Identification of stable RNA hairpins causing band compression in transcriptional sequencing and their elimination by use of inosine triphosphate

Sasaki, Nobuya; Izawa, Masaki; Sugahara, Yuichi; Tanaka, Takumi; Watahiki, Masanori; Ohara, Eiji; Funaki, Hiroko; Yoneda, Yuko; Ozawa, Kaori; Matsuura, Shuji; Muramatsu, Masami; Okazaki, Yasushi; Hayashizaki, Yoshihide

doi:10.1016/s0378-1119(98)00447-8

Cited by 17 publications

(13 citation statements)

References 15 publications

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“…We believe that fragments 1 and 2 are the products of a single cleavage event (see below) and that their actual sizes are 373 and 200 bases, respectively. We argue that these two fragments migrate anomalously, even in the presence of 7 M urea, because of the persistence of secondary structure after RNase IIIS cleavage (20). At higher ratios of enzyme to substrate the intensities of fragments 1 and 2 decrease, whereas those of fragments 3 and 4 increase (Fig.…”

Section: Cleavage Of the Rpso-pnp Transcript By Rnase Iiis-transcriptmentioning

confidence: 79%

The absB Gene Encodes a Double Strand-specific Endoribonuclease That Cleaves the Read-through Transcript of the rpsO-pnp Operon in Streptomyces coelicolor

Chang

Bralley

Jones

2005

Journal of Biological Chemistry

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The absB locus of Streptomyces coelicolor encodes a homolog of bacterial RNase III. We cloned and overexpressed the absB gene product and purified a decahistidine-tagged version of the protein.We show here that AbsB is active against double-stranded RNA transcripts derived from synthetic DNAs but is inactive with singlestranded homopolymers. We thus designate the absB product RNase IIIS. Using T7 RNA polymerase and a cloned template containing the rpsO-pnp intergenic region, we synthesized an RNA substrate representing a portion of the read-through transcript normally produced in S. coelicolor. This transcript contains the sequences that form the putative rpsO terminator and those that form an intergenic stem-loop structure thought to be the site for RNase IIIS processing of the read-through transcript. We show that RNase IIIS does cleave that model transcript, with primary and secondary cleavage sites in an internal loop in the stem-loop structure. We have identified the primary and secondary cleavage sites by primer extension and demonstrate the further processing of the initial cleavage products. Thus, as is the case in Escherichia coli, the read-through transcript from rpsO-pnp is cleaved by RNase IIIS in S. coelicolor. However, the cleavage sites are different in the two systems. The positions of the cleavage sites in the stem-loop of the S. coelicolor transcript are more akin to those identified in the processing of bacteriophage T7 mRNAs. A kinetic assay for RNase IIIS was developed, and kinetic parameters for the reaction utilizing the model transcript from rpsO-pnp were determined.Ribonuclease III is a double strand-specific endoribonuclease found in bacteria and eukaryotes. In Escherichia coli, RNase III is involved in the processing and maturation of viral, ribosomal, and messenger RNAs. Studies of the processing of bacteriophage T7 RNAs demonstrated an important role for RNase III and identified sequence features that determine the specificity of cleavage (1-3). RNase III is involved in the processing of the 30 S ribosomal RNA precursor to pre-16 S and pre-23 S intermediates in E. coli, and the 30 S precursor only accumulates in RNase III-deficient mutants (4, 5). Of particular relevance to the present study, RNase III is involved in the processing of transcripts from the rpsO-pnp operon in E. coli. The operon is transcribed from two promoters, PrpsO, situated upstream of rpsO, the gene for ribosomal protein S15, and Ppnp, a promoter located in the intergenic region between rpsO and pnp, the gene for polynucleotide phosphorylase (6). Transcription initiating at PrpsO produces a read-through transcript containing both rpsO and pnp, whereas initiation at Ppnp produces a pnp transcript only (6). Secondary structure models and biochemical studies have confirmed the presence of a stem-loop structure in the rpsO-pnp intergenic region, upstream of the pnp translation start site (6, 7). This stem loop is present in both the read-through transcript and the transcript from Ppnp and is cleaved at identical site...

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Section: Cleavage Of the Rpso-pnp Transcript By Rnase Iiis-transcriptmentioning

confidence: 79%

The absB Gene Encodes a Double Strand-specific Endoribonuclease That Cleaves the Read-through Transcript of the rpsO-pnp Operon in Streptomyces coelicolor

Chang

Bralley

Jones

2005

Journal of Biological Chemistry

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“…To speed up and reduce the costs of such large-scale sequencing operations, direct sequencing would be extremely useful because it allows skipping the plasmid preparation step. Alternatively, the use of the transcriptional sequencing (12,13) would have similar advantages because it requires only a minimal quantity of template, isothermal amplification, and no template concentration adjustment.…”

Section: Introductionmentioning

confidence: 99%

Removal of PolyA Tails from Full-Length cDNA Libraries for High-Efficiency Sequencing

et al. 2001

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We have developed a method to overcome sequencing problems caused by the presence of homopolymer stretches, such as polyA/T, in cDNA libraries. PolyA tails are shortened by cleaving before cDNA cloning with type IIS restriction enzymes, such as GsuI, placed next to the oligo-dT used to prime the polyA tails of mRNAs. We constructed four rice Cap-Trapper-selected, full-length normalized cDNA libraries, of which the average residual polyA tail was 4 bases or shorter in most of the clones analyzed Because of the removal of homopolymeric stretches, libraries prepared with this method can be used for direct sequencing and transcriptional sequencing without the slippage observed for libraries prepared with currently available methods, thus improving sequencing accuracy, operations, and throughput.

