Characterization of the sequence specificity of the R1Bm endonuclease domain by structural and biochemical studies

R1Bm is a long interspersed element (LINE) inserted into a specific sequence within 28S rDNA of the silkworm genome. Of two open reading frames (ORFs) of R1Bm, ORF2 encodes a reverse transcriptase (RT) and an endonuclease (EN) domain which digests specifically both top and bottom strand of the target sequence in 28S rDNA. To elucidate the sequence specificity of EN domain of R1Bm (R1Bm EN), we examined the cleavage tendency for the target sequences, and found that 5′-A(G/C)(A/T)!(A/G)T-3′ is the consensus sequence (! = cleavage site). We also determined the crystal structure of R1Bm EN at 2.0 Å resolution. Its structure was basically similar to AP endonuclease family, but had a special β-hairpin at the edge of the DNA binding surface, which is a common feature among EN of LINEs. Point-mutations on the DNA binding surface of R1Bm EN significantly decreased the cleavage activities, but did not affect the sequence recognition in most residues. However, two mutants Y98A and N180A had altered cleavage patterns, suggesting an important role of these residues (Y98 and N180) for the sequence recognition of R1Bm EN. In addition, Y98A mutant showed another cleavage pattern, that implies de novo design of novel sequence-specific EN

Essential Motifs in the 3′ Untranslated Region Required for Retrotransposition and the Precise Start of Reverse Transcription in Non-Long-Terminal-Repeat Retrotransposon SART1

Non-long-terminal-repeat (non-LTR) retrotransposons amplify their copies by reverse transcribing mRNA from the 3′ end, but the initial processes of reverse transcription are still unclear. We have shown that a telomere-specific non-LTR retrotransposon of the silkworm, SART1, requires the 3′ untranslated region (3′ UTR) for retrotransposition. With an in vivo retrotransposition assay, we identified several novel motifs within the 3′ UTR involved in precise and efficient reverse transcription. Of 461 nucleotides (nt) of the 3′ UTR, the central region, from nt 163 to nt 295, was essential for SART1 retrotransposition. Of five putative stem-loops formed in RNA for the SART1 3′ UTR, the second stem-loop (nt 159 to 221) is included in this region. Loss of the 3′ region (nt 296 to 461) in the 3′ UTR and the poly(A) tract resulted in decreased and inaccurate reverse transcription, which starts mostly from several telomeric repeat-like GGUU sequences just downstream of the second stem-loop. These results suggest that short telomeric repeat-like sequences in the 3′ UTR anneal to the bottom strand of (TTAGG)(n) repeats. We also demonstrated that the mRNA for green fluorescent protein (GFP) could be retrotransposed into telomeric repeats when the GFP coding region is fused with the SART1 3′ UTR and SART1 open reading frame proteins are supplied in trans

Characterization of the sequence specificity of

the R1Bm endonuclease domain by structural and biochemical studie

Efficient TALEN construction for Bombyx mori gene targeting.

Engineered nucleases are artificial enzymes able to introduce double stranded breaks at desired genomic locations. The double stranded breaks start the error-prone repair process of non-homologous end-joining (NHEJ), which eventually leads to the induction of mutations at target sites. We showed earlier that ZFNs and TALENs are able to induce NHEJ mutations in the B. mori genome. In order to optimize our mutagenesis protocol, we modified one of the reported truncated TALEN scaffolds and optimized it for use in the B. mori embryo. We also established a novel B. mori somatic cell assay suitable for the preselection of highly efficient TALENs directly in the B. mori model system. We compared the efficiency of several TALEN pairs based on three different frameworks using the BmBLOS2 gene. The new active TALENs show one order of magnitude higher efficiency than those we used previously. We confirmed the utility of our improved protocol by mutagenesis of the autosomal gene, red egg (Bm-re) and showed that it allows obtaining homozygous mutants in G1. Our procedure minimizes the chance of failure in B. mori gene targeting experiments

TALEN-induced insertions/deletions in <i>BmBLOS2</i>.

<p>(A) Mutant alleles obtained from the somatic assay; (B) Mutant alleles obtained from G₁ individuals. Sequences of mutant alleles are aligned with the respective wild-type sequences. TALEN target sites in the wild-type sequences are highlighted. The number of individuals carrying identical mutation is indicated to the right of each sequence.</p