Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Direct RNA sequencing”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 73 records · Page 4Linked to original sources
PubMed1979

Sequence relationships among defective interfering influenza viral RNAs.

Each clone of ts-52 and ts+ WSN influenza virus, when serially passaged at high multiplicity, gives rise to defective interfering (DI) virus with a unique set of new RNA species. The new RNAs (DI RNA) from several DI viruses were examined by the technique of RNase T1 oligonucleotide two-dimensional electrophoresis. It was found that each DI RNA arises from a specific segment of standard viral RNA. All DI RNA studied arose from the viral polymerase genes (P1, P2, and P3). DI RNAs originating from the same polymerase gene were interrelated. Certain of these DI RNAs appeared to contain completely overlapping nucleotide sequences. Others contained both overlapping and nonoverlapping nucleotide sequences. The latter DI RNAs may be formed from the progenitor viral RNA segment by a mechanism other than a common initiation (or termination) point and a simple deletion from one end.

Base Sequence↗
PubMed1977

DNA complementary to parathyroid mRNA directs synthesis of pre-proparathyroid hormone in a linked transcription-translation system.

DNA complementary in sequence to the messenger RNA for pre-proparathyroid hormone was synthesised using reverse transcriptase. In a linked transcription-translation system using RNA polymerase and cell-free extract from wheat germ, the DNA directed the synthesis of a protein identified as pre-proparathyroid hormone by N-terminal sequencing and by electrophoretic and immunologic criteria.

Amino Acid Sequence↗
PubMed2025

Mapping Active RNA Polymerases in Proliferating and Quiescent Fission Yeast Cells Using Precision Run-On Sequencing.

The development of next-generation sequencing (NGS) approaches to investigate the functioning of RNA polymerases has led to groundbreaking advances in the field of transcriptional regulation. One powerful method, Precision nuclear Run-On sequencing (PRO-seq), maps the locations of RNA polymerase active sites genome-wide at high resolution. PRO-seq provides a snapshot of strand-specific transcriptional activity and does not rely on immunoprecipitation of the polymerase of interest. Notably, this technique has been utilized to investigate the control of the RNA polymerase II transcription cycle in a variety of model systems. However, the initially published PRO-seq method required significant amounts of starting sample and was technically challenging, both of which were deterrents for its broader use. Recently, an improved and simplified version called qPRO-seq that reduced the length of the experiment and the quantity of necessary input sample was developed for human and Drosophila cell lines. Here we provide an updated, step-by-step protocol in which we have validated and optimized qPRO-seq for the fission yeast Schizosaccharomyces pombe. Importantly, we have implemented this method for assessing RNA polymerase activity in nutrient-limiting conditions, for both proliferating and nitrogen-depleted quiescent cells.

Schizosaccharomyces↗
PubMed1979

Termination of transcription by bacteriophage T3 RNA polymerase: homogeneous 3'-terminal oligonucleotide sequence of in vitro T3 RNA polymerase transcripts.

RNA was synthesized in vitro from a T3 DNA template by T3 RNA polymerase and subsequently separated into seven discrete size classes (molecular weights ranging between 0.21 x 10(6) and 6.2 x 10(6)) by electrophoresis in polyacrylamide slab gels. RNase T1-generated 3'-terminal oligonucleotide fragments were then selectively isolated from either the unfractionated total RNA or the gel-purified specific transcripts by chromatography on columns of dihydroxyboryl-cellulose. Sequence analysis of these oligonucleotide products indicated that the unfractionated transcripts as well as all the individual major RNA species examined had a unique sequence, (Gp)UpUpUpUpUpGOH, at their 3' termini. The specificity of this sequence, as well as the total lack of any sequence heterogeneity at the ends of these transcripts, indicates a high degree of specificity of termination during transcription in this system.

Base Sequence↗
PubMed1979

The interaction of RNA polymerase and lac repressor with the lac control region.

We have examined the interactions of lac repressor and RNA polymerase with the DNA of the lac control region, using a method for direct visualization of the regions of DNA protected by proteins from DNAase attack. The repressor protects the operator essentially as reported by Gilbert and Maxam (1) with some small modifications. However, the evidence reported here concerning the binding of RNA polymerase to the DNA of the promoter mutant UV5 indicates that : 1) the RNA polymerase molecule binds asymmetrically to the promoter DNA, 2) RNA polymerase protects DNA sequences to within a few bases of the CAP binding site, suggesting direct interaction between polymerase and the CAP protein at this site, 3) RNA polymerase still binds to the promoter when repressor is bound to the operator, but fails to form the same extensive complex.

