A Method for Expressing and Imaging Abundant, Stable, Circular RNAs In Vivo Using tRNA Splicing.

Recent improvements in high-throughput sequencing technologies underscore the pervasiveness of circular RNA (circRNA) expression in animal cells. CircRNAs are distinct from their linear counterparts because they lack the 5' caps and 3' tails that typically help determine the cellular fate of a transcript. However, due to the lack of free ends, circRNAs are impervious to exonucleases and thus can evade normal RNA turnover mechanisms. Most circRNAs are derived from protein-coding pre-mRNAs, via a mechanism called "back-splicing." Existing methods of circRNA expression thus typically involve genes that have been engineered to contain sequence elements that promote back-splicing. We recently uncovered an anciently conserved mechanism of RNA circularization in metazoans that involves splicing of tRNA introns. This splicing mechanism is completely independent from that of pre-mRNAs. In this chapter, we detail an orthogonal method that involves splicing of intron-containing tRNAs in order to produce circRNAs in vivo. We utilize fluorescence-based RNA reporters to characterize the expression, localization, and stability of these so-called tRNA intronic circular RNAs. Because tRNA biogenesis is essential for all cellular life, this method provides a means to express ultrastable, high-copy, circRNA effectors in a wide variety of metazoan cell types.

Genetic analysis of nuclear bodies: from nondeterministic chaos to deterministic order.

The eukaryotic nucleus is a congested place, and macromolecular crowding is thought to have an important role in increasing the relative concentrations of nuclear proteins, thereby accelerating the rates of biochemical reactions. Crowding is also thought to provide the environment needed for formation of nuclear bodies/subcompartments, such as the Cajal body (CB) and the histone locus body (HLB), via self-organization. In this chapter, we contrast the theories of stochastic self-organization and hierarchical self-organization in their application to nuclear body assembly, using CBs and HLBs as paradigms. Genetic ablation studies in Drosophila on components of CBs and HLBs have revealed an order to the assembly of these structures that is suggestive of a hierarchical model of self-organization. These studies also show that functions attributed to the nuclear bodies are largely unaffected in their absence, reinforcing an emerging theme in the field that the purpose of these subdomains may be to enhance the efficiency and specificity of reactions.

FLASH is required for histone transcription and S-phase progression.

Cajal bodies are nuclear subdomains that are involved in maturation of small ribonucleoproteins and frequently associate with small nuclear RNA and histone gene clusters in interphase cells. We have recently identified FADD-like IL-1beta-converting enzyme (FLICE) associated huge protein (FLASH) as an essential component of Cajal bodies. Here we show that FLASH associates with nuclear protein, ataxia-telangiectasia, a component of the cell-cycle-dependent histone gene transcription machinery. Reduction of FLASH expression by RNA interference results in disruption of the normal Cajal body architecture and relocalization of nuclear protein, ataxia-telangiectasia. Furthermore, FLASH down-regulation results in a clear reduction of histone transcription and a dramatic S-phase arrest of the cell cycle. Chromatin immunoprecipitation reveals that FLASH interacts with histone gene promoter sequences. These results identify FLASH as an important component of the machinery required for histone precursor mRNA expression and cell-cycle progression.

Coilin forms the bridge between Cajal bodies and SMN, the spinal muscular atrophy protein.

Spinal muscular atrophy (SMA) is a genetic disorder caused by mutations in the human survival of motor neuron 1 gene, SMN1. SMN protein is part of a large complex that is required for biogenesis of various small nuclear ribonucleoproteins (snRNPs). Here, we report that SMN interacts directly with the Cajal body signature protein, coilin, and that this interaction mediates recruitment of the SMN complex to Cajal bodies. Mutation or deletion of specific RG dipeptide residues within coilin inhibits the interaction both in vivo and in vitro. Interestingly, GST-pulldown experiments show that coilin also binds directly to SmB'. Competition studies show that coilin competes with SmB' for binding sites on SMN. Ectopic expression of SMN and coilin constructs in mouse embryonic fibroblasts lacking endogenous coilin confirms that recruitment of SMN and splicing snRNPs to Cajal bodies depends on the coilin C-terminal RG motif. A cardinal feature of SMA patient cells is a defect in the targeting of SMN to nuclear foci; our results uncover a role for coilin in this process.

