Terminal Hairpin Structure Research Articles

Quantitative analysis of C. elegans transcripts by Nanopore direct-cDNA sequencing reveals terminal hairpins in non trans-spliced mRNAs

In nematodes and kinetoplastids, mRNA processing involves a trans-splicing step through which a short sequence from a snRNP replaces the original 5’ end of the primary transcript. It has long been held that 70% of C. elegans mRNAs are submitted to trans-splicing. Our recent work suggested that the mechanism is more pervasive but not fully captured by mainstream transcriptome sequencing methods. Here we use Oxford Nanopore’s long-read amplification-free sequencing technology to perform a comprehensive analysis of trans-splicing in worms. We demonstrate that spliced leader (SL) sequences at the 5’ end of the mRNAs affect library preparation and generate sequencing artefacts due to their self-complementarity. Consistent with our previous observations, we find evidence of trans-splicing for most genes. However, a subset of genes appears to be only marginally trans-spliced. These mRNAs all share the capacity to generate a 5’ terminal hairpin structure mimicking the SL structure and offering a mechanistic explanation for their non conformity. Altogether, our data provide a comprehensive quantitative analysis of SL usage in C. elegans.

Structure and transcription of the Helicoverpa armigera densovirus (HaDV2) genome and its expression strategy in LD652 cells

BackgroundDensoviruses (DVs) are highly pathogenic to their hosts. However, we previously reported a mutualistic DV (HaDV2). Very little was known about the characteristics of this virus, so herein we undertook a series of experiments to explore the molecular biology of HaDV2 further.ResultsPhylogenetic analysis showed that HaDV2 was similar to members of the genus Iteradensovirus. However, compared to current members of the genus Iteradensovirus, the sequence identity of HaDV2 is less than 44% at the nucleotide-level, and lower than 36, 28 and 19% at the amino-acid-level of VP, NS1 and NS2 proteins, respectively. Moreover, NS1 and NS2 proteins from HaDV2 were smaller than those from other iteradensoviruses due to their shorter N-terminal sequences. Two transcripts of about 2.2 kb coding for the NS proteins and the VP proteins were identified by Northern Blot and RACE analysis. Using specific anti-NS1 and anti-NS2 antibodies, Western Blot analysis revealed a 78 kDa and a 48 kDa protein, respectively. Finally, the localization of both NS1 and NS2 proteins within the cell nucleus was determined by using Green Fluorescent Protein (GFP) labelling.ConclusionThe genome organization, terminal hairpin structure, transcription and expression strategies as well as the mutualistic relationship with its host, suggested that HaDV2 was a novel member of the genus Iteradensovirus within the subfamily Densovirinae.

Limitations of encapsidation of recombinant self-complementary adeno-associated viral genomes in different serotype capsids and their quantitation.

We previously reported that self-complementary adeno-associated virus (scAAV) type 2 genomes of up to 3.3 kb can be successfully encapsidated into AAV2 serotype capsids. Here we report that such oversized AAV2 genomes fail to undergo packaging in other AAV serotype capsids, such as AAV1, AAV3, AAV6, and AAV8, as determined by Southern blot analyses of the vector genomes, although hybridization signals on quantitative DNA slot-blots could still be obtained. Recently, it has been reported that quantitative real-time PCR assays may result in substantial differences in determining titers of scAAV vectors depending on the distance between the primer sets and the terminal hairpin structure in the scAAV genomes. We also observed that the vector titers determined by the standard DNA slot-blot assays were highly dependent on the specific probe being used, with probes hybridizing to the ends of viral genomes being significantly overrepresented compared with the probes hybridizing close to the middle of the viral genomes. These differences among various probes were not observed using Southern blot assays. This overestimation of titer is a systemic error during scAAV genome quantification, regardless of viral genome sequences and capsid serotypes. Furthermore, different serotypes capsid and modification of capsid sequence may affect the ability of packaging intact, full-length AAV genomes. Although the discrepancy is modest with wild-type serotype capsid and short viral genomes, the measured titer could be as much as fivefold different with capsid mutant vectors and large genomes. Thus, based on our data, we suggest that Southern blot analyses should be performed routinely to more accurately determine the titers of recombinant AAV vectors. At the very least, the use of probes/primers hybridizing close to the mutant inverted terminal repeat in scAAV genomes is recommended to avoid possible overestimation of vector titers.

