RNA-guided DNA Research Articles

RNA‐guided DNA rearrangements in ciliates: Is the best genome defence a good offence?

Genomes, like crazy patchwork quilts, are stitched together over evolutionary time from diverse elements, including some unwelcome invaders. To deal with parasitic mobile elements, most eukaryotes employ a genome self-defensive manoeuvre to recognise and silence such elements by homology-dependent interactions with RNA-protein complexes that alter chromatin. Ciliated protozoa employ more 'offensive' tactics by actually unstitching and reassembling their somatic genomes at every sexual generation to eliminate transposons and their remnants, using as patterns the maternal genomes that were rearranged in the previous cycle. Genetic and genomic studies of the distant relatives Paramecium and Tetrahymena have begun to reveal how such events are carried out with remarkable precision. Whole genome, non-coding transcripts from the maternal genome are compared with transcripts from the zygotic genome that are processed through an RNA interference (RNAi)-related process. Sequences found only in the latter are targeted for elimination by the resulting short 'scanRNAs' in many thousand DNA splicing reactions initiated by a domesticated transposase. The involvement of widely conserved mechanisms and protein factors clearly shows the relatedness of these phenomena to RNAi-mediated heterochromatic gene silencing. Such malleability of the genome on a generational time scale also has profound evolutionary implications, possibly including the epigenetic inheritance of acquired adaptive traits.

Structural basis for CRISPR RNA-guided DNA recognition by Cascade.

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA(1)B(2)C(6)D(1)E(1)) and a 61-nucleotide CRISPR RNA (crRNA) with 5'-hydroxyl and 2',3'-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

DNA recombination through assembly graphs

Motivated by DNA rearrangements and DNA homologous recombination modeled in [A. Angeleska, N. Jonoska, M. Saito, L.F. Landweber, RNA-guided DNA assembly, Journal of Theoretical Biology, 248(4) (2007), 706–720], we investigate smoothings on graphs that consist of only 4-valent and 1-valent rigid vertices, called assembly graphs. An assembly graph can be seen as a representation of the DNA during certain recombination processes in which 4-valent vertices correspond to the alignment of the recombination sites. A single gene is modeled by a polygonal path in an assembly graph. A polygonal path makes a “right-angle” turn at every vertex, defining smoothings at the 4-valent vertices and therefore modeling the recombination process. We investigate the minimal number of polygonal paths visiting all vertices of a given graph exactly once, and show that for every positive integer n there are graphs that require at least n such polygonal paths. We show that there is an embedding in three-dimensional space of each assembly graph such that smoothing of vertices according to a given set of polygonal paths results in an unlinked graph. As some recombination processes may happen simultaneously, we characterize the subsets of vertices whose simultaneous smoothings keep a given gene in tact and give a characterization of all sequences of sets of vertices defining successive simultaneous smoothings that can realize complete gene rearrangement.

Structural and biochemical advances in mammalian RNAi

RNAi is a collection of processes mediated by small RNAs that silence gene expression in a sequence-specific manner. Studies of processes as divergent as post-transcriptional gene silencing, transcriptional silencing through RNA-directed DNA methylation, or heterochromatin formation, and even RNA-guided DNA elimination have converged on a core pathway. This review will highlight recent structural and mechanistic studies illustrating siRNA and miRNA processing, RISC formation, the execution of RNAi by RISC, and the regulation of these pathways, with a specific focus on vertebrate systems.

RNA-Guided DNA Deletion in Tetrahymena: An RNAi-Based Mechanism for Programmed Genome Rearrangements

Ciliated protozoan are unicellular eukaryotes. Most species in this diverse group display nuclear dualism, a special feature that supports both somatic and germline nuclei in the same cell. Probably due to this unique life style, they exhibit unusual nuclear characteristics that have intrigued researchers for decades. Among them are large-scale DNA rearrangements, which restructure the somatic genome to become drastically different from its germline origin. They resemble the classical phenomenon of chromatin diminution in some nematodes discovered more than a century ago. The mechanisms of such rearrangements, their biological roles, and their evolutionary origins have been difficult to understand. Recent studies have revealed a clear link to RNA interference, and begin to shed light on these issues. Using the simple ciliate Tetrahymena as a model, this chapter summarizes the physical characterization of these processes, describes recent findings that connect them to RNA interference, and discusses the details of their mechanisms, potential roles in genome defense, and possible occurrences in other organisms.