RNA-mediated interaction of Cajal bodies and U2 snRNA genes.

Cajal bodies (CBs) are nuclear structures involved in RNA metabolism that accumulate high concentrations of small nuclear ribonucleoproteins (snRNPs). Notably, CBs preferentially associate with specific genomic loci in interphase human cells, including several snRNA and histone gene clusters. To uncover functional elements involved in the interaction of genes and CBs, we analyzed the expression and subcellular localization of stably transfected artificial arrays of U2 snRNA genes. Although promoter substitution arrays colocalized with CBs, constructs containing intragenic deletions did not. Additional experiments identified factors within CBs that are important for association with the native U2 genes. Inhibition of nuclear export or targeted degradation of U2 snRNPs caused a marked decrease in the levels of U2 snRNA in CBs and strongly disrupted the interaction with U2 genes. Together, the results illustrate a specific requirement for both the snRNA transcripts as well as the presence of snRNPs (or snRNP proteins) within CBs. Our data thus provide significant insight into the mechanism of CB interaction with snRNA loci, strengthening the putative role for this nuclear suborganelle in snRNP biogenesis.

Residual Cajal bodies in coilin knockout mice fail to recruit Sm snRNPs and SMN, the spinal muscular atrophy gene product.

Cajal bodies (CBs) are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). In addition to snRNPs, they are highly enriched in basal transcription and cell cycle factors, the nucleolar proteins fibrillarin (Fb) and Nopp140 (Nopp), the survival motor neuron (SMN) protein complex, and the CB marker protein, p80 coilin. We report the generation of knockout mice lacking the COOH-terminal 487 amino acids of coilin. Northern and Western blot analyses demonstrate that we have successfully removed the full-length coilin protein from the knockout animals. Some homozygous mutant animals are viable, but their numbers are reduced significantly when crossed to inbred backgrounds. Analysis of tissues and cell lines from mutant animals reveals the presence of extranucleolar foci that contain Fb and Nopp but not other typical nucleolar markers. These so-called "residual" CBs neither condense Sm proteins nor recruit members of the SMN protein complex. Transient expression of wild-type mouse coilin in knockout cells results in formation of CBs and restores these missing epitopes. Our data demonstrate that full-length coilin is essential for proper formation and/or maintenance of CBs and that recruitment of snRNP and SMN complex proteins to these nuclear subdomains requires sequences within the coilin COOH terminus.

Self-association of coilin reveals a common theme in nuclear body localization.

We have found that coilin, the marker protein for Cajal bodies (coiled bodies, CBs), is a self-interacting protein, and we have mapped the domain responsible for this activity to the amino-terminus. Together with a nuclear localization signal, the self-interaction domain is necessary and sufficient for localization to CBs. Overexpression of various wild-type and mutant coilin constructs in HeLa cells results in disruption of both CBs and survival motor neurons (SMN) gems. Additionally, we have identified a cryptic nucleolar localization signal (NoLS), within the coilin protein, which may be exposed in specific coilin phospho-isoforms. The implications of these findings are discussed in light of the fact that other proteins known to localize within nuclear bodies (e. g., PML, SMN and Sam68) can also self-associate. Thus protein self-interaction appears to be a general feature of nuclear body marker proteins.

NPAT links cyclin E-Cdk2 to the regulation of replication-dependent histone gene transcription.

In eukaryotic cells, histone gene expression is one of the major events that mark entry into S phase. While this process is tightly linked to cell cycle position, how it is regulated by the cell cycle machinery is not known. Here we show that NPAT, a substrate of the cyclin E-Cdk2 complex, is associated with human replication-dependent histone gene clusters on both chromosomes 1 and 6 in S phase. We demonstrate that NPAT activates histone gene transcription and that this activation is dependent on the promoter elements (SSCSs) previously proposed to mediate cell cycle-dependent transcription. Cyclin E is also associated with the histone gene loci, and cyclin E-Cdk2 stimulates the NPAT-mediated activation of histone gene transcription. Thus, our results both show that NPAT is involved in a key S phase event and provide a link between the cell cycle machinery and activation of histone gene transcription.