Mobilizing the Genome of Lepidoptera through Novel Sequence Gains and End Creation by Non-autonomous Lep1 Helitrons

Transposable elements (TEs) can affect the structure of genomes through their acquisition and transposition of novel DNA sequences. The 134-bp repetitive elements, Lep1, are conserved non-autonomous Helitrons in lepidopteran genomes that have characteristic 5′-CT and 3′-CTAY nucleotide termini, a 3′-terminal hairpin structure, a 5′- and 3′-subterminal inverted repeat (SIR), and integrations that occur between AT or TT nucleotides. Lep1 Helitrons have acquired and propagated sequences downstream of their 3′-CTAY termini that are 57–344-bp in length and have termini composed of a 3′-CTRR preceded by a 3′-hairpin structure and a region complementary to the 5′-SIR (3′-SIRb). Features of both the Lep1 Helitron and multiple acquired sequences indicate that secondary structures at the 3′-terminus may have a role in rolling circle replication or genome integration mechanisms, and are a prerequisite for novel end creation by Helitron-like TEs. The preferential integration of Lep1 Helitrons in proximity to gene-coding regions results in the creation of genetic novelty that is shown to impact gene structure and function through the introduction of novel exon sequence (exon shuffling). These findings are important in understanding the structural requirements of genomic DNA sequences that are acquired and transposed by Helitron-like TEs.

104. The Adeno-Associated Virus Terminal Repeat Hairpin Structure Effects Expression from Transfected DNA, but Not Recombination Frequency

104. The Adeno-Associated Virus Terminal Repeat Hairpin Structure Effects Expression from Transfected DNA, but Not Recombination Frequency

Host-dependent effects of the 3′ untranslated region of turnip crinkle virus RNA on accumulation in Hibiscus and Arabidopsis

The 3' untranslated region (UTR) of turnip crinkle virus (TCV) RNA is 253 nt long (nt 3798-4050) with a 27 nt hairpin structure near its 3' terminus. In this study, the roles of the 3' UTR in virus accumulation were investigated in protoplasts of Hibiscus cannabinus L. and Arabidopsis thaliana (L.) Heynh. Our results showed that, in Hibiscus protoplasts, the minimal 3' UTR essential for TCV accumulation extends from nt 3922 to 4050, but that maintenance of virus accumulation at wild-type (wt) levels requires the full-length 3' UTR. However, in Arabidopsis protoplasts, only 33 nt (nt 4018-4050) at the 3' extremity of the UTR is required for wt levels of accumulation, whereas other parts of the 3' UTR are dispensable. The 27 nt hairpin within the 33 nt region is essential for virus accumulation in both Hibiscus and Arabidopsis protoplasts. However, transposition of nucleotides in base pairs within the upper or lower stems has no effect on virus accumulation in either Hibiscus or Arabidopsis protoplasts, and alterations of the loop sequence also fail to affect replication. Disruption of the upper or lower stems and deletion of the loop sequence reduce viral accumulation in Arabidopsis protoplasts, but abolish virus accumulation in Hibiscus protoplasts completely. These results indicate that strict conservation of the hairpin structure is more important for replication in Hibiscus than in Arabidopsis protoplasts. In conclusion, both the 3' UTR primary sequence and the 3'-terminal hairpin structure influence TCV accumulation in a host-dependent manner.

Complete coding sequences of the rabbitpox virus genome

Rabbitpox virus (RPXV) is highly virulent for rabbits and it has long been suspected to be a close relative of vaccinia virus. To explore these questions, the complete coding region of the rabbitpox virus genome was sequenced to permit comparison with sequenced strains of vaccinia virus and other orthopoxviruses. The genome of RPXV strain Utrecht (RPXV-UTR) is 197 731 nucleotides long, excluding the terminal hairpin structures at each end of the genome. The RPXV-UTR genome has 66.5 % A + T content, 184 putative functional genes and 12 fragmented ORF regions that are intact in other orthopoxviruses. The sequence of the RPXV-UTR genome reveals that two RPXV-UTR genes have orthologues in variola virus (VARV; the causative agent of smallpox), but not in vaccinia virus (VACV) strains. These genes are a zinc RING finger protein gene (RPXV-UTR-008) and an ankyrin repeat family protein gene (RPXV-UTR-180). A third gene, encoding a chemokine-binding protein (RPXV-UTR-001/184), is complete in VARV but functional only in some VACV strains. Examination of the evolutionary relationship between RPXV and other orthopoxviruses was carried out using the central 143 kb DNA sequence conserved among all completely sequenced orthopoxviruses and also the protein sequences of 49 gene products present in all completely sequenced chordopoxviruses. The results of these analyses both confirm that RPXV-UTR is most closely related to VACV and suggest that RPXV has not evolved directly from any of the sequenced VACV strains, since RPXV contains a 719 bp region not previously identified in any VACV.

Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.

The human parvovirus adeno-associated virus (AAV) is unique in its ability to target viral integration to a specific site on chromosome 19 (ch-19). Recombinant AAV (rAAV) vectors retain the ability to integrate but have apparently lost this ability to target. In this report, we characterize the terminal-repeat-mediated integration for wild-type (wt), rAAV, and in vitro systems to gain a better understanding of these differences. Cell lines latent for either wt or rAAV were characterized by a variety of techniques, including PCR, Southern hybridization, and fluorescence in situ hybridization analysis. More than 40 AAV-rAAV integration junctions were cloned, sequenced, and then subjected to comparison and analysis. In both immortalized and normal diploid human cells, wt AAV targeted integration to ch-19. Integrated provirus structures consisted of head-to-tail tandem arrays with the majority of the junction sequences involving the AAV inverted terminal repeats (ITRs). No complete viral ITRs were directly observed. In some examples, the AAV p5 promoter sequence was found to be fused at the virus-cell junction. Data from dot blot analysis of PCR products were consistent with the occurrence of inversions of genomic and/or viral DNA sequences at the wt integration site. Unlike wt provirus junctions, rAAV provirus junctions mapped to a subset of non-ch-19 sequences. Southern analysis supported the integration of proviruses from two independent cell lines at the same locus on ch-2. In addition, provirus terminal repeat sequences existed in both the flip and flop orientations, with microhomology evident at the junctions. In all cases with the exception of the ITRs, the vector integrated intact. rAAV junction sequence data were consistent with the occurrence of genomic rearrangement by deletion and/or rearrangement-translocation at the integration locus. Finally, junctions formed in an in vitro system between several AAV substrates and the ch-19 target site were isolated and characterized. Linear AAV substrates typically utilized the end of the virus DNA substrate as the point of integration, whereas products derived from AAV terminal repeat hairpin structures in the presence or absence of Rep protein resembled AAV-ch-19 junctions generated in vivo. These results describing wt AAV, rAAV, and in vitro integration junctions suggest that the viral integration event itself is mediated by terminal repeat hairpin structures via nonviral cellular recombination pathways, with specificity for ch-19 in vivo requiring additional viral components. These studies should have an important impact on the use of rAAV vectors in human gene therapy.

Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.

Cellular recombination pathways and viral terminal repeat hairpin structures are sufficient for adeno-associated virus integration in vivo and in vitro.

Mutational Analysis of Conserved Tyrosines in the NS-1 Protein of the Parvovirus Minute Virus of Mice

