RNase cleavage

Another technique, RNase cleavage (M4), uses the enzyme ribonuclease A to cut RNA-DNA hybrids wherever there is a mismatch between a nucleotide in the RNA... 

Figure 13.14 Schematic representation of RNA Interference. Small interfering RNA (siRNA) is generated by Dicer (RNase) cleavage of a short hairpin RNA (shRNA) into small double-stranded RNA (dsRNA) of 21-25 nucleotide lengths, or transfected into the cell. The transfected siRNA is phosphorylated at 5 -ends by an endogenous kinase. The 5 -phosphorylated siRNA is incorporated into RNA-induced silencing complex (RISC) and unfolded. The antisense strand targets the RISC to homologous mRNA (sequence complementary to the siRNA guide) which is then cleaved by an endonuclease in the RISC complex (termed Sheer). The mRNA initially cleaved by Sheer is degraded by exonucleases and thus silenced...

Recently, the related phenomenon of RNA interference (RNAi) has attracted much attention [5]. RNAi occurs when a short (generally 21 nucleotides in length) double-stranded RNA (dsRNA) catalyticaUy represses the translation of a fully complementary mRNA sequence. The process appears to proceed via a complex formed between the antisense RNA strand and a protein with RNase activity [6]. Upon binding to the target mRNA sequence, the ribonucleoprotein complex initiates cleavage of the mRNA transcript thus preventing translation of intact protein. After dissociation from the truncated mRNAs, the ribonucleoprotein complex is free to act on other intact mRNAs. Such small interfering RNAs (siRNAs) have... 

Longer incubation times may improve RNase H cleavage but may also result in partial degradation. 

Expose to a phosphor-imager screen or a film. Three bands should appear two that are similar in size to the cleavage products (5 and 3 to the RNase H cleavage site), and a longer band similar in size to the full-length transcript that represents the remainder of an uncut mRNA (Figs. 9.1 and 9.2). 

Fig. 1A-F The two-dimensional structures of various ribozymes. The ribozyme or intron portion is printed in black. The substrate or exon portion is printed in gray. Arrows indicate sites of cleavage by ribozymes A (left) the two-dimensional structure of a hammerhead ribozyme and its substrate. Outlined letters are conserved bases that are involved in catalysis right) The y-shaped structure of the hammerhead ribozyme-sub-strate complex B-F the two-dimensional structures of a hairpin ribozyme, the genomic HDV ribozyme, a group I ribozyme from Tetrahymena, a group II ribozyme from S. cer-evisiae (aiy5), and the ribozyme of RNase P from E. coli...

Ribonuclease II [EC 3.1.13.1], also called exoribo-nuclease II, catalyzes the exonucleolytic cleavage of the polynucleic acid, preferring single-stranded RNA, in the 3 - to 5 -direction to yield 5 -phosphomononucleotides. The enzyme processes 3 -terminal extra-nucleotides of monomeric tRNA precursors, following the action of ribonuclease P. Similar enzymes include RNase Q, RNase BN, RNase PHI, and RNase Y. Ribonuclease T2 [EC 3.1.27.1] is also known as ribonuclease II. 

This enzyme [EC 3.1.26.3], also known as RNase O and RNase D, catalyzes the endonucleolytic cleavage of RNA to 5 -phosphomonoesters. The enzyme cleaves multimeric tRNA precursors at the spacer region and is also involved in the processing of precursor rRNA, hnRNA, and early T7-mRNA. This enzyme can also act on double-stranded DNA. 

M. J. Damha, Efficient RNase H-directed cleavage of RNA promoted by antisense DNA or 2 F-ANA constructs containing acyclic nucleotide inserts, J. Am. Chem. Soc., 125 (2003) 654-661. 

J. Li and R. M. Wartell, RNase HI can catalyze RNA/DNA hybrid formation and cleavage with stable hairpin or duplex DNA oligomers, Biochemistry, 37 (1998) 5154-5161. 

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