Alpha satellite DNA variant-specific oligoprobes differing by a single base can distinguish chromosome 15 homologs.

The ability to distinguish homologous chromosomes is a powerful cytogenetic tool. However, traditional techniques can only distinguish extreme physical variants and are highly dependent on sample preparation. We have previously reported oligonucleotide probes, specific for human chromosome 17 alpha satellite DNA sequence variants, that distinguish cytogenetically normal homologous chromosomes by FISH. Here we report the development of similar oligoprobes, differing at a single nucleotide position, that not only distinguish homologous chromosomes 15 but can be used to follow the transmission of a chromosome from parents to their offspring. We also identified a novel array-size polymorphism in another family. The alphoid array of one chromosome is quite small and below the detection threshold for our oligoprobes, although it is detectable by conventional FISH probes. This size polymorphism provides an additional FISH-based method for distinguishing homologs. Most importantly, this work illustrates the potential applicability of the technique to the entire human chromosome complement.

Structure and characterization of the murine p80 coilin gene, Coil.

Cajal bodies (coiled bodies, CBs) are nuclear organelles of unknown function and are characterized by a wide variety of components including various basal transcription and cell cycle proteins, the nucleolar proteins fibrillarin and Nopp140, numerous small nuclear ribonucleoproteins, the survival motor neuron protein complex, and the marker protein, p80 coilin. To gain insight into the role of p80 coilin in CBs, we have cloned the murine gene Coil and have mapped it to the distal portion of chromosome band 11D. The approximately 2.6-kb transcript is detectable in all tissues analyzed, with the highest levels in brain and testis. Sequence analysis shows that, like its human counterpart, the mouse coilin gene is composed of seven exons and spans nearly 30 kb of genomic DNA. The predicted amino acid sequence reveals two conserved N- and C-terminal domains, and comparison with the Xenopus SPH-1 protein reveals that these three genes are indeed orthologous. These results should facilitate gene disruption experiments aimed at creating a genetic model system to study CBs.

Cell cycle-dependent localization of the CDK2-cyclin E complex in Cajal (coiled) bodies.

We have found that CDK2 and cyclin E, but not cyclin A, accumulates within Cajal bodies (CBs) in a cell cycle-dependent fashion. In the absence of cyclin E, CDK2 is not enriched in the CB compartment, suggesting that the translocation of CDK2 to CBs is dependent on cyclin E. CDK2 and cyclin E could be recruited to CBs as a functional complex or CBs may serve as 'docking stations' for CDK2-cyclin E activation by CAKs during the G(1)/S transition. Notably, CDK7-cyclin H-Mat1 complexes are known to accumulate in CBs. Treatment of cells with inhibitors of either CDKs (olomoucine, 200 microM) or RNA polymerase I (actinomycin D, 0.05 microgram/ml), results in a striking reorganization of CDK2 and p80 coilin to the nucleolar periphery. Furthermore, we demonstrate that p80 coilin can be phosphorylated by purified CDK2-cyclin E complexes in vitro. Thus coilin and other CB proteins appear to be downstream targets of CDK2-cyclin E complex-mediated signaling pathways regulating cell cycle progression and controlling aspects of CB function. Possible roles for CDK2 and cyclin E in the well-documented association of CBs, histone gene clusters and RNA 3' end processing factors are discussed.

Nuclear domains enriched in RNA 3'-processing factors associate with coiled bodies and histone genes in a cell cycle-dependent manner.

