Direct Sensing of Single Native RNA with a Single-Biomolecule Interface of Aerolysin Nanopore.

RNA sensing is of vital significance to advance our comprehension of gene expression and to further benefit medical diagnostics. Taking advantage of the excellent sensing capability of the aerolysin nanopore as a single-biomolecule interface, we for the first time achieved the direct characterization of single native RNA of Poly(A)4 and Poly(U)4. Poly(A)4 induces ∼10% larger blockade current amplitude than Poly(U)4. The statistical duration of Poly(A)4 is 18.83 ± 1.08 ms, which is 100 times longer than that of Poly(U)4. Our results demonstrated that the capture of RNA homopolymers is restricted by the biased diffusion. The translocation of RNA needs to overcome a lower free-energy barrier than that of DNA. Moreover, the strong RNA-aerolysin interaction is attributed to the hydroxyl in pentose, which prolongs the translocation time. This study opens an avenue for aerolysin nanopores to directly achieve RNA sensing, including discrimination of RNA epigenetic modification and selective detection of miRNA.

Ebola Virus Infections in Nonhuman Primates Are Temporally Influenced by Glycoprotein Poly-U Editing Site Populations in the Exposure Material.

Recent experimentation with the variants of the Ebola virus that differ in the glycoprotein's poly-uridine site, which dictates the form of glycoprotein produced through a transcriptional stutter, has resulted in questions regarding the pathogenicity and lethality of the stocks used to develop products currently undergoing human clinical trials to combat the disease. In order to address these concerns and prevent the delay of these critical research programs, we designed an experiment that permitted us to intramuscularly challenge statistically significant numbers of naïve and vaccinated cynomolgus macaques with either a 7U or 8U variant of the Ebola virus, Kikwit isolate. In naïve animals, no difference in survivorship was observed; however, there was a significant delay in the disease course between the two groups. Significant differences were also observed in time-of-fever, serum chemistry, and hematology. In vaccinated animals, there was no statistical difference in survivorship between either challenge groups, with two succumbing in the 7U group compared to 1 in the 8U challenge group. In summary, survivorship was not affected, but the Ebola virus disease course in nonhuman primates is temporally influenced by glycoprotein poly-U editing site populations.

pre-miRNA profiles obtained through application of locked nucleic acids and deep sequencing reveals complex 5'/3' arm variation including concomitant cleavage and polyuridylation patterns.

Recent research hints at an underappreciated complexity in pre-miRNA processing and regulation. Global profiling of pre-miRNA and its potential to increase understanding of the pre-miRNA landscape is impeded by overlap with highly expressed classes of other non coding (nc) RNA. Here, we present a data set excluding these RNA before sequencing through locked nucleic acids (LNA), greatly increasing pre-miRNA sequence counts with no discernable effect on pre-miRNA or mature miRNA sequencing. Analysis of profiles generated in total, nuclear and cytoplasmic cell fractions reveals that pre-miRNAs are subject to a wide range of regulatory processes involving loci-specific 3'- and 5'-end variation entailing complex cleavage patterns with co-occurring polyuridylation. Additionally, examination of nuclear-enriched flanking sequences of pre-miRNA, particularly those derived from polycistronic miRNA transcripts, provides insight into miRNA and miRNA-offset (moRNA) production, specifically identifying novel classes of RNA potentially functioning as moRNA precursors. Our findings point to particularly intricate regulation of the let-7 family in many ways reminiscent of DICER1-independent, pre-mir-451-like processing, introduce novel and unify known forms of pre-miRNA regulation and processing, and shed new light on overlooked products of miRNA processing pathways.

Meta-analysis of small RNA-sequencing errors reveals ubiquitous post-transcriptional RNA modifications.

Recent advances in DNA-sequencing technology have made it possible to obtain large datasets of small RNA sequences. Here we demonstrate that not all non-perfectly matched small RNA sequences are simple technological sequencing errors, but many hold valuable biological information. Analysis of three small RNA datasets originating from Oryza sativa and Arabidopsis thaliana small RNA-sequencing projects demonstrates that many single nucleotide substitution errors overlap when aligning homologous non-identical small RNA sequences. Investigating the sites and identities of substitution errors reveal that many potentially originate as a result of post-transcriptional modifications or RNA editing. Modifications include N1-methyl modified purine nucleotides in tRNA, potential deamination or base substitutions in micro RNAs, 3' micro RNA uridine extensions and 5' micro RNA deletions. Additionally, further analysis of large sequencing datasets reveal that the combined effects of 5' deletions and 3' uridine extensions can alter the specificity by which micro RNAs associate with different Argonaute proteins. Hence, we demonstrate that not all sequencing errors in small RNA datasets are technical artifacts, but that these actually often reveal valuable biological insights to the sites of post-transcriptional RNA modifications.

