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

Abstract

A method to investigate the structure of RNA molecules within intact plant tissues has been developed. The RNA structures are analyzed using dimethyl sulfate (DMS), which modifies substituents of adenine and cytosine residues within single-stranded regions of RNA molecules. Reactive sites are identified by primer extension analysis. Using this procedure, an analysis of the secondary structure of the cytoplasmic 18S ribosomal RNA in soybean seedling leaves has been completed. DMS modification data are in good agreement with the phylogenetic structure predicted for soybean 18S rRNA. However, there are a few notable exceptions where residues thought to be involved in double-stranded regions in all 18S rRNAs are strongly modified in soybean leaf samples. These data taken together with the phylogenetic structure suggest that alternate structures may exist in vivo. The further applicability of this technique is demonstrated by comparing the modification pattern obtained in vivo to that obtained in vitro for a particular mRNA molecule encoding the small subunit of ribulose-1,5-bisphosphate carboxylase. The results obtained are compared to a predicted minimum energy secondary structure. The data indicate that the conformation of RNA molecules within the cell may not be reflected in a structural analysis of purified mRNA molecules.

Determination of in vivo RNA structure in low-abundance transcripts

“…Among RNA structural probing reagents, dimethyl sulfate (DMS) is highly versatile and useful for in vivo probing 9 , owing to its ability to penetrate cells and modify RNA in numerous organisms [10][11][12][13][14][15][16] . Recently, in vivo SHAPE reagents were developed and have been used to probe the highly abundant 5S rRNA in bacteria, yeast, fly and mammalian cells 17 .…”

“…In cellular systems, structures of high-abundance RNAs such as rRNA can be assessed in vivo by a DMS/SHAPE-RT approach 11,12,14,17 . However, the vast majority of RNAs is of low abundance in vivo and cannot be explored by an RT-based approach.…”

“…Moreover, the effects of RNA-binding proteins on in vivo RNA structures are also largely unexplored. This lack of in vivo RNA structural information becomes even more evident in plants, where only a few in vivo RNA structural probing studies have been conducted, and these have been on high-abundance rRNA and photosynthesis-related RNAs 11,13 .…”

Degradation of the Soybean Ribulose-1,5-Bisphosphate Carboxylase Small-Subunit mRNA, SRS4, Initiates with Endonucleolytic Cleavage

“…The size of the arrow indicates the relative intensity of the band. Solid dots and open circles indicate strong and weak DMS-modified bases, respectively (27 to the control lane). These data suggest that in the in vitro extract, one activity was responsible for the generation of the major degradation products.…”

RNA folding in living cells

“…DMS is the most successfully used chemical to probe RNA structure in a variety of organisms, ranging from bacteria to eukaryotes. 120,[123][124][125][126][127][128][129][130][131] A major convenience of DMS is that it rapidly penetrates all compartments of cells without prior cell permeabilization. After uptake, DMS methylates N7 of guanines, N1 of adenines and N3 of cytosines, if they are not involved in H-bonding or protected by proteins.…”