Sequence analysis of RNA

Soon after the discovery of RNase Ti, it was suggested (70, 71) that it would become an important tool for the elucidation of nucleotide sequence in RNA. Indeed, since 1962 several workers have tried to use the enzyme for the nucleotide sequence analysis of RNA, especially in highly purified specific tRNA s. Finally, the brilliant research of Holley and his associates in 1965 resulted in the first elucidation of the complete nucleotide sequence of an RNA, alanine specific yeast tRNA, using RNase Ti as a main tool (29). Since then many successful elucidations of nucleotide sequence of various RNA s, using RNase Ti as a main tool, followed, and now the enzyme is well-known as an essential tool for the structural analysis of RNA. 

Although, because of its complicated specificity, it is not easy to use the enzyme with a probable expectation for the nucleotide sequence analysis of RNA, sometimes it has been used with success (115, 116). It may be useful especially to digest the large fragments produced by RNase T,. In this case, it usually splits 3 -adenylyl and 3 -uridylyl bonds but not 3 -cytidylyl bonds. 

Thiede, B., Urlaub, H., Neubauer, H., and Wittmann-Liebold, B. (1999) Identification and sequence analysis of RNA-protein contad sites by N-terminal sequencing and MALDI-MS. Methods Mol. Biol., 118, 63-72. 

Structure. The mononucleotides of RNA consist of ribose phosphorylated at C3, and linked by an N-gly-cosidic bond to one of four bases adenine, guanine, cytosine or uracil. Many other bases (chiefly methylated bases) also occur, but are less common (see Rare nucleic acid components). The mononucleotide units form a linear chain via 3, 5 phosphodiesler bonds (see Nucleic acids). Sequence analysis of RNA has become a standard technique. In many cases the amino acid sequences of proteins are predicted from the sequence of the corresponding mRNA (or DNA) because it is much easier to clone the nucleic acid than to isolate the protein. 

Randerath, E., Gupta, R. C., and Randerath, E. (1980). Tritium and phosphorus-32 derivative methods for base composition and sequence analysis of RNA. Methods Enzy-mol. 65 638-680. 

Zarlenga, D.S., Lichtenfels, J.R. and Stringfellow, F. (1994a) Cloning and sequence analysis of the small subunit ribosomal RNA gene from Nematodirus battus. Journal of Parasitology 80, 342-344. 

The knowledge of the primary structure was the basis for the construction of models of the secondary structure of the RNA molecules. Different approaches have been used in several laboratories to get experimental support for developing secondary structure models for example, chemic modification of the RNA, treatment with single- or double-strand-specihc nucleases, intramolecular RNA cross-linking, isolation and sequence analysis of double-stranded RNA, and, last but not least, comparison of ribosomal RNA sequences from different organisms (reviewed by Brimacombe et al., 1983). 

The second approach to sequencing RNAs, via their cDNA transcripts, is exemplified by the sequence analysis of the influenza virus haemagglutin gene (Sleigh et al., 1979). In this method a double-stranded (dsDNA) copy of the RNA genome was inserted, after digestion with nuclease Si, into the double-stranded bacterial plasmid pBR322 which was subsequently used to transform a... 

Northern analysis of RNA from cyc S49 cells (Fig. 11) shows absence of as coding sequences, in agreement with the absence of as polypeptides in these cells. The levels of as mRNA in different tissues is variable. Very likely, as subunits are under developmental and hormonal control and experiments exploring this should be forthcoming shortly. 

A structural analysis of the 18 S rRNA from rabbit has also been published18 the sequence within this region is only 60% similar to that of the plant RNAs (Figs. 3 and 5A). However, one of the lowest energy structures proposed for this segment is very similar to that proposed herein for the four plant rRNAs (Fig. 5A). The in vivo DMS modification data reported for the plant RNAs support the proposed secondary structures of the four plant RNA segments, and, in fact, they are more consistent with the proposed structure than the in vitro DMS modification data on rabbit rRNA for this region. However, for the analysis of RNA secondary structure to have its full impact on studies in molecular evolution, it must be possible to quantify observed differences. 