The NS-1 gene of the parvovirus minute virus of mice encodes a multifunctional protein essential for viral DNA replication and gene expression. In addition to possessing DNA helicase and ATPase activities, NS-1 forms a covalent linkage with the 5′ ends of viral DNA and is a strong candidate for the site-specific nicking-closing enzyme postulated to be involved in the resolution of concatemers and terminal hairpin structures that arise during parvoviral DNA replication. Since the covalent linkage between NS-1 and the 5′ terminus of MVM DNA resists alkali and mild acid treatment, a tyrosine phosphodiester is likely to be involved. To map domains responsible for this activity, mutations converting tyrosine to phenylalanine were introduced into the NS-1 gene using oligonucleotide-directed mutagenesis and their effect on the DNA replication and transcriptional activation functions of NS-1 was examined in transient in vivo transfection assays. Replacement of Tyr-188, Tyr-197, Tyr-210, Tyr-310, Tyr-422, or Tyr-550 with phenylalanine greatly reduced the ability of NS-1 to complement the replication of the target genome ins 20B in COS-7 cells. However, a Ser-545 to Thr-545 substitution in the Phe-550 mutant restored DNA replication activity. Replacement of 5 other tyrosines in NS-1 with phenylalanine either enhanced (Phe-6), had a moderate inhibitory effect (Phe-209) or had no effect (Phe-47, Phe 227 and Phe-543) on its DNA replication activity. Two of the 11 phenylalanine substitution mutations, Phe-188 and Phe-197, also greatly reduced the ability of NS-1 to transactivate the p38 promoter and displayed a dominant negative phenotype with respect to transactivation. Since the remaining tyrosines in MVM NS-1, Tyr-152, Tyr-252, Tyr-374, and Tyr-595, are not conserved among the NS-1 proteins encoded by porcine and feline parvoviruses, they are presumed to be nonessential for the normal functioning of NS-1. The results point to a role for either Tyr-188, Tyr-197, Tyr-210, Tyr-310, or Tyr-422 in forming a covalent linkage with viral DNA and further suggest a regulatory role for several tyrosines in other DNA replication and transcriptional activation functions of NS-1.

In vitro resolution of adeno-associated virus DNA hairpin termini by wild-type Rep protein is inhibited by a dominant-negative mutant of rep.

An adeno-associated virus (AAV) genome with a Lys-to-His (K340H) mutation in the consensus nucleotide triphosphate binding site of the rep gene has a dominant-negative DNA replication phenotype in vivo. We expressed both wild-type (Rep78) and mutant (Rep78NTP) proteins in two helper-free expression systems consisting of either recombinant baculoviruses in insect cells or the human immunodeficiency virus type 1 long terminal repeat promoter in human 293 cell transient transfections. We analyzed nuclear extracts from both expression systems for the ability to complement uninfected HeLa cell cytoplasmic extracts in an in vitro terminal resolution assay in which a covalently closed AAV terminal hairpin structure is converted to an extended linear duplex. Although both Rep78 and Rep78NTP bound to AAV terminal hairpin DNA in vitro, Rep78 but not Rep78NTP complemented the terminal resolution assay. Furthermore, Rep78NTP was trans dominant for AAV terminal resolution in vitro. We propose that the dominant-negative replication phenotype of AAV genomes carrying the K340H mutation is mediated by mutant Rep proteins binding to the terminal repeat hairpin.

The minute virus of mice capsid specifically recognizes the 3' hairpin structure of the viral replicative-form DNA: mapping of the binding site by hydroxyl radical footprinting

The terminal hairpin structures of the DNA of minute virus of mice (MVM) are essential for viral replication. Here we show that the hairpin 3' terminus of MVM replicative-form DNA binds specifically to empty MVM capsids. Binding of the same terminal DNA sequence in its linear double-stranded (extended) conformation was not observed. After heat denaturation and quick cooling of 3'-terminal extended-form fragments, not only the virion strand but also the complementary strand was found to bind to the capsid, presumably because each strand re-formed a similar hairpin structure. No binding affinity for the capsid was found to be associated with hairpin or extended 5' termini or with any other region of the viral DNA. Hydroxyl radical footprinting analyses revealed three protected nucleotide stretches forming a binding site at the branch point of the two 3'-terminal hairpin arms looping out from the DNA stem (T structure). Single base changes within this site did not affect the binding. In band shift experiments, specific binding to the T structure was demonstrated for VPI but not for VP2.

Presence of poly(A) in a flavivirus: significant differences between the 3' noncoding regions of the genomic RNAs of tick-borne encephalitis virus strains

A poly(A) tail was identified on the 3' end of the prototype tick-borne encephalitis (TBE) virus strain Neudoerfl. This is in contrast to the general lack of poly(A) in the genomic RNAs of mosquito-borne flaviviruses analyzed so far. Analysis of several closely related strains of TBE virus, however, revealed the existence of two different types of 3' noncoding (NC) regions. One type (represented by strain Neudoerfl) is only 114 nucleotides long and carries a 3'-terminal poly(A) structure. This was also found in several TBE virus strains isolated from different geographic regions over a period of almost 30 years. The other type (represented by strain Hypr) is 461 nucleotides long and not polyadenylated. The sequence homology between the two types of TBE virus 3' NC regions terminates at a specific position 81 nucleotides after the stop codon. The second type of 3' NC region more closely resembles the common flavivirus pattern, including the potential for the formation of a 3'-terminal hairpin structure. However, it lacks primary sequence elements that are conserved among other flavivirus genomes.

