Probing the structure of RNAs in solution

Abstract

During these last years, a powerful methodology has been developed to study the secondary and tertiary structure of RNA molecules either free or engaged in complex with proteins. This method allows to test the reactivity of every nucleotide towards chemical or enzymatic probes. The detection of the modified nucleotides and RNase cleavages can be conducted by two different paths which are oriented both by the length of the studied RNA and by the nature of the probes used. The first one uses end-labeled RNA molecule and allows to detect only scissions in the RNA chain. The second approach is based on primer extension by reverse transcriptase and detects stops of transcription at modified or cleaved nucleotides. The synthesized cDNA fragments are then sized by electrophoresis on polyacrylamide:urea gels. In this paper, the various structure probes used so far are described, and their utilization is discussed.

A guanosine quadruplex and two stable hairpins flank a major cleavage site in insulin-like growth factor II mRNA

“…The SmaI site is used for linearisation in the production of hum316 RNA. (CMCT), and kethoxal (12). The sites of strand cleavage or modification can, with the exception of the guanosine N-7 position, be determined by reverse transcription from a 5' endlabelled primer.…”

Ribosomal binding to the internal ribosomal entry site of classical swine fever virus

“…A fundamental aspect of initiation on the CSFV IRES is its ability to bind 40S subunits in the absence of factors to form stable binary complexes+ Sites at which 40S subunits bind the IRES were identified by footprinting, using an approximately threefold molar excess of 40S subunits, buffer conditions appropriate for CSFV translation, and either RNase V 1 (which cleaves basepaired or stacked RNA) or RNase T 1 (which cleaves RNA after unpaired G residues) (Ehresmann et al+, 1987)+ Enzymatic cleavage of RNA is detected by arrest of primer extension at the nucleotide on the 39 side of the cleaved bond, and numbering refers to this nucleotide+ Results of this analysis are summarized in Figure 1+ 40S subunits protected the IRES from RNase V 1 cleavage at G 263 and UC The accessibility of sites in the CSFV pseudoknot to cleavage by RNase T 1 at sites that are also cleaved by RNase V 1 is consistent with an earlier report regarding cleavage of the HCV pseudoknot by these two RNAses (Wang et al+, 1995)+ Taken together, these observations suggest that the pseudoknot in these IRESs is not a static structure+…”

Inhibition of Escherichia coli RNase P by oligonucleotide directed misfolding of RNA

“…DEPC modifies purine N7s (Ehresmann et al 1987;Doudna and Cech 1995), and therefore is a probe for changes in tertiary structure. As seen in Figures 1 and 5, m(CAGCCUACCCGG) enhances modification at A residues 65-67, 79, 81, and 98-99.…”