Nuclear domains, called cleavage bodies, are enriched in the RNA 3'-processing factors CstF 64 kDa and and CPSF 100 kDa. Cleavage bodies have been found either overlapping with or adjacent to coiled bodies. To determine whether the spatial relationship between cleavage bodies and coiled bodies was influenced by the cell cycle, we performed cell synchronization studies. We found that in G1 phase cleavage bodies and coiled bodies were predominantly coincident, whereas in S phase they were mostly adjacent to each other. In G2 cleavage bodies were often less defined or absent, suggesting that they disassemble at this point in the cell cycle. A small number of genetic loci have been reported to be juxtaposed to coiled bodies, including the genes for U1 and U2 small nuclear RNA as well as the two major histone gene clusters. Here we show that cleavage bodies do not overlap with small nuclear RNA genes but do colocalize with the histone genes next to coiled bodies. These findings demonstrate that the association of cleavage bodies and coiled bodies is both dynamic and tightly regulated and suggest that the interaction between these nuclear neighbors is related to the cell cycle-dependent expression of histone genes.

Nuclear bodies: multifaceted subdomains of the interchromatin space.

Higher-eukaryotic nuclei contain numerous morphologically distinct substructures that are collectively called nuclear bodies. Although the precise functions of these subdomains remain unknown, elucidation of their molecular composition has been the subject of a great deal of research in recent years. Changes in the constitution of these nuclear inclusions are associated with disease phenotypes. The wide variety of components that concentrate within these subdomains makes them a likely interface for multiple cellular processes, including transcription, RNA processing, transport, RNP assembly, protein modification, apoptosis and cell-cycle control. This review discusses the different types of nuclear bodies, with emphasis on the two most prominent subtypes - the coiled and PML bodies.

Assignment of a novel bifurcated SET domain gene, SETDB1, to human chromosome band 1q21 by in situ hybridization and radiation hybrids.

We have identified a human gene encoding an unusual bifurcated SET domain protein containing a large "insertion" between the most highly conserved parts of the SET domain. The existence of an evolutionarily related C. elegans gene encoding a similarly bifurcated SET domain suggests that SET domains may generally be composed of two functionally distinct subdomains. We mapped this gene, called SETDB1, to human chromosome 1q21. This region is targeted by a large number of recurrent translocations, suggesting that like the SET domain protein MLL, mutant forms of SETDB1 may be associated with human neoplasias.

Coiled bodies preferentially associate with U4, U11, and U12 small nuclear RNA genes in interphase HeLa cells but not with U6 and U7 genes.

Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle-regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for chi2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed.

RNA splicing: more clues from spinal muscular atrophy.

Spinal muscular atrophy is caused by mutations in the SMN1 gene, the product of which is part of a multi-component complex involved in the assembly of small nuclear ribonucleoproteins. A recent study indicates that SMN may also play a role in pre-mRNA splicing.

Association of snRNA genes with coiled bodies is mediated by nascent snRNA transcripts.

BACKGROUND: Coiled bodies are nuclear organelles that are highly enriched in small nuclear ribonucleoproteins (snRNPs) and certain basal transcription factors. Surprisingly, coiled bodies not only contain mature U snRNPs but also associate with specific chromosomal loci, including gene clusters that encode U snRNAs and histone messenger RNAs. The mechanism(s) by which coiled bodies associate with these genes is completely unknown. RESULTS: Using stable cell lines, we show that artificial tandem arrays of human U1 and U2 snRNA genes colocalize with coiled bodies and that the frequency of the colocalization depends directly on the transcriptional activity of the array. Association of the genes with coiled bodies was abolished when the artificial U2 arrays contained promoter mutations that prevent transcription or when RNA polymerase II transcription was globally inhibited by alpha-amanitin. Remarkably, the association was also abolished when the U2 snRNA coding regions were replaced by heterologous sequences. CONCLUSIONS: The requirement for the U2 snRNA coding region indicates that association of snRNA genes with coiled bodies is mediated by the nascent U2 RNA itself, not by DNA or DNA-bound proteins. Our data provide the first evidence that association of genes with a nuclear organelle can be directed by an RNA and suggest an autogenous feedback regulation model.