Base Sequence↗
PubMed1978

The attenuator of the tryptophan operon in E.coli: rho-mediated release of RNA polymerase from a transcription termination complex in vitro.

In vivo, termination of transcription at the attenuator site of the tryptophan (trp) operon of E. coli is influenced by the protein termination factor rho. In vitro, termination does not depend on rho factor, and is very efficient in a purified system consisting only of RNA polymerase, the DNA template, nucleoside triphosphates, and buffer. The extent of termination in this system is unaffected over a wide range of salt and nucleoside triphosphate concentration. However, there is a 10-fold stimulation of trp leader mRNA synthesis if rho factor is present during the transcription reaction. This stimulation occurs only at low molar ratios of polymerase to template, and can be blocked by rifampicin. It is thus most likely due to the recycling of RNA polymerase molecules that have been released from the attenuator site by rho factor. In fact, transcription of the trp leader region in vitro results in the fomration of a stable termination complex which can be observed on sucrose gradients or by binding to nitrocellulose filters. These data indicate that a major function of rho at the trp attenuator is to release completed transcripts from a pre-formed termination complex, rather than to cause the cessation of elongation.

Base Sequence↗
PubMed1978

DNA sequences of promoter regions for the str and spc ribosomal protein operons in E. coli.

The DNA sequences have been determined for promoter regions of two ribosomal protein operons in E. coli, the str operon and the spc operon. The site of in vitro transcription initiation within each of these promoter regions has been determined. The start site of the str operon occurs 69 bases upstream from the initiation codon of the S12 gene. The start site of the spc operon occurs 72 bases upstream from the L14 gene, and only 91 bases downstream from the termination codon of the S17 gene (which is in the preceding S10 operon). Both promoters are similar to other sequenced promoters in that they each have an identifiable "Pribnow box" sequence 5 bases upstream from the transcription start site. The spc promoter has a long sequence of 2 fold symmetry centered within the Pribnow box; the str promoter has a shorter but similar symmetry. At positions -69 through -40 in the spc operon, another long region of symmetry is present which may be the termination signal of the preceding S10 operon. Extensive sequence similarity between the str and spc promoter regions is found downstream from the Pribnow box-that is, in a transcribed region preceding the translation start sites.

Bacterial Proteins↗
PubMed1977

Terminally repeated sequences in the avian sarcoma virus RNA genome.

The initiation of DNA synthesis in vitro by RNA-directed DNA polymerase (deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase, EC 2.7.7.7) of avian oncornaviruses requires a tRNAtrp primer molecule located close to the 5' end of the viral RNA genome. DNA transcripts, 100 nucleotides in length, initiated on the tRNAtrp primer molecule contain nucleotide sequences complementary to a large (25 nucleotides) RNase T1 oligonucleotide, T-13, located at the 5' terminus of the avian sarcoma virus RNA genome. tRNAtrp-initiated DNA transcripts with a length of about 70 nucleotides contain substantially fewer nucleotide sequences complementary to this 5'-terminal oligonucleotide, suggesting that the tRNAtrp primer associated with the avian sarcoma virus RNA is located approximately 100 nucleotides from the 5' end of the RNA. In addition, we present evidence to demonstrate that DNA transcribed from avian sarcoma virus RNA sequences located at the 3' end, immediately adjacent to the poly(A), contains nucleotide sequences that are complementary to the 5'-terminal T1 oligonucleotide T-13. These data indicate that the 5' end of the viral genome contains nucleotide sequences that are repeated at the 3' end of the genome. We conclude that the avian oncornavirus RNA genome is terminally redundant.

Avian Sarcoma Viruses↗
PubMed1979

An MSV-specific subgenomic mRNA in MSV-transformed G8-124 cells.

An intracellular subgenomic RNA species from MSV-transformed G8-124 cells was characterized by electron microscopy of RNA:cDNA heteroduplexes using long cDNAs both MSV and MuLV. This subgenomic RNA, 3.1 kb long, consisted of 5'-derived sequences of about 0.4 kb joined to 2.7 kb of RNA derived from the 3' end of the RNA genome. The 3'-derived sequences included the residual sequences from the MuLV pol region and the acquired cellular sequences of MSV. The genome of MSV was shown to retain approximately 0.13 kb from the 5' end of the MuLV env region, including sequences which span the point in the MuLV env mRNA. No subgenomic MSV RNA could be detected, however, which consisted of a 5'-derived leader sequence spliced to the retained env region sequences. Nor could a subgenomic MSV RNA be detected in which a 5'-derived leader sequence was joined directly to the acquired cellular sequences. Although its translation products are unknown, the subgenomic MSV RNA was present in preparations of poly(A)+ polysomal RNA, consistent with this RNA functioning as a messenger. The structure of this 3.1 kb MSV subgenomic RNA suggests a possible role in the expression of 3'-encoded MSV information, possibly including transformation-specific sequences.

Cell Transformation, Viral↗
PubMed2026

YAP Promotes Microtubule Growth to Facilitate Sarcomere Disassembly in Adult Cardiomyocytes.

BACKGROUND: Mature mammalian cardiomyocytes (CMs) develop compact sarcomeric structures that inhibit proliferation. Consequently, CMs must dedifferentiate to a fetus-like state, which is accompanied by sarcomere disassembly, to enable successful cytokinesis. However, the regulation and coordination of CM dedifferentiation, cell cycle progression, and sarcomere reorganization remain unclear. METHODS: We generated adenovirus and adeno-associated virus (MyoAAV) vectors expressing YAP5SA and YAP5SA-S94A under Xon control for LMI070-inducible protein expression. We also developed MyoAAV-cTnT-Tuba1b-shRNA-miR30 for cardiomyocyte-specific knockdown of Tuba1b. These tools were used to investigate CM dedifferentiation and proliferation and sarcomere disassembly. We also performed Cleavage Under Targets and Release Using Nuclease to map the genome-wide binding sites of YAP5SA and YAP5SA-S94A in combination with RNA sequencing to identify YAP target genes. In addition, time-course live-imaging analysis was used to evaluate microtubule and sarcomere dynamics in adult CMs. RESULTS: We show that microtubule expression and network density decline with cardiac maturation. Overexpression of YAP5SA, a constitutively active YAP mutant, promotes microtubule growth by stabilizing microtubule dynamics, leading to CM dedifferentiation, cell cycle re-entry, and sarcomere disassembly. In contrast, colchicine blocks these processes and significantly attenuates YAP-induced cardiac regeneration. Live imaging reveals a distinct mode of sarcomere disassembly driven by enhanced microtubule polymerization, wherein microtubule plus-ends directly interact with α-actinin and displace α-actinin fragments, thereby facilitating sarcomere breakdown. Furthermore, the YAP5SA-S94A mutation, which disrupts the YAP and TEA domain interaction, significantly reduces YAP5SA-induced microtubule growth, sarcomere disassembly, and cell cycle activity. Mechanistically, cleavage under targets and release using nuclease combined with RNA sequencing identified direct YAP targets, including Ajuba and Tuba1b, which are critical for microtubule growth. CM-specific knockdown of Tuba1b attenuates YAP-driven sarcomere disassembly. CONCLUSIONS: These findings identify microtubule networks as an essential regulator modulating CM dedifferentiation and sarcomere reorganization, which is critical for CM cytokinesis and cardiac regenerative repair.

Animals↗
PubMed2008

Isolation and characterization of Plasmodium falciparum UAP56 homolog: evidence for the coupling of RNA binding and splicing activity by site-directed mutations.

UAP56 (U2AF65 associated protein) is a member of the DEAD-box helicase family. Helicases are essential enzymes generally involved in the metabolism of nucleic acids. The gene encoding a member of DEAD-box family was cloned and characterized from the human malaria parasite Plasmodium falciparum. PfU52 is homologous to UAP56 and contains the RNA-dependent ATPase, RNA helicase and RNA binding activities. Using the parasite extract we report that PfU52 is involved in splicing reaction. Site-directed mutagenesis studies indicate that the conserved residues glycine 181, isoleucine 182 and arginine 206 are involved in RNA binding and this activity is required for the enzymatic activities of PfU52. PfU52 is expressed in all the intraerythrocytic developmental stages of the parasite. In the present study we have reported the detailed characterization of PfU52 from P. falciparum and these results advance the knowledge regarding the function of UAP56 in general.

Adenosine Triphosphatases↗