Platelet function in the newborn

The transition from lineform DNA to cruciform DNA (cruciformation) within the cloned telomere sequences of the Leporipoxvirus Shope fibroma virus (SFV) has been studied. The viral telomere sequences have been cloned in recombination-deficient Escherichia coli as a 322 base-pair, imperfect palindromic insert in pUC13. The inverted repeat configuration is equivalent to the arrangement of the telomere structures observed within viral DNA replicative intermediates. A major cruciform structure in the purified recombinant plasmid has been identified and mapped using, as probes, the enzymes AflII, nuclease S1 and bacteriophage T7 endonuclease I. It was extruded from the central axis of the cloned viral inverted repeat and, by unrestricted branch migration, attained a size commensurate with the superhelical density of the plasmid molecule at native superhelical densities. This major cruciform extrusion event was the only detectable duplex DNA perturbation, induced by negative superhelical torsion, in the insert viral sequences. No significant steady-state pool of extruded cruciform was identified in E. coli. However, the identification of a major deletion variant generated even in the recombination-deficient E. coli strain DB1256 (recA recBC sbcB) suggested that the cruciform may be extruded transiently in vivo. The lineform to cruciform transition has been further characterized in vitro using two-dimensional agarose gel electrophoresis. The transition was marked by a high energy of formation (ΔGf = 44 kcal/mol), and an apparently low activation energy that enabled facile transitions at physiological temperatures provided there was sufficient torsional energy. By comparing cruciformation in a series of related bidirectional central axis deletions of the telomeric insert, it has been concluded that the presence of extrahelical bases in the terminal hairpin structures contributes substantially to the high ΔGf value. Also, viral sequences flanking the extruded cruciform were shown to influence the measured ΔGf value. Several general features of poxvirus telomere structure that would be expected to influence the facility of cruciform extrusion are discussed along with the implications of the observed cruciform transition event on the replicative process of poxviruses in vivo.

Molecular cloning and sequence of the concatemer junction from vaccinia virus replicative DNA: Viral nuclease cleavage sites in cruciform structures

The concatemer junction from replicative forms of vaccinia virus DNA was cloned into plasmid vectors and shown to be a precise duplex copy of the viral terminal hairpin structure, with each strand corresponding to one of the alternative sequence isomers. The plasmids were relaxed circles with extruded cruciforms representing two copies of the vaccinia telomere hairpin structure. Head-to-head dimers containing two copies of the vaccinia virus concatemer junction were observed to contain only one set of stem-loop structures per molecule, suggesting that the initial formation of a small cruciform, and not branch migration, was the rate-limiting step in cruciform formation. The plasmids containing the concatemer junction were converted into nicked circular, linear and cross-linked linear molecules by a nuclease isolated from vaccinia virions. The region-specific cleavage near the border of the hairpin loop and the formation of DNA cross-links in some of the molecules is consistent with the nuclease acting as a nicking-closing enzyme that participates in the resolution of mature termini from replicative concatemer intermediates.

Cruciform extrusion in plasmids bearing the replicative intermediate configuration of a poxvirus telomere

