Structure and sequence at an RNA template 5' end influence insertion of transgenes by an R2 retrotransposon protein.

Autor:	Palm SM; Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA., Horton CA; Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA., Zhang X; Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA., Collins K; Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA kcollins@berkeley.edu.
Jazyk:	angličtina
Zdroj:	RNA (New York, N.Y.) [RNA] 2024 Aug 16; Vol. 30 (9), pp. 1227-1245. Date of Electronic Publication: 2024 Aug 16.
DOI:	10.1261/rna.080031.124
Abstrakt:	R2 non-long terminal repeat retrotransposons insert site-specifically into ribosomal RNA genes (rDNA) in a broad range of multicellular eukaryotes. R2-encoded proteins can be leveraged to mediate transgene insertion at 28S rDNA loci in cultured human cells. This strategy, p recise R NA-mediated in sertion of t ransgenes (PRINT), relies on the codelivery of an mRNA encoding R2 protein and an RNA template encoding a transgene cassette of choice. Here, we demonstrate that the PRINT RNA template 5' module, which as a complementary DNA 3' end will generate the transgene 5' junction with rDNA, influences the efficiency and mechanism of gene insertion. Iterative design and testing identified optimal 5' modules consisting of a hepatitis delta virus-like ribozyme fold with high thermodynamic stability, suggesting that RNA template degradation from its 5' end may limit transgene insertion efficiency. We also demonstrate that transgene 5' junction formation can be either precise, formed by annealing the 3' end of first-strand complementary DNA with the upstream target site, or imprecise, by end-joining, but this difference in junction formation mechanism is not a major determinant of insertion efficiency. Sequence characterization of imprecise end-joining events indicates surprisingly minimal reliance on microhomology. Our findings expand the current understanding of the role of R2 retrotransposon transcript sequence and structure, and especially the 5' ribozyme fold, for retrotransposon mobility and RNA-templated gene synthesis in cells. (© 2024 Palm et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
Databáze:	MEDLINE
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