of the Bunyaviridae.

Animals↗
PubMed2026

Beyond Canonical Neoantigens: Emerging Technologies for Identification of Noncanonical Antigens and Implications for Personalized Cancer Vaccines.

Over the past decade, advances in sequencing technologies and computational pipelines enabled the development of personalized cancer vaccines (PCVs). Current PCV strategies primarily target cancer neoantigens generated by non-synonymous DNA mutations, which can result in altered amino acid sequences capable of eliciting tumor-specific immune responses. More recently, a distinct class of tumor-specific antigens (TSA), termed noncanonical or cryptic antigens, has emerged as an additional source of immunogenic targets. Unlike canonical neoantigens, noncanonical antigens typically cannot be identified by tumor/normal whole-exome sequencing, as they do not arise from classical DNA mutations. Instead, they are often associated with less well recognized and/or aberrant processes in the pathways from DNA to human leukocyte antigen (HLA)-presented peptides. Examples include transposable elements, circular RNA, translation of alternative open reading frames and/or long non-coding RNA, among others. Emerging evidence suggests that noncanonical antigens represent a substantial portion of the tumor-specific immunopeptidome and, similar to canonical neoantigens, are absent during thymic selection and can evade central tolerance and elicit T cell responses. Technological advances have increasingly facilitated the identification of noncanonical antigens. Long-read RNA sequencing reveals noncanonical transcripts by improving transcriptome assembly, while ribosome profiling provides genome-wide maps of actively translated regions, facilitating the discovery of peptides from aberrant translation events. Specialized molecular approaches enable enrichment and sequencing of circular RNAs, and immunopeptidomics using mass spectrometry allows for direct characterization of HLA-presented peptides. Together, these technological advances have led to an increasing interest in prioritizing and targeting noncanonical antigens in the next generation of PCVs. This review provides an overview of the diverse origins of TSAs beyond classical neoantigens and discusses emerging approaches that may enable the integration of these antigens in future clinical trials.

circular RNA↗
PubMed2001

Deletion or substitution of the aphthovirus 3' NCR abrogates infectivity and virus replication.

The 3' noncoding region (NCR) of the genomic picornaviral RNA is believed to contain major cis-acting signals required for negative-strand RNA synthesis. The 3' NCR of foot-and-mouth disease virus (FMDV) was studied in the context of a full-length infectious clone in which the genetic element was deleted or exchanged for the equivalent region of a distantly related swine picornavirus, swine vesicular disease virus (SVDV). Deletion of the 3' NCR, while maintaining the intact poly(A) tail as well as its replacement for the SVDV counterpart, abrogated virus replication in susceptible cells as determined by infectivity and Northern blot assays. Nevertheless, the presence of the SVDV sequence allowed the synthesis of low amounts of chimeric viral RNA at extended times post-transfection as compared to RNAs harbouring the 3' NCR deletion. The failure to recover viable viruses or revertants after several passages on susceptible cells suggests that the presence of specific sequences contained within the FMDV 3' NCR is essential to complete a full replication cycle and that FMDV and SVDV 3' NCRs are not functionally interchangeable.

3' Untranslated Regions↗
PubMed1998

Yellow fever 5' noncoding region as a potential element to improve hepatitis C virus production through modification of translational control.

The lengthy 5' noncoding region (5' NCR) of hepatitis C virus (HCV) RNA forms a highly ordered secondary structure, very conserved among different strains. It includes an internal ribosome entry site (IRES) element, responsible for the cap-independent translation initiation of HCV RNA. Similarly to the IRES of hepatitis A virus (HAV), another human hepatitis virus, HCV IRES, activity in internal initiation of translation is weak. Furthermore, both viruses exhibit a poor growth phenotype that may result at least partially from an inhibitory control of translation. To enhance HCV translation, as a preliminary step in designing constructs for improvement in viral production, we sought to evaluate a chimeric construct containing the yellow fever virus (YFV) 5' NCR fused to the initiation codon of the HCV coding sequence. YF viral RNA, as the majority of eukaryotic messenger RNAs, is translated by a ribosome scanning mechanism in a cap-dependent manner. The efficiency of translation initiation of the parental HCV construct was compared in vitro in rabbit reticulocyte lysates with that of the chimeric construct containing YFV 5' NCR. Surprisingly, the related distanced YFV 5' NCR was fivefold more active than was the wild-type HCV IRES in directing that function. Furthermore, chimeric transcripts were shown to be effective in vivo after transfection of eukaryotic cells. Taken together, these results raise the following question: why has the HCV genus evolved to the acquisition of an IRES element within its 5' NCR among the Flaviviridae family?

5' Untranslated Regions↗
PubMed2002

Nucleotide sequence shows that Bean leafroll virus has a Luteovirus-like genome organization.

The complete nucleotide sequence of the Bean leafroll virus (BLRV) genomic RNA and the termini of its smallest subgenomic RNAs were determined to better understand its mechanisms of gene expression and replication and its phylogenetic position within the Luteoviridae: The number and placement of open reading frames (ORFs) within the BLRV genome was Luteovirus-like. The nucleotide and predicted amino acid sequences of BLRV were most similar to those of Soybean dwarf virus (SbDV). Phylogenetic analyses employing the neighbour-joining method and sister-scanning analysis indicated that the BLRV nonstructural proteins were closely related to those of Barley yellow dwarf virus-PAV (BYDV-PAV), a luteovirus: The region surrounding the frameshift at the junction between ORFs 1 and 2 also contained sequences very similar to those of BYDV-PAV and a Dianthovirus, Red clover necrotic mosaic virus. Similar analyses showed that the structural proteins were most similar to those of the Polerovirus genus. The 3'-noncoding regions downstream of ORF5 contained sequences similar to translational control elements identified in the BYDV-PAV genome. These data suggest that BLRV, like SbDV, is derived either through selection from a common ancestor with BYDV-PAV or that BLRV is the product of two recombination events between luteovirus-like and polerovirus-like ancestors where the 5' 2900 nt and 3' 700 nt of the BLRV genome are from a Luteovirus and the intervening sequences are derived from a Polerovirus:

3' Untranslated Regions↗
PubMed1986

U6 small nuclear RNA is transcribed by RNA polymerase III.

A DNA fragment homologous to U6 small nuclear RNA was isolated from a human genomic library and sequenced. The immediate 5'-flanking region of the U6 DNA clone had significant homology with a potential mouse U6 gene, including a "TATA box" at a position 26-29 nucleotides upstream from the transcription start site. Although this sequence element is characteristic of RNA polymerase II promoters, the U6 gene also contained a polymerase III "box A" intragenic control region and a typical run of five thymines at the 3' terminus (noncoding strand). The human U6 DNA clone was accurately transcribed in a HeLa cell S100 extract lacking polymerase II activity. U6 RNA transcription in the S100 extract was resistant to alpha-amanitin at 1 microgram/ml but was completely inhibited at 200 micrograms/ml. A comparison of fingerprints of the in vitro transcript and of U6 RNA synthesized in vivo revealed sequence congruence. U6 RNA synthesis in isolated HeLa cell nuclei also displayed low sensitivity to alpha-amanitin, in contrast to U1 and U2 RNA transcription, which was inhibited greater than 90% at 1 microgram/ml. In addition, U6 RNA synthesized in isolated nuclei was efficiently immunoprecipitated by an antibody against the La antigen, a protein known to bind most other RNA polymerase III transcripts. These results establish that, in contrast to the polymerase II-directed transcription of mammalian genes for U1-U5 small nuclear RNAs, human U6 RNA is transcribed by RNA polymerase III.

Amanitins↗