A synergistic network of interactions promotes the formation of in vitro processing bodies and protects mRNA against decapping

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

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U2.3 Precursor Small Nuclear RNA in vitro Processing Assay

BIO-PROTOCOL ◽

10.21769/bioprotoc.4142 ◽

2021 ◽

Vol 11 (17) ◽

Author(s):

Chan Lin ◽

Yujie Feng ◽

Xueyan Peng ◽

Jiaming Wu ◽

Weili Wang ◽

...

Keyword(s):

Small Nuclear Rna ◽

In Vitro Processing ◽

Nuclear Rna

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Ribozyme, antisense RNA, and antisense DNA inhibition of U7 small nuclear ribonucleoprotein-mediated histone pre-mRNA processing in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4479-4487.1989 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4479-4487

Author(s):

M Cotten ◽

G Schaffner ◽

M L Birnstiel

Keyword(s):

Antisense Rna ◽

Nuclear Extract ◽

Mrna Processing ◽

Rna Molecules ◽

Small Nuclear Ribonucleoprotein ◽

In Vitro Processing ◽

Antisense Dna ◽

Nuclear Ribonucleoprotein ◽

Fold Excess

A comparative analysis of ribozyme, antisense RNA, and antisense DNA inhibitors of the in vitro small nuclear ribonucleoprotein U7-dependent histone pre-mRNA processing reaction was performed. RNA molecules complementary to the U7 sequence inhibited in vitro processing of histone pre-mRNA at a sixfold excess over U7. Single-stranded DNA complementary to the entire U7 sequence inhibited the reaction at a 60-fold excess over U7, while a short, 18-nucleotide DNA molecule complementary to the 5' end of U7 inhibited the processing reaction at a 600-fold excess. A targeted ribozyme was capable of specifically cleaving the U7 small nuclear ribonucleoprotein in a nuclear extract and inhibited the U7-dependent processing reaction, but in our in vitro system it required a 1,000-fold excess over U7 for complete inhibition of processing.

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Expression in Escherichia coli and in vitro processing of HIV-1 p24 fusion protein

Journal of Biotechnology ◽

10.1016/0168-1656(93)90163-h ◽

1993 ◽

Vol 31 (2) ◽

pp. 225-232 ◽

Cited By ~ 4

Author(s):

Ilona Marczinovits ◽

Imre Boros ◽

Fouad El Jarrah ◽

György Füst ◽

János Molnár

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Expression In Escherichia Coli ◽

In Vitro Processing ◽

Hiv 1

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Accurate and efficient 3' processing of U2 small nuclear RNA precursor in a fractionated cytoplasmic extract

Molecular and Cellular Biology ◽

10.1128/mcb.7.9.3131-3137.1987 ◽

1987 ◽

Vol 7 (9) ◽

pp. 3131-3137

Author(s):

A M Kleinschmidt ◽

T Pederson

Keyword(s):

Rna Polymerase Ii ◽

Small Nuclear Rna ◽

Rnase Protection ◽

In Vitro Processing ◽

S100 Extract ◽

Processing Activity ◽

Precursor Molecules ◽

Trna Precursor ◽

Rna Precursor

The small nuclear RNAs U1, U2, U4, and U5 are cofactors in mRNA splicing and, like the pre-mRNAs with which they interact, are transcribed by RNA polymerase II. Also like mRNAs, mature U1 and U2 RNAs are generated by 3' processing of their primary transcripts. In this study we have investigated the in vitro processing of an SP6-transcribed human U2 RNA precursor, the 3' end of which matches that of authentic human U2 RNA precursor molecules. Although the SP6-U2 RNA precursor was efficiently processed in an ammonium sulfate-fractionated HeLa cytoplasmic S100 extract, the product RNA was unstable. Further purification of the processing activity on glycerol gradients resolved a 7S activity that nonspecifically cleaved all RNAs tested and a 15S activity that efficiently processed the 3' end of pre-U2 RNA. The 15S activity did not process the 3' end of a tRNA precursor molecule. As demonstrated by RNase protection, the processed 3' end of the SP6-U2 RNA maps to the same nucleotides as does mature HeLa U2 RNA.

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