A new G-tailing method for the determination of the poly(A) tail length applied to hepatitis A virus RNA

Kusov, Yuri; Shatirishvili, Gocha; Dzagurov, Georgy; Gauss‐Müller, Verena

doi:10.1093/nar/29.12.e57

Cited by 39 publications

(46 citation statements)

References 16 publications

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“…A new and improved variation of the 39-RACE PAT involving G-tailing of mRNA was employed to show both the presence of the poly(A) tail and its length, as described elsewhere (Kusov et al, 2001).…”

Section: Methodsmentioning

confidence: 99%

“…For this, we employed a recently described PCR-based PAT assay, where the 39 end of the mRNA is polyguanylated using yeast poly(A) polymerase, as described earlier (Kusov et al, 2001). With this step, a poly(A)-oligo(G) junction is generated, which serves as specific target for the amplification of the 39 end of the transcriptome with the universal reverse oligo(dC 9 T 6 ) anchor primer and a gene-specific forward primer.…”

Section: Cdr1 Mrna Poly(a) Tail Length Is Longer In Ar Isolatesmentioning

confidence: 99%

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PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans

et al. 2010

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Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates ofCandida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 39 untranslated region (39 UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 39 UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 39 UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was~35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-a, were recovered from AS (MTL-a/MTL-a), while a single type of PAP1 allele (PAP1-a) was recovered from AR isolates (MTL-a/MTL-a). Among the heterozygous deletions of PAP1-a (Dpap1-a/PAP1-a) and PAP1-a (PAP1-a/Dpap1-a), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.

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Section: Methodsmentioning

confidence: 99%

Section: Cdr1 Mrna Poly(a) Tail Length Is Longer In Ar Isolatesmentioning

confidence: 99%

PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans

et al. 2010

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“…This primer contains an oligo(dT) 17 sequence that can anneal to poly(A) tails. cDNA amplification used gene-specific forward primers and reverse primer AD20 ( Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…The polyguanylation method used in 3Ј tail analysis was adapted from Kusov et al (17). Fifteen micrograms of total RNA was dissolved in 14 l water, heated for 5 min at 65°C, and then snap chilled on ice.…”

Section: Methodsmentioning

confidence: 99%

“…Lane 1 of the figure shows a size standard, oligo(dT) 18 To confirm the presence of poly(A) tails and to analyze those tails for sequence heterogeneity, we cloned cDNAs using two RT-PCR protocols. In the first method an oligo(dT) 17 primer designed to hybridize to poly(A) tail sequences was used for reverse transcription of total RNA from B. halodurans. This primer (ADoT; Table 1) also contained an adaptor sequence identical to the reverse primer AD20 that was subsequently used along with gene-specific primers in PCR amplification as described in Materials and Methods.…”

Section: Liv]-[liv]-g-[rk]-f-x-[liv]-h-[hlq]-[liv] (Alternative Aminomentioning

confidence: 99%

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A Phylogeny of Bacterial RNA Nucleotidyltransferases: Bacillus halodurans Contains Two tRNA Nucleotidyltransferases

2005

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We have analyzed the distribution of RNA nucleotidyltransferases from the family that includes poly(A) polymerases (PAP) and tRNA nucleotidyltransferases (TNT) in 43 bacterial species. Genes of several bacterial species encode only one member of the nucleotidyltransferase superfamily (NTSF), and if that protein functions as a TNT, those organisms may not contain a poly(A) polymerase I like that of Escherichia coli. The genomes of several of the species examined encode more than one member of the nucleotidyltransferase superfamily. The function of some of those proteins is known, but in most cases no biochemical activity has been assigned to the NTSF. The NTSF protein sequences were used to construct an unrooted phylogenetic tree. To learn more about the function of the NTSFs in species whose genomes encode more than one, we have examined Bacillus halodurans. We have demonstrated that B. halodurans adds poly(A) tails to the 3 ends of RNAs in vivo. We have shown that the genes for both of the NTSFs encoded by the B. halodurans genome are transcribed in vivo. We have cloned, overexpressed, and purified the two NTSFs and have shown that neither functions as poly(A) polymerase in vitro. Rather, the two proteins function as tRNA nucleotidyltransferases, and our data suggest that, like some of the deep branching bacterial species previously studied by others, B. halodurans possesses separate CC-and A-adding tRNA nucleotidyltransferases. These observations raise the interesting question of the identity of the enzyme responsible for RNA polyadenylation in Bacillus.RNA nucleotidyltransferases play an important role in the maturation, processing, and degradation of RNAs. tRNA nucleotidyltransferases (TNT) and poly(A) polymerases (PAP) are members of a superfamily of RNA nucleotidyltransferases (13, 39). tRNA nucleotidyltransferases add CCA residues to the 3Ј ends of immature tRNAs and repair the 3Ј ends of damaged tRNAs (36). tRNA nucleotidyltransferases (TNTs) are found in bacteria, archaeae, and eukaryotes, suggesting conservation of structure and function of this class of enzymes over the course of evolution (39). Polyadenylation of RNA 3Ј ends, once thought to occur exclusively in eukaryotic cells, has now been shown definitively to occur in bacterial systems as well (7,25,30,31). Escherichia coli cells contain at least two enzymes capable of adding poly(A) tails to the 3Ј ends of RNA molecules. The major polyadenylating enzyme, poly(A) polymerase I (PAP I), was originally identified by virtue of its role in regulating plasmid copy number and is a product of the pcnB gene (6, 12). PAP I can utilize any of the three nucleoside triphosphates as substrates in vitro, but only A residues are added by the enzyme to RNA 3Ј ends in vivo (38). Messenger RNAs and 16S and 23S ribosomal RNAs are all polyadenylated in E. coli, and this modification is an important element of the RNA degradation pathway in bacterial systems (7,25,30,31). RNA 3Ј ends are generated in E. coli via endonucleolytic cleavage by RNase E and RNase III, thes...

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Deadenylation by the CCR4‐NOT complex contributes to the turnover of hairy‐related mRNAs in the zebrafish segmentation clock

et al. 2018

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In the zebrafish segmentation clock, hairy/enhancer of split‐related genes her1, her7, and hes6 encodes components of core oscillators. Since the expression of cyclic genes proceeds rapidly in the presomitic mesoderm (PSM), these hairy‐related mRNAs are subject to strict post‐transcriptional regulation. In this study, we demonstrate that inhibition of the CCR4‐NOT deadenylase complex lengthens poly(A) tails of hairy‐related mRNAs and increases the amount of these mRNAs, which is accompanied by defective somite segmentation. In transgenic embryos, we show that EGFP mRNAs with 3′UTRs of hairy‐related genes exhibit turnover similar to endogenous mRNAs. Our results suggest that turnover rates of her1, her7, and hes6 mRNAs are differently regulated by the CCR4‐NOT deadenylase complex possibly through their 3′UTRs in the zebrafish PSM.

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A new G-tailing method for the determination of the poly(A) tail length applied to hepatitis A virus RNA

Cited by 39 publications

References 16 publications

PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans

PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans

A Phylogeny of Bacterial RNA Nucleotidyltransferases: Bacillus halodurans Contains Two tRNA Nucleotidyltransferases

Deadenylation by the CCR4‐NOT complex contributes to the turnover of hairy‐related mRNAs in the zebrafish segmentation clock

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