A unique, thermostable dimer linkage structure of RD114 retroviral RNA

Abstract

Retroviruses package their genome as RNA dimers linked together primarily by base-pairing between palindromic stem-loop (psl) sequences at the 59 end of genomic RNA. Retroviral RNA dimers usually melt in the range of 55°C-70°C. However, RNA dimers from virions of the feline endogenous gammaretrovirus RD114 were reported to melt only at 87°C. We here report that the high thermal stability of RD114 RNA dimers generated from in vitro synthesized RNA is an effect of multiple dimerization sites located in the 59 region from the R region to sequences downstream from the splice donor (SD) site. By antisense oligonucleotide probing we were able to map at least five dimerization sites. Computational prediction revealed a possibility to form stems with autocomplementary loops for all of the mapped dimerization sites. Three of them were located upstream of the SD site. Mutant analysis supported a role of all five loop sequences in the formation and thermal stability of RNA dimers. Four of the five psls were also predicted in the RNA of two baboon endogenous retroviruses proposed to be ancestors of RD114. RNA fragments of the 59 R region or prolonged further downstream could be efficiently dimerized in vitro. However, this was not the case for the 39 R region linked to upstream U3 sequences, suggesting a specific mechanism of negative regulation of dimerization at the 39 end of the genome, possibly explained by a long double-stranded RNA region at the U3-R border. Altogether, these data point to determinants of the high thermostability of the dimer linkage structure of the RD114 genome and reveal differences from other retroviruses.

Thermodynamics of the pseudo‐knot in helix 18 of 16S ribosomal RNA

“…For example, dimerizations through a loop–loop “kissing” interactions and loop–linear interactions were observed in the studies of RNA folding . Also, high RNA concentration could induce dimerization . Thus we first tested various concentrations of RNA and it turned out that in all concentrations at room temperature two bands were present at nondenaturing PAGE (Supporting Information Figure S7).…”

“…[58][59][60][61] Also, high RNA concentration could induce dimerization. [62] Thus we first tested various concentrations of RNA and it turned out that in all concentrations at room temperature two bands were present at nondenaturing PAGE (Supporting Information Figure S7). Therefore, we suspected that helix 18 forms thermodynamically stable dimers and further proceeded to confirm this observation.…”

Retroviral RNA Dimerization: From Structure to Functions

“…Finally, in vitro analysis of the gRNA dimer of feline endogenous RD-114, another gamma-retrovirus, revealed the presence of several palindromic stem-loops (psl-1 to psl-5) leading to RNA dimer formation ( Kharytonchyk and Pedersen, 2010 ). Interestingly, and similarly to MuLV, the RD-114 DLS also contains two GACG tetraloops that are close to the crucial DIS elements and contribute to in vitro RNA dimerization ( Kharytonchyk and Pedersen, 2010 ).…”