We describe a technique for the detection and localization of RNA transcripts in living cells. The method is based on fluorescent-protein complementation regulated by the interaction of a split RNA-binding protein with its corresponding RNA aptamer. In our design, the RNA-binding protein is the eukaryotic initiation factor 4A (eIF4A). eIF4A is dissected into two fragments, and each fragment is fused to split fragments of the enhanced green fluorescent protein (EGFP). Coexpression of the two protein fusions in the presence of a transcript containing eIF4A-interacting RNA aptamer resulted in the restoration of EGFP fluorescence in Escherichia coli cells. We also applied this technique to the visualization of an aptamer-tagged mRNA and 5S ribosomal RNA (rRNA). We observed distinct spatial and temporal changes in fluorescence within single cells, reflecting the nature of the transcript.

Saccharomyces cerevisiae cells expressing both a-and a-mating-type (MAT) genes (termed mating-type heterozygosity) exhibit higher rates of spontaneous recombination and greater radiation resistance than cells expressing only MATa or MATa. MAT heterozygosity suppresses recombination defects of four mutations involved in homologous recombination: complete deletions of RAD55 or RAD57, an ATPase-defective Rad51 mutation (rad51-K191R), and a C-terminal truncation of Rad52, rad52-D327. We investigated the genetic basis of MAT-dependent suppression of these mutants by deleting genes whose expression is controlled by the Mata1-Mata2 repressor and scoring resistance to both campothecin (CPT) and phleomycin. Haploid rad55D strains became more damage resistant after deleting genes required for nonhomologous end-joining (NHEJ), a process that is repressed in MATa/MATa cells. Surprisingly, NHEJ mutations do not suppress CPT sensitivity of rad51-K191R or rad52-D327. However, rad51-K191R is uniquely suppressed by deleting the RME1 gene encoding a repressor of meiosis or its coregulator SIN4; this effect is independent of the meiosis-specific homolog, Dmc1. Sensitivity of rad52-D327 to CPT was unexpectedly increased by the MATa/MATa-repressed gene YGL193C, emphasizing the complex ways in which MAT regulates homologous recombination. The rad52-D327 mutation is suppressed by deleting the prolyl isomerase Fpr3, which is not MATregulated. rad55D is also suppressed by deletion of PST2 and/or YBR052C (RFS1, rad55 suppressor), two members of a three-gene family of flavodoxin-fold proteins that associate in a nonrandom fashion with chromatin. All three recombination-defective mutations are made more sensitive by deletions of Rad6 and of the histone deacetylases Rpd3 and Ume6, although these mutations are not themselves CPT or phleomycin sensitive. D NA repair in budding yeast is strongly influenced by the cell's mating status. Saccharomyces cells can be of three mating types: those able to mate expressing only MATa or only MATa and nonmating cells expressing both MATa and MATa. MATa/MATa diploid cells are more radiation resistant and recombination proficient than diploids expressing only MATa or MATa (Friis and Roman 1968;Heude and Fabre 1993;Fasullo and Dave 1994;Lowell et al. 2003). A similar increase in radioresistance and resistance to radiomimetic drugs is seen in haploid cells that express both mating-type alleles. Coexpression of MATa and MATa, either in diploids or in haploids, leads to the formation of the Mata1-Mata2 corepressor that turns off the expression of haploid-specific genes and induces expression of diploid-specific genes. The most striking effect of a1-a2 repression is a severe disabling of nonhomologous end-joining (NHEJ) by the repression of NEJ1 (Å strö m et al. 1999;Lee et al. 1999; FrankVaillant and Marcand 2001;Kegel et al. 2001;Ooi and Boeke 2001;Valencia et al. 2001). Whether any of the other targets of a1-a2 repression affect homologous recombination (HR) is not known.A double-strand break (DSB) c...