[Analysis of polyA, polyU and double-stranded complex polyA x polyU via Raman spectroscopy].

The Raman spectra of PolyA, PolyU and their double-stranded complex were measured, and the spectral changes upon the formation of double-stranded complex were studied. The experimental results show: (1) Under the experimental conditions used in the present work (0.14 mol x L(-1) NaCl, 1 mmol x L(-1) Tris solution, neutral pH and 15 degrees C), PolyU, PolyA and PolyA x PolyU occur as random-coiled, A-single-stranded helical and A-double-stranded helical conformations, respectively. One of the main spectral differences between the latter two conformations and the former one is the Raman band near 814 cm(-1) of ordered structures. Another difference is in the full width at half the maximum (i.e. FWHM) of the band near 1100 cm(-1). The FWHM of the band 1100 cm(-1) of PolyA is the same as that of PolyA x PolyU, while the band of PolyU shows remarkable broadening. In addition, we found that the conformation of PolyA is somewhat not so ordered as that of its duplex, which can be concluded from the value of I814/I1100 of the two polynucleotides. (2) The formation of duplex makes base-base stacking interactions much stronger, and the conformation of the backbone more ordered, which leads to obvious Raman hypochromic effect with some corresponding band shift. In this process, PolyU underwent more significant spectral changes than PolyA. As spectral markers, these results can be of great importance in Raman spectral signal detection of gene-chips.

Rampant polyuridylylation of plastid gene transcripts in the dinoflagellate Lingulodinium.

Dinoflagellate plastid genes are believed to be encoded on small generally unigenic plasmid-like minicircles. The minicircle gene complement has reached saturation with an incomplete set of plastid genes (18) compared with typical functional plastids (60-200). While some of the missing plastid genes have recently been found in the nucleus, it is still unknown if additional genes, not located on minicircles, might also contribute to the plastid genome. Sequencing of tailed RNA showed that transcripts derived from the known minicircle genes psbA and atpB contained a homogenous 3' polyuridine tract of 25-40 residues. This unusual modification suggested that random sequencing of a poly(dA) primed cDNA library could be used to characterize the plastid transcriptome. We have recovered only 12 different polyuridylylated transcripts from our library, all of which are encoded on minicircles in several dinoflagellate species. The correspondence of all polyuridylylated transcripts with previously described minicircle genes thus supports the dinoflagellate plastid as harbouring the smallest genome of any functional chloroplast. Interestingly, northern blots indicate that the majority of transcripts are modified, suggesting that polyuridylylation is unlikely to act as a degradation signal as do the heterogeneous poly(A)-rich extensions of transcripts in cyanobacteria and other plastids.

Elastic properties of a single-stranded charged homopolymeric ribonucleotide.

We have investigated the elastic properties of poly(U), homopolymeric single-stranded RNA molecules that lack any base pairing and stacking interactions and conform to a random-coil structure. Using single-molecule stretching experiments we show that the elastic properties are described by a wormlike chain model for polymer elasticity rather than by a freely jointed chain model as is commonly used for single-stranded DNA. At low [Na+], introduction of a scale-dependent persistence length is required to account for electrostatic contributions.

Expression of acinar and ductal products in Capan-1 cells growing in synthetic serum and serum-free media.