The transition from lineform DNA to cruciform DNA (cruciformation) within the cloned telomere sequences of the Leporipoxvirus Shope fibroma virus (SFV) has been studied. The viral telomere sequences have been cloned in recombination-deficient Escherichia coli as a 322 base-pair, imperfect palindromic insert in pUC13. The inverted repeat configuration is equivalent to the arrangement of the telomere structures observed within viral DNA replicative intermediates. A major cruciform structure in the purified recombinant plasmid has been identified and mapped using, as probes, the enzymes AflII, nuclease S 1 and bacteriophage T7 endonuclease I. It was extruded from the central axis of the cloned viral inverted repeat and, by unrestricted branch migration, attained a size commensurate with the superhelical density of the plasmid molecule at native superhelical densities. This major cruciform extrusion event was the only detectable duplex DNA perturbation, induced by negative superhelical torsion, in the insert viral sequences. No significant steady-state pool of extruded cruciform was identified in E. coli. However, the identification of a major deletion variant generated even in the recombination-deficient E. coli strain DB1256 ( recA recBC sbcB) suggested that the cruciform may be extruded transiently in vivo. The lineform to cruciform transition has been further characterized in vitro using two-dimensional agarose gel electrophoresis. The transition was marked by a high energy of formation ( ΔG f = 44 kcal/mol), and an apparently low activation energy that enabled facile transitions at physiological temperatures provided there was sufficient torsional energy. By comparing cruciformation in a series of related bidirectional central axis deletions of the telomeric insert, it has been concluded that the presence of extrahelical bases in the terminal hairpin structures contributes substantially to the high ΔG f value. Also, viral sequences flanking the extruded cruciform were shown to influence the measured ΔG f value. Several general features of poxvirus telomere structure that would be expected to influence the facility of cruciform extrusion are discussed along with the implications of the observed cruciform transition event on the replicative process of poxviruses in vivo.

The genomic RNA of diabetogenic encephalomyocarditis virus: characterization and molecular cloning

The RNA of a diabetogenic variant of encephalomyocarditis (EMC) virus (D variant, ifp − phenotype) and a nondiabetogenic variant of EMC virus (B variant, ifp + phenotype) which were derived from the same virus stock (M strain) have been compared. The size of both genomes is estimated at 7.7 kb. The poly(C) tract of EMC-D is estimated at 144 bases, whereas that of EMC-B is 141 bases in length. The untranslated 5′ terminal 103 nucleotides are identical for B and D with preservation of a stable terminal hairpin structure. The entire open reading frame of both variants has been cloned and the restriction maps of 12 different enzymes are identical. These maps were compared to a computer-generated restriction map of another strain of EMC virus which has been cloned and sequenced by Palmenberg et al. (1984 , Nucleic Acids Res. 12, 2969–2985). Approximately 50% of the restriction sites of the B and D variants had similar locations in this strain of EMC. We conclude that significant genetic variation exists between the M strain (B and D variants) and the Palmenberg strain of EMC virus. However, the B and D variants are very similar at the molecular level and comparison of their nucleotide sequences will be necessary to reveal the basis for their different biological properties.

Replication of the adeno-associated virus DNA termini in vitro.

The validity of the model proposed by Cavalier-Smith for the replication of linear, single-stranded DNA molecules was tested by using subgenomic DNA termini isolated from adeno-associated virus (AAV), a defective parvovirus. In purified DNA replication systems, the terminal hairpin structure of AAV DNA with a 3'-OH end was shown to prime the synthesis of the daughter DNA strand, which was covalently linked to the parental strand. This resulted in the synthesis of a duplex DNA structure with a terminal hairpin. The availability of such a hairpin structure should facilitate the isolation and characterization of the pivotal enzyme, the putative nickase, for the replication of AAV DNA.

Replication of the B19 parvovirus in human bone marrow cell cultures.

The B19 parvovirus is responsible for at least three human diseases. The virus was successfully propagated in suspension cultures of human erythroid bone marrow from patients with hemolytic anemias; release of newly synthesized virus into the supernatants of infected cultures was observed. This culture system allowed study at a molecular level of events associated with the B19 life cycle. The B19 parvovirus replicated through high molecular weight intermediate forms, linked through a terminal hairpin structure. B19 replication in vitro was highly dependent on the erythropoietic content of cultures and on addition of the hormone erythropoietin.

Electron microscopic observations of heterogeneous nuclear RNA of chicken reticulocytes

Secondary structures of annealed, snapped-back and untreated chicken reticulocyte poly(A)-containing nuclear RNA were investigated by electron microscopy. Three shapes of molecules were observed: ‘coat-hanger’ shaped, loop-bearing and filamentous molecules. The former two shaped molecules consist of a small and large fold-back or loop plus a short hook or tail, corresponding to the poly(A) segment at the apparent middle of the structure. The third shaped molecule, mainly seen after snap-back treatment, possesses a small fold-back structure at one end only. It is suggested that the small and large fold-backs or loops of the molecule represent the 5′- and 3′-terminal hairpin structures, respectively. Two distinct sizes of molecule, small and large, irrespective of molecule shape, were observed.