Analysis of RNA-protein complexes by oligonucleotide-targeted RNase H digestion.
Oligonucleotide-targeted RNase H protection assays are powerful means to analyze protein binding domains in ribonucleoprotein particles (RNPs). In such an assay, the RNA component of a RNP and, in an essential control reaction, the corresponding deproteinized RNA are targeted with an antisense DNA oligonucleotide and RNase H. If the oligonucleotide is able to anneal to the complementary sequence of the RNA, RNase H will cleave the RNA within the double-stranded DNA/RNA region. However, protein binding to a specific RNA sequence may prevent hybridization of the DNA oligonucleotide, thereby protecting the RNA molecule from endonucleolytic cleavage. An RNase H protection analysis can usually be carried out with crude cell extract and does not require further RNP purification. On the other hand, purified RNP fractions are preferable when a crude extract contains RNase activity or a heterogenous RNP population of a specific RNA. The cleavage pattern of RNase H digestion can be analyzed by Northern blotting or primer-extension assays. In addition, the investigation of RNP fragments, for example, by native gel electrophoresis, may reveal important structural information about a RNP. In this article, we describe procedures for RNP and RNA preparation, the oligonucleotide-targeted RNase H protection assay, and methods for the analysis of RNA and RNP cleavage products. As an example, we show oligonucleotide-targeted RNase H protection of the Trypanosoma brucei U1 small nuclear RNP.
Günzl
Arthur
A
Zoologisches Institut der Universität Tübingen, Abteilung Zellbiologie, Auf der Morgenstelle 28, 72076, Germany.
Palfi
Zsofia
Z
Bindereif
Albrecht
A
D019921:Biotinylation; D002845:Chromatography; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000494:pharmacology; D008957:Models, Genetic; D009841:Oligonucleotides / Q000737:chemistry; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011485:Protein Binding; D012313:RNA / Q000737:chemistry*; D016601:RNA-Binding Proteins / Q000737:chemistry*; D016914:Ribonuclease H / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism
D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear; D000431:Ethanol; D012313:RNA; D016914:Ribonuclease H