Bacteria have a complex internal organization with specific localization of many proteins and DNA, which dynamically move during the cell cycle and in response to changing environmental stimuli. Much less is known, however, about the localization and movements of RNA molecules. By modifying our previous RNA labeling system, we monitor the expression and localization of a model RNA transcript in live Escherichia coli cells. Our results reveal that the target RNA is not evenly distributed within the cell and localizes laterally along the long cell axis, in a pattern suggesting the existence of ordered helical RNA structures reminiscent of known bacterial cytoskeletal cellular elements.espite their relatively small dimensions, bacterial cells show a remarkable, rich internal subcellular organization that has captured the interest of researchers over the past decade (1-4). Many cytoplasmic and membrane proteins, particularly those involved in cell division, DNA replication, and chromosome segregation, have specific subcellular localizations that can change quickly over time in response to cell cycle progression, motility, and environmental cues. This dynamic and organized behavior is also true for bacterial chromosomal DNA. The use of GFP fusions and in situ fluorescence hybridization (FISH) have shown that every chromosomal locus has a defined subcellular address and is replicated and segregated into the new cell as part of an active and directed process (4, 5). Bacterial plasmids, both low and high copy, also have specific cellular addresses and segregate in a fashion that is unique for a given plasmid (6-8).Little is known, however, about RNA dynamics in bacteria. With the advent of new methods to label RNA in live cells, the transcription kinetics, localization, and movement of RNA in the bacterium Escherichia coli has begun to be discerned only recently (9-13).To understand RNA dynamics in live cells better, it would be useful to develop RNA labeling methods that would allow direct visualization and real-time quantitation of RNAs with low background levels. We recently reported a system based on protein complementation that uses binding of a split and inactive protein complex to a short interacting sequence on a target RNA. The marker protein re-associates and becomes fluorescent only upon binding to RNA (13), which makes this approach more desirable than alternative techniques relying on expression of full-size fluorescent proteins. Briefly, the method consists of fusing the Nterminal fragment of EGFP to the N-terminal domain of an RNA-binding protein, the eukaryotic initiation factor 4A (eIF4A), via a polypeptide linker. Similarly, the C-terminal fragment of EGFP is fused to the C-terminal domain of eIF4A. The target RNA is tagged at the 3Ј end with an aptamer sequence known to bind eIF4A with high affinity (14) (Fig. 1A). Expression of the labeling components in E. coli cells generates a fluorescent signal only in the presence of the target RNA, caused by the reassociation of the two EGFP fragments and forma...

The immune systems of jawed vertebrates assemble a diverse array of immunoglobulin molecules and T-cell receptors by the process known as V(D)J recombination (1). This sitespecific recombination reaction is initiated by the lymphoidspecific proteins, RAG1 and RAG2, which together compose the V(D)J recombinase. RAG1/2 bind recombination signal sequences (RSSs) 1 flanking the borders of gene segments and create a double-strand break via a two-step transesterification mechanism common to several transposases and retroviral integrases (2, 3). Two distinct types of DNA ends are generated from this mechanism of cleavage: blunt, 5Ј-phosphoryl, 3Ј-hydroxyl signal ends and covalently sealed hairpin-coding ends (2, 4, 5). Coding joints are then formed by the imprecise fusion of coding ends, whereas signal joints are formed by joining signal ends without nucleotide loss or addition. Like other double-strand breaks in mammalian cells, repair of RAG1/2-induced breaks requires ubiquitously expressed factors of the non-homologous end joining (NHEJ) repair pathway (6).However, the repair phase of V(D)J recombination is a specialized case of NHEJ, where the repair of double-strand breaks is dependent not only on components of the NHEJ pathway but also on the RAG proteins themselves. Mutations in both RAG1 and RAG2 have been identified that interfere with V(D)J joining but not V(D)J cleavage in vivo and in vitro (7-10). Furthermore, in vitro reconstitution of the joining phase of V(D)J recombination shows a dependence on RAG1 and RAG2 (11). Although the precise role of the RAG proteins in the post-cleavage events of V(D)J recombination is not known, RAG1 and RAG2 do remain associated with the DNA ends in a stable, heparin-resistant, synaptic complex postcleavage in vitro (12-15). Moreover, nuclease-resistant complexes containing the RAG proteins and cleaved

This unit describes a method allowing RNA visualization in live cells. The method is based on fluorescent protein complementation regulated by RNA‐aptamer/RNA‐binding protein interactions. Based on these two principles, a fluorescent ribonucleoprotein complex is assembled inside the cell only in response to the presence of the aptamer sequence on the target RNA. Curr. Protoc. Cell Biol. 37:17.11.1‐17.11.20. © 2007 by John Wiley & Sons, Inc.