Throughout the 2003 mesocosm study, water (50-500 ml) was collected every third day onto 0.8 gm Supor membrane filters (Pall corp.) and stored at -80°C until analysis. Following enumeration of Phaeocystis sp. single cells and colonies, two samples were chosen for molecular determination of the composition of the Phaeocystis sp. community. Gene sequence analysis of Phaeocystis sp. small subunit ribosomal RNA (rRNA) genes from samples collected from a fertilized enclosure early in the experiment when only single cells were present (5 March 2003 single cells 15 ml"1,... 

Shine, J. Dalgamo, L. (1975). Terminal-sequence analysis of bacterial ribosomal RNA. Correlation between the 3 -tenninal-polypyrimidine sequence of 16-S RNA and translational specificity of the ribosome. Eur J Biochem 57,221-30. 

Bhattacharya D, Elwood HJ, Goff LJ, Sogin ML Phylogeny of GracUaria lemaneiformis (Rhodophyta) based on sequence analysis of its small subunit ribosomal RNA coding region. JPhycol 1990 26 181-186. 

Similarly, RNA molecules can be separated by gel electrophoresis, and specific sequences can be identified by hybridization subsequent to their transfer to nitrocellulose. This analogous technique for the analysis of RNA has been whimsically termed Northern blotting. A further play on words accounts for the term Western blotting, which refers to a technique for detecting a particular protein by staining with specific antibody (Section 4.3.4). Southern, Northern, and Western blots are also known respectively as DNA, RNA, and protein blots. 

A variety of powerful methods is available for fractionating short oligonucleotides. Work on nucleotide sequence analysis of small RNA molecules (n=80 or 120) and more recently viral RNA molecules (n= 3000) and even ribosomal precursor RNAs (up to 45 s) (Holley et al. 1965a Brownlee 1972 Maden et al. 1972) has stimulated the development of these procedures. They can also be used for synthetic oligonucleotides (e.g. Hachman and Khorana, 1969). The methods depend largely on chromatography and electrophoresis on filter papers, diethylaminoethyl(DEAE) paper, cellulose acetate, thin layers of cellulose or polyethyleneimine (PEI)-cellulose, or columns. 

In contrast to the lack of detailed structural information for the 3, 5 -cyclic nucleotide phosphodiesterase, staphylococcal (or micrococcal) nuclease (SNase), an extracellular nuclease produced by Staphylococcus aureus, is well characterized. SNase is an extraordinarily efficient catalyst for the endo- and exonucleo-lytic hydrolysis of single-stranded DNA and RNA, with the rate acceleration for DNA being approximately 10 relative to the uncatalyzed rate the final products of the reaction are 3 -mononucleotides. The sequence of 149 amino acids that constitute the enzyme has been determined both by classical degradation procedures and by sequence analysis of the cloned gene. A highly refined 1.65-A X-ray structure determined in the presence of Ca and the competitive inhibitor thymidine 3, 5 -bisphosphate (pdTp) was recently completed (87) this structure differs only slightly from a less refined 1.5-A structure that was reported in 1979... 

In a method of analysis of RNA splicing, an oligodeoxyribonucleotide complementary to the exon sequence (or sequences) upstream of a known 5 -splice site is hybridized to the RNA, and the RNA strand of the hybrid(s) cleaved with RNase H. 14 a second primer, labelled with phosphorus -32, and complementary to an exon sequence downstream of a 3 -splice site, is then hybridized to the cleaved RNA, and primer extension performed using reverse transcriptase, a process terminated by the site(s) of RNase H-catalyzed cleavage. The length(s) of the... 

Such mutations may be detected by way of theh effects. Intronic mutations that affect splicing can be revealed tlirough the analysis of RNA. Polymorphisms (benign sequence variants) within a mRNA transcript can be used to assess relative levels of expression of the two alleles of the gene. Deletions of one or a few exons can be detected by quantitative hybridization, quantitative PCR or Southern blotting. Large deletions should be detectable with FISH methods. A method called Conversion may be used to simplify the detection of refractory mutations. 

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