Use of in Vitro 32P Labeling in the Sequence Analysis of Nonradioactive tRNAs

Silberklang, Melvin; Gillum, Amanda M.; RajBhandary, Uttam L.

doi:10.1016/b978-0-12-504180-5.50020-1

Cited by 49 publications

(64 citation statements)

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“…The pre-tRNAsI'u were digested with T1 RNase, and the digests were fractionated on DBAE-cellulose columns. The bound oligonucleotides were analyzed by fingerprinting (33 two-dimensional thin-layer chromatography (27).…”

Section: Discussionmentioning

confidence: 99%

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo.

Ganguly¹,

Sharp²,

RajBhandary³

1988

Mol. Cell. Biol.

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We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNALU gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNAI"U gene. Extracts from HeLa or CV1 cells transcribed both tRNALeU genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNALu.Surprisingly, when the same tRNAI"U genes were introduced into CV1 cells, only pre-tRNAsLu were produced. The pre-tRNAsI"u made in vivo were of the same size and contained the same 5'-leader and 3'-trailer sequences as did pre-tRNAsLu made in vitro. Furthermore, the pre-tRNAsI"u made in vivo were processed to mature tRNAIeU when incubated with HeLa cell extracts. A tRNALeU gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNALu in CV1 cells is blocked at the level of 5'-and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAslu synthesized in CV1 cells are not transported to the appropriate site.

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Section: Discussionmentioning

confidence: 99%

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo.

Ganguly¹,

Sharp²,

RajBhandary³

1988

Mol. Cell. Biol.

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“…We wanted to investigate the correlation between adenylation and uridylation on the 39 end of small RNAs+ Initially, we used HeLa cell extracts capable of uridylating the endogenous U6 snRNA present in HeLa cell extracts (Gu et al+, 1997)+ Subsequently, we developed an in vitro system utilizing HeLa cell extract (Weil et al+, 1979) in which uridylation is dependent on the addition of in vitro-transcribed U6 RNA substrate (Fig+ 4A)+ The HeLa cell extract itself was capable of uridylating endogenous U6 snRNA and 5S rRNA, and can be readily detected upon incubation in the presence of (England et al+, 1980) and the labeled RNA was digested to completion with T2 RNase and fractionated by two-dimensional chromatography (Silberklang et al+, 1979) Fig+ 4B, lane 3)+ Although equal quantities of 5S rRNA of discrete size were added to the incubation mixture, in addition to the correct size 5S RNA band, a minor labeled RNA with slightly faster migration was consistently observed (Fig+ 4B, lanes 3 and 4)+ We presume that this faster migrating band is a 5S RNA truncated at its 59 end+…”

Section: U6 Snrna With 39 a Is Not Uridylated In Vitromentioning

confidence: 99%

“…32 P]-UTP were mixed in a total volume of 50 mL and incubated at 30 8C for 90 min+ The amount of in vitrosynthesized RNAs used as substrates for uridylation assay was ;1 mg (20 pmol) for each reaction+ Labeled RNAs were extracted using the phenol/chloroform procedure, purified, and fractionated on 10% polyacrylamide gels containing 7 M urea+ Whenever necessary, labeled RNAs were excised from the gel, eluted, and purified+ These RNAs were then digested with T2 RNase and analyzed by two-dimensional chromatography on cellulose plates as described by Silberklang et al+ (1979)+ The first dimension solvent was isobutyric acid/water/ ammonium hydroxide (66:33:1, v/v/v) and the second dimension solvent was 0+1 M sodium phosphate buffer (pH 6+8)/ ammonium sulfate/n-propanol (100:60:2, v/w/v)+ The dried plates were subjected to autoradiography or phosphorimaging+…”

Section: Preparation Of Hela Cell S-100 Extract and In Vitro Labelingmentioning

confidence: 99%

Effect of 3′ terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes

Chen

Sinha

Perumal

et al. 2000

RNA

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It is known that several small RNAs including human and Xenopus signal recognition particle (SRP) RNA, U2 small nuclear RNA (snRNA) and 7SK RNAs are posttranscriptionally adenylated, whereas U6 snRNA and ribosomal 5S RNA are posttranscriptionally uridylated on their 39 ends. In this study, we provide evidence that a small fraction of U6 snRNA and 5S ribosomal RNA molecules from human as well as Xenopus oocytes contain a single posttranscriptionally added adenylic acid residue on their 39 ends. These data show that U6 snRNA and 5S rRNAs are posttranscriptionally modified on their 39 ends by both uridylation and adenylation. Although the SRP RNA, 7SK RNA, 5S RNA, and U6 snRNA with the uridylic acid residue on their 39 ends were readily uridylated, all these RNAs with posttranscriptionally added adenylic acid residue on their 39 ends were not uridylated in vitro, or when U6 snRNA with 39 A OH was injected into Xenopus oocytes. These results show that the presence of a single posttranscriptionally added adenylic acid residue on the 39 end of SRP RNA, U6 snRNA, 5S rRNA, or 7SK RNA prevents 39 uridylation. These data also show that adenylation and uridylation are two competing processes that add nucleotides on the 39 end of some small RNAs and suggest that one of the functions of the 39 adenylation may be to negatively affect the 39 uridylation of small RNAs.

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“…Samples isolated from RPC-5 columns (see above) were completely digested with nuclease P1 by incubating in 50 mL containing 50 mM ammonium acetate, pH 5+3, at 37 8C for 16 h+ Three microliters of each digest were applied to a cellulose TLC plate (20 ϫ 20 cm) and the resulting 59-monophosphate nucleosides resolved by two-dimensional chromatography for 8 h in each direction using solvent A and C of Silberklang et al+ (1979)+ Autoradiographs were prepared from developed plates by exposing for about 12 h+ Modified bases were identified by cochromatographing the standards, pA, pC, pU, and pG (Sigma)+ Standards were detected by UV irradiation and the relative mobility rates (Rf ) of each modified base were calculated and compared with those of known modified bases (Silberklang et al+, 1979;Diamond et al+, 1993;Choi et al+, 1994)+…”

Section: Minor-base Analysismentioning

confidence: 99%

Methylation of the ribosyl moiety at position 34 of selenocysteine tRNA[Ser]Sec is governed by both primary and tertiary structure

Kim

Matsufuji

et al. 2000

RNA

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The selenocysteine (Sec) tRNA A) at positions 37, 55, and 58, respectively. As methylation of the ribose at position 34 is influenced by the intracellular selenium status and the presence of this methyl group dramatically alters tertiary structure, we investigated the effect of the modifications at other positions as well as tertiary structure on its formation. Mutations were introduced within a synthetic gene encoded in an expression vector, transcripts generated and microinjected into Xenopus oocytes, and the resulting tRNA products analyzed for the presence of modified bases. The results suggest that efficient methylation of mcmU to yield mcmUm requires the prior formation of each modified base and an intact tertiary structure, whereas formation of modified bases at other positions, including mcmU, is not as stringently connected to precise primary and tertiary structure. These results, along with the observations that methylation of mcmU is enhanced in the presence of selenium and that this methyl group affects tertiary structure, further suggest that the mcmUm isoacceptor must have a role in selenoprotein synthesis different from that of the mcmU isoacceptor.

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Use of in Vitro 32P Labeling in the Sequence Analysis of Nonradioactive tRNAs

Cited by 49 publications

References 0 publications

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo.

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo.

Effect of 3′ terminal adenylic acid residue on the uridylation of human small RNAs in vitro and in frog oocytes

Methylation of the ribosyl moiety at position 34 of selenocysteine tRNA[Ser]Sec is governed by both primary and tertiary structure

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