BACKGROUND: Capan-1 is a human pancreatic adenocarcinoma cell line of presumed ductal origin. This is based on the histologic appearance of the tumor from which it arose. Yet considerable controversy exists regarding the actual cell of origin for these exocrine carcinomas. Two acinar antigens, ribonuclease and trypsin, were analyzed in cells growing in synthetic serum. METHODS: Capan-1 cells were adapted to grow in basal medium supplemented with synthetic serum, because fetal bovine serum (FBS) normally used to culture cells contains bovine ribonuclease, which can interfere with measurements of the ribonuclease secretion. These cells were also adapted to grow in different serum-free media, allowing us to determine its minimal growth requirements. The presence of ribonuclease in Capan-1 and PANC-1 conditioned media was monitored by activity. Other acinar and ductal markers were monitored using Northern blot analysis. RESULTS: Capan-1, PANC-1, IBF-CP3, and MDAAmp-7 cell lines were successfully adapted to grow in synthetic serum by means of the adaptation protocol reported here. The adaptation of Capan-1 to serum-free media showed that the cells are capable of growing in a medium containing insulin, transferrin, selenium, a nonprotein carrier, and lipoic and linoleic acids. Northern blot analysis showed the expression of carbonic anhydrase II, cytokeratin 18, ribonuclease, and trypsin in Capan-1 cells growing in FBS and synthetic serum. No changes in morphology, karyotype, or gene expression were observed in these cells as a result of the adaptation process. CONCLUSION: The cell line Capan-1 is expressing some ductal as well as acinar products despite its supposed ductal origin. The expression of trypsin at the mRNA level and ribonuclease at mRNA and protein levels is shown in Capan-1 cells. The protein expression will be further investigated as the cell line has been adapted to grow in synthetic serum and serum-free media with no apparent changes with respect to their growth in FBS.

The 5' end of coronavirus minus-strand RNAs contains a short poly(U) tract.

A radiolabeled oligodeoxynucleotide primer that anneals near the common 5' end of bovine coronavirus minus-strand RNAs was extended with reverse transcriptase, and a major product suggesting poly(U) tracts of 8 to 20 nucleotides was found. The extended primer molecules were ligated head to tail, amplified by the polymerase chain reaction, cloned, and sequenced, and poly(U) tracts of 9 to 26 nucleotides were found. Poly(A) tails of 100 to 130 nucleotides on the 3' end of coronavirus plus-strand mRNAs and genome must, therefore, be generated by a mechanism that uses only a short poly(U) template. This pattern contrasts with that of other cytoplasmic, polyadenylated, plus-strand animal RNA viruses which utilize a full-length poly(U) template for poly(A) synthesis.

High-performance ion-exchange chromatography of oligoribonucleotides using linear and hyperbolic salt gradients.

A method for separation and chain length determination of oligo- and polynucleotides by high-performance anion-exchange chromatography was developed, which allows resolution of individual fragments according to their chain length n, up to n approximately 10 by linear gradient of sodium chloride and up to n approximately 30 by an hyperbolic gradient of this salt. The hyperbolic relationship between n and the salt concentration at which elution of the fragment occurs allows determination of the degree of polymerization of oligo- and polynucleotides with unknown n. The method proposed can be used for estimation of the effective charge of nucleic acids with complex structure.

Occurrence and sequence complexity of polyadenylated RNA in squid axoplasm.

Axoplasmic RNA from the giant axon of the squid (Loligo pealii) comprises polyadenylated [poly (A)+] RNA, as judged, in part, by hybridization to [3H]polyuridine and by in situ hybridization analyses using the same probe. The polyadenylate content of axoplasm (0.24 ng/microgram of total RNA) suggests that the poly(A)+ RNA population makes up approximately 0.4% of total axoplasmic RNA. Axoplasmic poly(A)+ RNA can serve as a template for the synthesis of cDNA using a reverse transcriptase and oligo(deoxythymidine) as primer. The size of the cDNA synthesized is heterogeneous, with most fragments greater than 450 nucleotides. The hybridization of axoplasmic cDNA to its template RNA reveals two major kinetic classes: a rapidly hybridizing component (abundant sequences) and a slower-reacting component (moderately abundant and rare sequences). The latter component accounts for approximately 56% of the total cDNA mass. The rapidly and slowly hybridizing kinetic components have a sequence complexity of approximately 2.7 kilobases and 3.1 X 10(2) kilobases, respectively. The diversity of the abundant and rare RNA classes is sufficient to code for one to two and 205, respectively, different poly(A)+ RNAs averaging 1,500 nucleotides in length. Overall, the sequence complexity of axoplasmic poly(A)+ RNA represents approximately 0.4% that of poly(A)+ mRNA of the optic lobe, a complex neural tissue used as a standard. Taken together, these findings indicate that the squid giant axon contains a heterogeneous population of poly(A)+ RNAs.

Part of the 23S RNA located in the 11S RNA fragment is a constituent of the ribosomal peptidyltransferase centre.

