Alan M. Lambowitz

A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility

The mobility (homing) of the yeast mitochondrial DNA group II intron al2 occurs via target DNA-primed reverse transcription at a double-strand break in the recipient DNA. Here, we show that the site-specific DNA endonuclease that makes the double-strand break is a ribonucleoprotein complex containing the al2-encoded reverse transcriptase protein and excised al2 RNA. Remarkably, the al2 RNA catalyzes cleavage of the sense strand of the recipient DNA, while the al2 protein appears to cleave the antisense strand. The RNA-catalyzed sense strand cleavage occurs via a partial reverse splicing reaction in which the protein component stabilizes the active intron structure and appears to confer preference for DNA substrates. Our results demonstrate a biologically relevant ribozyme reaction with a substrate other than RNA.

Group I and group II introns.

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.— Saldanha, R., Mohr, G., Belfort, M., and Lambowitz, A. M. Group I and group II introns. FASEB J. 7: 15‐24; 1993.

Involvement of aminoacyl-tRNA synthetases and other proteins in group I and group II intron splicing

Group I and group II introns catalyse their own splicing, but depend on protein factors for efficient splicing in vivo. Some of these proteins, termed maturases, are encoded by the introns themselves and may also function in intron mobility. Other proteins are encoded by host chromosomal genes and include aminoacyl-tRNA synthetases and various proteins that function in protein synthesis. The splicing factors identified thus far appear to be idiosyncratic, even in closely related organisms. We suggest that some of these protein-assisted splicing reactions evolved relatively recently, possibly reflecting the recent dispersal of the introns themselves.

A protein required for splicing group I introns in Neurospora mitochondria is mitochondrial tyrosyl-tRNA synthetase or a derivative thereof

The nuclear cyt-18 mutants of Neurospora crassa are defective in splicing a number of group I introns in mitochondria. Here, cloning and sequencing of the cyt-18 gene show that it contains an open reading frame having significant homology to bacterial tyrosyl-tRNA synthetases. Biochemical and genetic experiments lead to the conclusions that the cyt-18 gene encodes mitochondrial tyrosyl-tRNA synthetase, that mutations in this gene inhibit splicing directly, and that mitochondrial tyrosyl-tRNA synthetase or a derivative of this protein is related to the soluble activity that functions in splicing the mitochondrial large rRNA intron and possibly other group I introns. Analysis of partial revertants provides direct evidence that the cyt-18 gene encodes a protein or proteins with two activities, splicing and aminoacylation, that can be partially separated by mutation. Our findings may be relevant to the evolution of introns and splicing mechanisms in eukaryotes.

18 Group I and Group II Ribozymes as RNPs: Clues to the Past and Guides to the Future

Group I and group II introns are not only catalytic RNAs, but also mobile genetic elements. The success of these introns as mobile elements almost certainly relates to their innate self-splicing capability, which enables them to propagate by inserting into host genes while only minimally impairing gene expression. Nevertheless, both types of introns have become dependent on proteins for efficient splicing in vivo to help fold the intron RNA into the catalytically active structure. Although group I and group II introns have very different structures and splicing mechanisms (Chapter 13), there are striking parallels in the evolution of their protein-assisted splicing reactions. For example, the splicing factors for both types of introns include intron-encoded as well as cellular proteins, and the intron-encoded proteins, DNA endonucleases for group I introns and reverse transcriptases (RTs) for group II introns, also function in intron mobility. In addition, excised group I and group II intron RNAs remain associated with splicing factors in RNP particles, which can then cleave and insert into cellular RNA or DNA target sites by reverse splicing. The need to control this deleterious ribozyme activity may have been an evolutionary driving force favoring mutations that impaired self-splicing activity and resulted in dependence on protein factors (Nikolcheva and Woodson 1997). In this chapter, we review protein-assisted reactions of group I and group II introns. These studies illustrate how proteins facilitate RNA folding and catalysis and provide unique insights into how splicing mechanisms evolve. A recurring theme, first developed in a previous review...

