RNase, double-stranded

Dicer represents the key enzyme in the RNAi pathway. Dicer is also known as Helicase with RNAse motif, heRNA, Helicase-moi, K12H4.8-like, or KIAA0928. Dicer produces cleaves long double-stranded RNA into small pieces of about 21-23 nucleotides. These so-called siRNA duplexes produced by the action of Dicer contain 5 -phosphates and free 3 -hydroxylgroups... 

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... 

This enzyme is associated with the virions of RNA tumor viruses such as the Rous sarcoma virus (RSV). The enzyme has remarkable enzymatic activity in that it can catalyze several seemingly diverse steps in the synthesis of double-stranded DNA from the single-stranded RNA viral genome. The enzyme uses a tRNA for tryp-tophan as a primer to make a copy of DNA that is complementary to the viral RNA. The resulting RNA-DNA hybrid is converted to a double-stranded DNA molecule by ribon-uclease (RNase)H and DNA-dependent DNA polymerase activities that are intrinsic to reverse transcriptase. 

This protocol uses propidium iodide (PI) as the fluorescent tracer for DNA content (3-6). PI binds to both double-stranded DNA and double-stranded RNA. Therefore, RNase will be used to reduce the double-stranded RNA resulting in only DNA staining. For alternative DNA-specific ligands and dyes, see Chapter 30. 

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. 

The reverse transcriptase enzyme (RT) is the primary enzyme responsible for the conversion of the viral single-strand RNA to the double-strand DNA. The reverse transcriptase enzyme is a component of the virion and is encoded by the pol gene. The RT is manufactured in the HIV-infected cells as a gag-pol fusion polyprotein. The RT is not the only enzyme necessary for the translation of RNA to DNA. The other enzymes for this conversion include RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H (Gilboa and Mitra, 1978 Prasad and Gogg, 1990). The reverse transcriptase enzyme has a high error rate (1 in 2000 bases), which produces higher incidents of mutation. Some of these mutations make the virus resistant to NNRTI treatment. 

Like all other retroviruses, human immunodeficiency virus type 1 (HIV-1) contains the multifunctional enzyme reverse transcriptase (RT). Retroviral RTs have a DNA polymerase activity that can use either an RNA or a DNA template and an RNase H activity. HIV-1 RT is essential for the conversion of single-stranded viral RNA into a linear double-stranded DNA that is subsequently integrated into the host cell chromosomes [1-4]. In this conversion process HIV-1 RT catalyzes the incorporation of approximately... 

Double-stranded cytoplasmic-polyhedrosis-virus RNA obtained from silkworm was scarcely split by RNase U2 (106). 

Unlike the double-stranded nature of DNA, RNA molecules usually occur as single strands. This does not mean they are unable to base-pair as DNA can. Complementary regions within an RNA molecule often base-pair and form complex tertiary structures, even approaching the three-dimensional nature of proteins. Some RNA molecules, such as transfer RNA (tRNA) possess several helical areas and loops as the strand interacts with itself in complementary sections. Other hybrid molecules such as the enzyme RNase P contain protein and RNA portions. The RNA part is highly complex with many circles, loops, and helical regions creating a convoluted structure. 

Martens, H., Novotny, J., Oberstrass, J., Steck, T.L., Postlethwait, P. and Nellen, W. (2002) RNAi in Dictyostelium the role of RNA-directed RNA polymerases and double-stranded RNase. Molecular Biology of the Cell 1 3, 445 153. 

Figure 6. Amplification of RNA molecules by assays that are sequence- insensitive. The first assay (upper part) combines the polymerase chain reaction (PCR) of DNA templates with reverse transcription and transcription. Commonly used enzymes are TAQ-polym-erase, HIV reverse transcriptase and bacteriophage T7 RNA polymerase. The assay requires a temperature program applying higher temperatures for double strand dissociation. The second assay (lower part) shows the self-sustained sequence replication reaction (3SR) which can be carried out isothermally because double strand dissociation is replaced by enzymatic digestion of the RNA strand in the RNA-DNA duplex. The enzymes used are HIV reverse transcriptase, RNase H and T7 RNA polymerase.

The second assay makes use of the isothermal self-sustained sequence replication reaction of RNA (3SR Fahy et al., 1991). Instead of double strand melting to yield single strands the RNA DNA hybrid obtained through reverse transcription is converted into single stranded DNA by RNA digestion making use of RNase H. DNA double strand synthesis and transcription complete the cycle. Here, transcription by T7 polymerase represents the amplification step. 

It is possible to permeabilize the outer membrane of normal cells (with detergent or alcohol) in order to allow propidium iodide to enter the nuclei. If we then treat the normal cells with RNase in order to ensure that any fluorescence results from their DNA content (without a contribution from double-stranded RNA), we find that the nuclei fluoresce red with an intensity that is more or less proportional to their DNA content. By the use of a red filter and a linear amplifier on the photomultiplier tube, we can detect that red fluorescence. The channel number of the fluorescence intensity will be proportional to the DNA content of the cells. The method is simple and takes about 10 minutes. Flow cytometric analysis of the red fluorescence from the particles in this preparation of nuclei from normal, nondividing cells will result in a histogram with a single, narrow peak (see the first histogram in Fig. 8.1) all the particles emit very nearly the same amount of red fluorescence. This supports our knowledge that all... 

Propidium iodide Propidium iodide is a probe that can be used to measure quantitatively the amount of double-stranded nucleic acid that is present in a cell. After treatment of cells with RNase, it will measure the amount of DNA present. Because it does not cross an intact cell membrane, cells need to be treated with detergent or ethanol before it can be used to determine their DNA content. It can also be used to assess the viability of cells. 

Like other DNA polymerases, reverse transcriptases are primer-and template-dependent. They also possess an RNase H activity (H stands for hybrid) that can degrade the RNA template after it is used for synthesis of the first DNA strand. The enzyme then can copy the first strand of DNA to make a double-stranded molecule. 

One possibility is that the double-strand RNA-processing activity of RNase III, an endoribonuclease involved in the rate-limiting first cleavage step of mRNA degradation (Chang et al., 1991), is potentially lethal to E. coli, and the... 

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