Conformation of yeast 18S rRNA. Direct chemical probing of the 5′ domain in ribosomal subunits and in deproteinized RNA by reverse transcriptase mapping of dimethyl sulfate-accessible sites

Abstract

The structure of the 5' domain of yeast 18S rRNA has been probed by dimethyl sulfate (DMS), either in "native" deproteinized molecules or in the 40S ribosomal subunits. DMS-reacted RNA has been used as a template for reverse transcription and a large number of reactive sites, corresponding to all types of bases have been mapped by a primer extension procedure, taking advantage of blocks in cDNA elongation immediately upstream from bases methylated at atom positions involved in the base-pair recognition of the template. Since the same atom positions are protected from DMS in base-paired nucleotides, the secondary structure status of each nucleotide can be directly assessed in this procedure, thus allowing to evaluate the potential contribution of proteins in modulating subunit rRNA conformation. While the DMS probing of deproteinized rRNA confirms a number of helical stems predicted by phylogenetic comparisons, it is remarkable that a few additional base-pairings, while proven by the comparative analysis, appear to require the presence of the bound ribosomal subunit proteins to be stabilized.

In vivo analysis of plant RNA structure: soybean 18S ribosomal and ribulose-1,5-bisphosphate carboxylase small subunit RNAs

“…This same phenomenon has been observed in structural analysis of rRNA from other species [26,30,36]. Presumably, additional interactions such as protein binding prevent modification of all single-stranded bases.…”

“…Modified nucleotides result in a termination of the primer extension reaction at one base preceding the modified site. Similar analyses have been used to determined modified bases in 18S rRNA from rabbit [36], yeast [26] and in the analysis of 16S rRNA from Escherichia coli [30].…”

“…In order to study the structure/function relationship within the cell, we wished to develop a method to analyze secondary structure of a variety of RNAs in vivo. Chemical modification with dimethyl sulfate (DMS) methylates adenine and cytosine at the N1 and N3 positions on the nucleotide, respectively [26]. Modifications at these positions inhibit the progress of reverse transcriptase during primer extension reactions.…”

Detection of RNA modifications

“…Arrest of primer elongation during reverse transcription is sensitive to a variety of modifications in addition to the aforementioned 2'-OMe. Generally, all modifications on the Watson-Crick face of the nucleobase (see G, [51][52][53] ) are prone to block the incorporation of a complementary deoxynucleotide, while small modifications at the Hoogsteen edge often allow unimpeded readthrough (see Fig. 1A and C for Ψ, m 5 U, m 5 C, m 2 G).…”

High-throughput sequencing for 1-methyladenosine (m1A) mapping in RNA

“…by dimethylsulfate or other methylating agents. This methylation reaction was extensively used in the past for structural probing of RNA in solution, followed by detection of accessible m 1 A sites by the reverse transcription blocks [9,10].…”