The Brown Algae Pl.LSU/2 Group II Intron-Encoded Protein Has Functional Reverse Transcriptase and Maturase Activities

Group II introns are self-splicing mobile elements found in prokaryotes and eukaryotic organelles. These introns propagate by homing into precise genomic locations, following assembly of a ribonucleoprotein complex containing the intron-encoded protein (IEP) and the spliced intron RNA. Engineered group II introns are now commonly used tools for targeted genomic modifications in prokaryotes but not in eukaryotes. We speculate that the catalytic activation of currently known group II introns is limited in eukaryotic cells. The brown algae Pylaiella littoralis Pl.LSU/2 group II intron is uniquely capable of in vitro ribozyme activity at physiological level of magnesium but this intron remains poorly characterized. We purified and characterized recombinant Pl.LSU/2 IEP. Unlike most IEPs, Pl.LSU/2 IEP displayed a reverse transcriptase activity without intronic RNA. The Pl.LSU/2 intron could be engineered to splice accurately in Saccharomyces cerevisiae and splicing efficiency was increased by the maturase activity of the IEP. However, spliced transcripts were not expressed. Furthermore, intron splicing was not detected in human cells. While further tool development is needed, these data provide the first functional characterization of the PI.LSU/2 IEP and the first evidence that the Pl.LSU/2 group II intron splicing occurs in vivo in eukaryotes in an IEP-dependent manner.II型内含子（Group II intron）是一类自我剪接型可移动遗传元件，广泛存在于原核生物与真核生物细胞器中。这类内含子通过归巢作用精准插入基因组特定位点实现增殖，该过程需先组装由内含子编码蛋白（intron-encoded protein，IEP）与剪接后内含子RNA组成的核糖核蛋白复合物。经工程改造的II型内含子目前已成为原核生物靶向基因组修饰的常用工具，但尚未能应用于真核生物体系。我们推测，当前已知的II型内含子的催化激活过程在真核细胞中存在显著局限。褐藻（Pylaiella littoralis）来源的Pl.LSU/2 II型内含子是目前已知唯一可在生理浓度镁离子条件下展现体外核酶活性的II型内含子，但该内含子的功能特性仍未得到充分解析。本研究纯化并鉴定了重组表达的Pl.LSU/2 IEP。与绝大多数IEP不同，Pl.LSU/2 IEP在缺乏内含子RNA的情况下仍可展现逆转录酶活性。经工程改造的Pl.LSU/2内含子可在酿酒酵母（Saccharomyces cerevisiae）中实现精准剪接，且IEP的成熟酶（maturase）活性可提升其剪接效率，但经剪接产生的转录本无法正常表达。此外，在人类细胞中未检测到该内含子的剪接现象。尽管仍需进一步开发相关研究工具，本研究的数据首次完成了Pl.LSU/2 IEP的功能特性鉴定，并首次证实Pl.LSU/2 II型内含子可在真核生物体内以IEP依赖的方式完成剪接。