The Lactococcus lactis group II intron Ll.ltrB is similar to mobile yeast mtDNA group II introns, which encode reverse transcriptase, RNA maturase, and DNA endonuclease activities for site-specific DNA insertion. Here, we show that the Lactococcal intron can be expressed and spliced efficiently in Escherichia coli. The intron-encoded protein LtrA has reverse transcriptase and RNA maturase activities, with the latter activity shown both in vivo and in vitro, a first for any group II intron-encoded protein. As for the yeast mtDNA introns, the DNA endonuclease activity of the Lactococcal intron is associated with RNP particles containing both the intron-encoded protein and the excised intron RNA. Also, the intron RNA cleaves the sense-strand of the recipient DNA by a reverse splicing reaction, whereas the intron-encoded protein cleaves the antisense strand. The Lactococcal intron endonuclease can be obtained in large quantities by coexpression of the LtrA protein with the intron RNA in E. coli or reconstituted in vitro by incubating the expressed LtrA protein with in vitro-synthesized intron RNA. Furthermore, the specificity of the endonuclease and reverse splicing reactions can be changed predictably by modifying the RNA component. Expression in E. coli facilitates the use of group II introns for the targeting of specific foreign sequences to a desired site in DNA.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in Type I and II CRISPR systems by the acquisition of short segments of DNA (“spacers”) from invasive elements. In several Type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we show that an RT-Cas1 fusion enables the acquisition of RNA spacers in vivo in an RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze ligation of RNA segments into the CRISPR array, followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA.

Many group II introns encode reverse transcriptase-like proteins that potentially function in intron mobility and RNA splicing. We compared 34 intron-encoded open reading frames and four related open reading frames that are not encoded in introns. Many of these open reading frames have a reverse transcriptase-like domain, followed by an additional conserved domain X, and a Zn(2+)-finger-like region. Some open reading frames have lost conserved sequence blocks or key amino acids characteristic of functional reverse transcriptases, and some lack the Zn(2+)-finger-like region. The open reading frames encoded by the chloroplast tRNA(Lys) genes and the related Epifagus virginiana matK open reading frame lack a Zn(2+)-finger-like region and have only remnants of a reverse transcriptase-like domain, but retain a readily identifiable domain X. Several findings lead us to speculate that domain X may function in binding of the intron RNA during reverse transcription and RNA splicing. Overall, our findings are consistent with the hypothesis that all of the known group II intron open reading frames evolved from an ancestral open reading frame, which contained reverse transcriptase, X, and Zn(2+)-finger-like domains, and that the reverse transcriptase and Zn(2+)-finger-like domains were lost in some cases. The retention of domain X in most proteins may reflect an essential function in RNA splicing, which is independent of the reverse transcriptase activity of these proteins.

Group I and group II introns are two types of RNA enzymes, ribozymes, that catalyze their own splicing by different mechanisms. In this review, we summarize current information about the structures of group I and group II introns, their RNA-catalyzed reactions, the facilitation of RNA-catalyzed splicing by protein factors, and the ability of the introns to function as mobile elements. The RNA-based enzymatic reactions and intron mobility provide a framework for considering the role of primordial catalytic RNAs in evolution and the origin of introns in higher organisms.

The DEAD-box proteins in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N-and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended α-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted α-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the α-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two subregions of Mss116p's C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions.