Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits

Abstract

The 18S rRNA of the small eukaryotic ribosomal subunit contains several expansion segments. Electron microscopy data indicate that two of the largest expansion segments are juxtaposed in intact 40S subunits, and data from phylogenetic sequence comparisons indicate that these two expansion segments contain complementary sequences that could form a direct tertiary interaction on the ribosome. We have investigated the secondary structure of the two expansion segments in the region around the putative tertiary interaction. Ribosomes from yeast, wheat, and mouse-three organisms representing separate eukaryotic kingdoms-were isolated, and the structure of ES3 and part of the ES6 region were analyzed using the single-strand-specific chemical reagents CMCT and DMS and the double-strand-specific ribonuclease V1. The modification patterns were analyzed by primer extension and gel electrophoresis on an ABI 377 automated DNA sequencer. The investigated sequences were relatively exposed to chemical and enzymatic modification. This is in line with their indicated location on the surface at the solvent side of the subunit. The complementary ES3 and ES6 sequences were clearly inaccessible to single-strand modification, but available for cleavage by double-strand-specific RNase V1. The results are compatible with a direct helical interaction between bases in ES3 and ES6. Almost identical results were obtained with ribosomes from the three organisms investigated.

Genome-wide profiling of mouse RNA secondary structures reveals key features of the mammalian transcriptome

“…Our data are in agreement with previous reports showing DMS reactivity with G/U residues [36,[45][46][47] and CMCT reactivity with cytosines [48][49][50]. We observed a significant increase in the accuracy of the de novo prediction of structures when considering also these non-canonical reactivities as both the canonical and non-canonical reactivities lay within single-stranded regions ( Figure S3C in Additional file 1).…”

Expression of distinct maternal and somatic 5.8S, 18S, and 28S rRNA types during zebrafish development

“…One way eukaryotic mRNAs can interact with ribosomes is via sequence complementarity to rRNA, in particular 18S rRNA (Tranque et al 1998;Alkemar and Nygård 2004). The interactions by base-pairing of mRNAs to the 18S rRNA are located within the translated (Tranque et al 1998;Martin et al 2016) as well as in the untranslated regions (UTRs) of the mRNAs (Barendt et al 2012;Pánek et al 2013), and it seems that they can both favor (Barendt et al 2012) and inhibit translation (Verrier and Jean-Jean 2000).…”

Analysis of the Secondary Structure of Expansion Segment 39 in Ribosomes from Fungi, Plants and Mammals

“…15 If so, sequences in ES39 and ES7 could be in direct contact by forming tertiary interactions, as suggested for ES3 and ES6 in the small ribosomal subunit. 27,37 Materials and Methods Chemicals DMS and CMCT were from Sigma-Aldrich. Kethoxal was from ICN Pharmaceuticals.…”

“…44 At the pH used, CMCT occasionally modifies cytosine bases. 27,34 Modification with kethoxal was done essentially according to Stern et al, 45 except that the stock solution of kethoxal was 34 mg/ml in 99% (v/v) ethanol. Samples were incubated for 60 min at 4 8C in the presence of 9 mM and 23 mM kethoxal.…”

“…7 We have used this technique to analyse the structure of ES3/ES6 and ES15 in 18 S and 28 S rRNA, respectively. 26,27 Here, we have used experimental data obtained by RNA structure-specific modifying reagents combined with thermodynamic energy minimization to determine the secondary structure for ES39 and to generate secondary structure models for this expansion segment in yeast, wheat, mouse, rat and rabbit, five organisms representing three different eukaryotic kingdoms. These models will be useful in future three-dimensional modelling of eukaryotic ribosomes.…”