Retrohoming of a Bacterial Group II Intron: Mobility via Complete Reverse Splicing, Independent of Homologous DNA Recombination

The mobile group II intron of Lactococcus lactis, Ll.LtrB, provides the opportunity to analyze the homing pathway in genetically tractable bacterial systems. Here, we show that Ll.LtrB mobility occurs by an RNA-based retrohoming mechanism in both Escherichia coli and L. lactis. Surprisingly, retrohoming occurs efficiently in the absence of RecA function, with a relaxed requirement for flanking exon homology and without coconversion of exon markers. These results lead to a model for bacterial retrohoming in which the intron integrates into recipient DNA by complete reverse splicing and serves as the template for cDNA synthesis. The retrohoming reaction is completed in unprecedented fashion by a DNA repair event that is independent of homologous recombination between the alleles. Thus, Ll.LtrB has many features of retrotransposons, with practical and evolutionary implications.

Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria

Mobile group II introns can be retargeted to insert into virtually any desired DNA target. Here we show that retargeted group II introns can be used for highly specific chromosomal gene disruption in Escherichia coli and other bacteria at frequencies of 0.1–22%. Furthermore, the introns can be used to introduce targeted chromosomal breaks, which can be repaired by transformation with a homologous DNA fragment, enabling the introduction of point mutations. Because of their wide host range, mobile group II introns should be useful for genetic engineering and functional genomics in a wide variety of bacteria.

A DEAD-Box Protein Functions as an ATP-Dependent RNA Chaperone in Group I Intron Splicing

The Neurospora crassa CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in splicing group I introns by inducing formation of the catalytically active RNA structure. Here, we identified a DEAD-box protein (CYT-19) that functions in concert with CYT-18 to promote group I intron splicing in vivo and vitro. CYT-19 does not bind specifically to group I intron RNAs and instead functions as an ATP-dependent RNA chaperone to destabilize nonnative RNA structures that constitute kinetic traps in the CYT-18-assisted RNA-folding pathway. Our results demonstrate that a DExH/D-box protein has a specific, physiologically relevant chaperone function in the folding of a natural RNA substrate.

General method for cloning Neurospora crassa nuclear genes by complementation of mutants

We have developed a sib selection procedure for cloning Neurospora crassa nuclear genes by complementation of mutants. This procedure takes advantage of a modified N. crassa transformation procedure that gives as many as 10,000 to 50,000 stable transformants per microgram of DNA with recombinant plasmids containing the N. crassa qa-2+ gene. Here, we describe the use of the sib selection procedure to clone genes corresponding to auxotrophic mutants, nic-1 and inl. The identities of the putative clones were confirmed by mapping their chromosomal locations in standard genetic crosses and using restriction site polymorphisms as genetic markers. Because we can obtain very high N. crassa transformation frequencies, cloning can be accomplished with as few as five subdivisions of an N. crassa genomic library. The sib selection procedure should, for the first time, permit the cloning of any gene corresponding to an N. crassa mutant for which an appropriate selection can be devised. Analogous procedures may be applicable to other filamentous fungi before the development of operational shuttle vectors.

RNA splicing in neurospora mitochondria: Self-splicing of a mitochondrial intron in vitro

We have used Neurospora nuclear mutant cyt-18-1, which accumulates a number of unspliced mitochondrial precursor RNAs, to identify rapidly mitochondrial introns that are self-splicing in vitro. Incubation of deproteinized whole mitochondrial RNA from the mutant with 32P-GTP resulted in strong labeling of a 1.3 kb RNA, subsequently identified as cytochrome b (cob) intron 1, and weaker labeling of additional RNAs. Self-splicing of cob intron 1, including precise cleavage and ligation, was confirmed using an in vitro transcript synthesized from the SP6 promoter. The in vitro splicing reaction was shown to be analogous to that for the Tetrahymena nuclear rRNA intron. Since splicing of cob intron 1 is inhibited in a recessive nuclear mutant, we infer that this essentially RNA-catalyzed splicing reaction must be facilitated by a protein in vivo.