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“…For comparison, lane 2 shows the migration position of the end-labeled rpsO-pnp transcript, but it should be noted that unlabeled transcript was used in the polyadenylation assays. The rpsO-pnp transcript migrated more rapidly than expected, presumably due to the presence of some secondary structure in the transcript even in the presence of urea (20).…”

Section: Resultsmentioning

confidence: 75%

Geobacter sulfurreducens Contains Separate C- and A-Adding tRNA Nucleotidyltransferases and a Poly(A) Polymerase

2009

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The genome of Geobacter sulfurreducens contains three genes whose sequences are quite similar to sequences encoding known members of an RNA nucleotidyltransferase superfamily that includes tRNA nucleotidyltransferases and poly(A) polymerases. Reverse transcription-PCR using G. sulfurreducens total RNA demonstrated that the genes encoding these three proteins are transcribed. These genes, encoding proteins designated NTSFI, NTSFII, and NTSFIII, were cloned and overexpressed in Escherichia coli. The corresponding enzymes were purified and assayed biochemically, resulting in identification of NTSFI as a poly(A) polymerase, NTSFII as a C-adding tRNA nucleotidyltransferase, and NTSFIII as an A-adding tRNA nucleotidyltransferase. Analysis of G. sulfurreducens rRNAs and mRNAs revealed the presence of heteropolymeric RNA 3 tails. This is the first characterization of a bacterial system that expresses separate C-and A-adding tRNA nucleotidyltransferases and a poly(A) polymerase.RNA nucleotidyltransferases play an important role in the maturation, processing, and degradation of RNAs. tRNA nucleotidyltransferases (TNTs) and poly(A) polymerases (PAPs) are both members of a superfamily of nucleotidyltransferases (13, 27). TNTs add C and A residues to the 3Ј ends of immature or damaged tRNA molecules and are found in bacteria, archaea, and eukaryotes (24). PAPs, which are present in both eukaryotes and bacteria, polyadenylate RNA 3Ј ends (27). With regard to the distribution of RNA nucleotidyltransferases in bacteria, with only a few exceptions, the genomes of the alpha-and epsilonproteobacteria and of the low-and high-GϩC-content gram-positive bacteria appear to encode only one member of this superfamily, presumably a CCA-adding TNT (1). Several of the so-called deeply branching bacterial species, including Aquifex aeolicus and Deinococcus radiodurans, and some cyanobacteria contain two RNA nucleotidyltransferases, which have been shown to function as separate C-and A-adding TNTs (22,23). Interestingly, at least one low-GϩC-content gram-positive species, Bacillus halodurans, has also been shown to contain separate C-and A-adding TNTs (1). The beta-and gammaproteobacteria, such as Escherichia coli, also contain two RNA nucleotidyltransferases, a CCA-adding TNT and a PAP, PAP I, that adds 3Ј tails to the ends of RNA molecules in cells of these organisms (8,11,18,19). mRNAs and 16S and 23S rRNAs are polyadenylated in E. coli and presumably in other beta-and gammaproteobacteria, and polyadenylation facilitates the exonucleolytic degradation of these RNAs by RNase II and polynucleotide phosphorylase (9,16,18,19).The genomes of several deltaproteobacteria also encode two members of the RNA nucleotidyltransferase superfamily (NTSF), and the genomes of a few deltaproteobacteria appear to encode three NTSF enzymes (1). In particular, the genomes of Geobacter sulfurreducens, Geobacter metallireducens, Pelobacter carbinolicus, and Desulfotalea psychrophila all contain three open reading frames with significant homology to open reading ...

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Identification of stable RNA hairpins causing band compression in transcriptional sequencing and their elimination by use of inosine triphosphate

Cited by 17 publications

References 15 publications

The absB Gene Encodes a Double Strand-specific Endoribonuclease That Cleaves the Read-through Transcript of the rpsO-pnp Operon in Streptomyces coelicolor

The absB Gene Encodes a Double Strand-specific Endoribonuclease That Cleaves the Read-through Transcript of the rpsO-pnp Operon in Streptomyces coelicolor

Removal of PolyA Tails from Full-Length cDNA Libraries for High-Efficiency Sequencing

Geobacter sulfurreducens Contains Separate C- and A-Adding tRNA Nucleotidyltransferases and a Poly(A) Polymerase

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