Upon irradiation, 3-[4-benzoylphenyl]propionyl-PhetRNA bound to the P-site of poly(U)-primed ribosomes is exclusively cross-linked to 23S RNA. It is shown that the photoreaction only occurs with pyrimidine nucleotides. The site of the cross-link is located within an 11S RNA fragment, which comprises the 1100 nucleotides at the 3'-end of 23S RNA. The cross-linked Phe tRNA derivative is still functionally active in peptide bond formation. The site labelled on the 11S fragment is therefore an integral part of the peptidyltransferase centre.

[DNA-binding proteins in human serum]. / DNK-sviazyvaiushchie belki syvorotki krovi cheloveka.

Fractions of acid and base blood serum proteins, separated by ion exchange chromatography on QAE-Sephadex (but not the proteins of the whole blood serum, which had the same charge), reacted with DNA preparations. Separate fractions of blood serum proteins were able to react with DNA after electrophoretic separation. Binding of blood serum proteins with DNA did not depend on ion strength within the range of NaCl concentration from 0.1 M to 0.5 M; it was also stable at pH 5.5 = 8.0. Interaction between acid DNA-binding proteins and native DNA was inhibited by denatured DNA, polyguanilic and polyinosinic acids and by other polyanions: dextran sulfate, polyvinyl sulfate, polyanetol sulphonate, polystyrol sulphonate and heparin. Acid DNA-binding proteins showed only a slight affinity to polyadenilic, polyuridilic and polycytidilic acids. The acid and base DNA-binding proteins appear to be contained in blood serum in the form of a loosely bound complex.

Characterization of a 70S polyuridylic acid polymerase isolated from foot-and-mouth disease virus-infected cells.

A polyuridylic acid polymerase complex isolated from foot-and-mouth disease virus-infected cells sedimented at 70S in a sucrose gradient and appeared in the exclusion volume of an agarose column whose molecular weight cutoff was 5 x 10(6). Phenol extraction of the complex yielded a heterogeneous band of virus-specific RNA and an apparently host cell-derived 4.5 to 5S RNA, both of which are essentially single stranded. Neither RNA served as a template in the cell-free enzyme reaction. Polyacrylamide gel analysis revealed five polypeptides with molecular weights of 50,000, 56,000, 60,000, 70,000, and 74,000 and with molar ratios of 1:2:2:1:1, respectively. Autoradiography showed P56 to be the only major virus-induced polypeptide; the other proteins are apparently of host cell origin. Electron microscopic examination suggested a cartwheel shape for the polymerase complex which was seen to dissociate as polyadenylic acid was added. Antibody previously shown to inhibit enzyme activity aggregated the 70S units.

A circular dichroism study of Escherichia coli Initiation Factor-1 binding to polynucleotides.

Binding of Escherichia coli Initiation Factor-1 protein to the nucleic acid lattice induces alterations in the secondary structures of a variety of purine and pyrimidine containing polynucleotides in both the single and double stranded conformations, as assessed by circular dichroism spectroscopy. The helical hairpin form of poly(U), the single-stranded stacked form of poly(C), and the duplex poly(A) x poly(U) (in the presence of Mg2+) are stoichiometrically converted by Initiation Factor-1 (IF-1) to structures spectrally indistinguishable from their partially or completely thermally denatured forms. By contrast, the binding of IF-1 to double stranded poly(C), single- and double-stranded poly(A) elicited spectral responses which were interpreted in terms of diminished base-base interaction, not equivalent to that induced by thermal means. Stoichiometric endpoints of 3-5 nucleotide residues/IF-1 were determined for polynucleotide structures in those cases where light scattering artifacts at low nucleotide residue to protein ratios were absent. In the absence of Mg2+ IF-1 was unable to elicit a conformation alteration effect in poly(A) x poly(U), while for poly(U) much less of an effect was observed than in the presence of this divalent ion. The functional significance of these results is briefly considered.

Oligomeric sequences in the cytoplasmic RNA of sea urchin embryos.

Oligomeric stretches of adenylate and uridylate and polymeric segments of adenylate have been shown to exist in sea urchin embryo hnRNA. It is demonstrated here that at least some oligo(U)-enriched sequences are conserved in sea urchin cytoplasmic RNA, whereas apparently few, if any, oligo(A) sequences are so conserved.