Globular domain structure and function of restriction-like-endonuclease LINEs: similarities to eukaryotic splicing factor Prp8

Abstract

BackgroundR2 elements are a clade of early branching Long Interspersed Elements (LINEs). LINEs are retrotransposable elements whose replication can have profound effects on the genomes in which they reside. No crystal or EM structures exist for the reverse transcriptase (RT) and linker regions of LINEs.ResultsUsing limited proteolysis as a probe for globular domain structure, we show that the protein encoded by the Bombyx mori R2 element has two major globular domains: (1) a small globular domain consisting of the N-terminal zinc finger and Myb motifs, and (2) a large globular domain consisting of the RT, linker, and type II restriction-like endonuclease (RLE). Further digestion of the large globular domain occurred within the RT. Mapping these RT cleavages onto an updated model of the R2Bm RT indicated that the thumb of the RT was largely protected from proteolytic cleavage. The crystal structure of the large globular domain of Prp8, a eukaryotic splicing factor, was a major template used in building the R2Bm RT model, particularly the thumb region. The large fragment of Prp8 consists not only of a RT similar to R2Bm, but also an RLE and a linker connecting the two regions. The linker sequences adjacent to the RLE in LINEs and Prp8 share a set of two important α-helices and a (presumptive) knuckle/ββα structural motif that are closely associated with the thumb. The RLEs of LINEs and Prp8 share a unique catalytic core residue spacing as well as other key residues.ConclusionsThe protein encoded by RLE LINEs consists of two major globular domains. The larger of the two globular domain contains the RT, linker, and RLE and is similar to the large fragment of the spliceosomal protein Prp8. The similarities are suggestive of possible common ancestry.Electronic supplementary materialThe online version of this article (10.1186/s13100-017-0097-9) contains supplementary material, which is available to authorized users.

Separable structural requirements for cDNA synthesis, nontemplated extension, and template jumping by a non-LTR retroelement reverse transcriptase

“…To guide structure/function analysis, we used multiple sequence alignments to define the boundaries of shared RT motifs in BoMoC. The resulting sequence annotations largely agree with those reported by the Eickbush and Christensen labs (10,12), with minor adjustments (Supporting Information Figure S1). In addition, we generated a protein structure prediction for the R2 RT domain using Phyre2 (Figure 1B).…”

“…RTs and also viral RNA-dependent RNA polymerases (RdRPs) share seven primary sequence motifs, 1-7 (8,9). RdRPs and non-viral RTs also share subsets of additional insertion motifs (Figure 1A), typically termed 2a, 3a, 4a, 6a and 7a, as well as an N-terminal extension (NTE) that can include motifs 0 and -1 (10)(11)(12). Structure determination and mutagenesis studies of a limited number of bacterial mobile group II intron RTs and eukaryotic telomerase RTs indicate that motif insertions are tailored by evolution to adapt RTs to their biological functions (13)(14)(15)(16)(17).…”

Structure of the R2 non-LTR retrotransposon initiating target-primed reverse transcription

“…The ZnF then links to the C-terminal RLE domain, which cleaves the target DNA. This domain arrangement closely resembles that of Prp8 ( 13 , 16 , 17 ), the core protein of the spliceosome, underscoring the close relationship between pre-mRNA splicing and retrotransposons.…”