Secondary Structures for 5′ Regions of R2 Retrotransposon RNAs Reveal a Novel Conserved Pseudoknot and Regions that Evolve under Different Constraints

Abstract

SummarySequences from the 5′ region of R2 retrotransposons of four species of silk moth are reported. In Bombyx mori, this region of the R2 messenger RNA contains a binding site for R2 protein and mediates interactions critical to R2 element insertion into the host genome. A model of secondary structure for this RNA fragment is proposed on the basis of binding to oligonucleotide microarrays, chemical mapping, and comparative sequence analysis. Five regions of conserved secondary structure are identified, including a novel pseudoknot. There is an apparent transition from an entirely RNA structure coding function in most of the 5′ segment of the fragment to a protein coding function in the 3′ segment. This suggests that regions evolved under separate functional constraints (structural, coding, or both).

Mapping Targetable Sites on Human Telomerase RNA Pseudoknot/Template Domain Using 2′-OMe RNA-interacting Polynucleotide (RIPtide) Microarrays

“…In addition, we incorporated 2Ј-OMe substituents on all of the RIPtides displayed on the microarray (Fig. 1, A and B), a modification known to increase oligonucleotide affinity for RNA targets (42,43) as well as stability toward nucleolytic degradation; this modification has previously been used by Turner and co-workers (13)(14)(15)(16)(17)(18) to increase the resolution of RNA structure-mapping by microarray screening.…”

“…Of particular note in the latter regard is the work of Turner and colleagues (15)(16)(17)(18), who employed microarrays of 2Ј-OMe ribonucleotides and locked nucleic acids to perform an independent experimental assessment of the predicted secondary structure for several biologically relevant RNAs. Efforts along these lines to date have involved sparse sampling of ligand sequence space, with at most 320 candidate sequences examined per study, these having been designed on the basis of Watson-Crick complementarity to prospective docking sequences on the RNA target of interest.…”

“…Here, we report the development of microarrays that comprehensively represent all sequences of 2Ј-OMe RNA-interacting polynucleotides (2Ј-OMe RIPtides) having the four natural nucleobases (A, C, G, and U), and ranging in length from 4-to 8-mers. This screening platform includes a total of 87,296 individual oligonucleotide analog probes, vastly exceeding the few hundreds of nucleic acid binders employed in previously reported microarray studies (13)(14)(15)(16)(17)(18).…”

The R2 retrotransposon RNA families

“…Further support for this model is the lack of a conserved initiation codon upstream of the identifiably conserved R2 ORF. 5 In silkmoth R2 28S-R2 co-transcript. Comparisons of possible secondary structures to the five known classes of self-cleaving ribozymes revealed that R2 RNA has remarkable similarities to the hepatitis delta virus (HDV) ribozyme (Fig.…”

“…The location of this structure and the utilization of RNA pseudoknots as internal ribosome entry sites (IRES) for some viral RNAs 6 suggests that this structure is important for R2 protein synthesis. 5 The R2 RNA interacts with the R2 protein during insertion into the host genome. The mechanism by which the R2 element inserts has been well documented for Bombyx mori and proceeds via an ordered sequence of DNA cleavage and synthesis.…”