Eukaryotic ribosomes are complex molecular machines whose assembly is tightly controlled to ensure faithful protein biosynthesis. Ribosome assembly is initiated in the nucleolus by the transcription and processing of ribosomal RNAs, which together with ribosomal proteins form pre-60S ribosomal particles. The pre-60S particles mature by transiently interacting with various assembly factors during cytosolic export. The nearly 5000 residues long maturation factor Rea1 is vital for the export of the pre-60S particles. Rea1 belongs to the AAA+ protein family and harnesses the energy of ATP hydrolysis to mechanically remove assembly factors from pre-60S particles. Despite its key importance for ribosome maturation, the Rea1 structure and mechanism are poorly understood. In this proposal, we will use cryo-electron microscopy as well as in-vitro and in-vivo activity assays to elucidate the Rea1 structure and mechanism in the context of pre60S particles.

Eukaryotic genome sequencing projects led to the astonishing discovery that the number of protein-coding genes shows a surprisingly moderate increase during evolution, although the genome size is rapidly growing in higher eukaryotes. This finding led to the recently emerging view that in addition to the protein-coding genes, the eukaryotic genomes contain another, thus far hidden layer of genetic information. Indeed, during that past decade it became apparent that the eukaryotic genome encodes a tremendous number of non-protein-coding or non-coding RNAs (ncRNAs) which function as regulatory RNAs in all aspects of gene expression and thereby, largely contribute to the biological complexity of eukaryotic organisms. Box H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse group of ncRNAs. The H/ACA RNAs function in pseudouridylation of various classes of cellular RNAs, nucleolytic processing of rRNAs, synthesis of telomeric DNA and they serve as substrates for microRNA processing. All H/ACA RNAs associate with four H/ACA core ribonucleoproteins, dyskerin, Nhp2, Nop10 and Gar1, and they accumulate either in the nucleolus or in the nucleoplasmic Cajal bodies. The Cajal body-specific H/ACA RNAs also associate with Wdr79. In our laboratory, recent characterization of human Wdr79-associated RNAs identified more than 400 novel putative H/ACA RNAs. Besides several canonical H/ACA RNAs, we identified 348 novel human H/ACA RNAs which are encoded by genomic Alu repetitive elements located within introns of protein-coding genes. We have demonstrated that the newly discovered Alu-derived H/ACA RNAs, termed AluACA RNAs, are synthesized and processed from pre-mRNA intronic Alu sequences in an H and ACA box-dependent manner. The mature AluACA RNPs associate with the four H/ACA core proteins and Wdr79, but unexpectedly, they co-localize with spliceosomal small nuclear (sn)RNAs in the nucleoplasm. The Alu elements are the most abundant repetitive elements in the human genome with largely unknown function. Originated from the 7SL signal recognition particle RNA gene, Alu elements were propagated by retrotransposition in the genomes of primates. Thus, the newly discovered AluACA RNAs represent a novel, abundant, human- or maybe primate-specific subclass of H/ACA RNAs. Here, I propose a research program to dissect the biogenesis, subnuclear trafficking and the cellular function of this fascinating group of human ncRNPs.

Cellular responses to environmental stimuli often require rapid transcriptional induction of specific gene subsets, which are somehow “targeted” within minutes despite representing a tiny fraction of the entire DNA sequence within the nucleus. The genome is highly spatially organized within eukaryotic cell nuclei, and a large body of work has correlated gene locations relative to nuclear landmarks, or certain genome configurations, with regulation of DNA metabolism. Active and inactive compartments form topologically separate domains exhibiting signatures of chromatin chemical modifications and factors recruited by them. Within the domains, enhancers can contact promoters specifically or selectively over short or long genomic distances, usually in cis. Enhancers, including super-enhancers, are known to interact stochastically, a feature particularly relevant during cellular differentiation. However it is still unclear whether transcription per se drives spatial reorganization and how we can use this information to gain a better understanding of cell responses in normal and pathological contexts. Moreover, the dynamics of such processes are poorly understood and mainly correlative. By live cell imaging we have recently demonstrated that transcription initiation rapidly confines the transcribed locus and that chromatin dynamics occur in large domains of coordinated movement with abrupt boundaries only in actively transcribing cells. To achieve this, we have developed two complementary new methods, one for particle tracking, the proprietary ANCHOR technology to fluorescently tag up to three specific DNA loci, and one for flow determination of fluorescent proteins and DNA in the entire nucleus. These approaches give us an unprecedented opportunity to assess the mechanism by which chromatin topology evolves and contributes to transcriptional control in living cells. We propose a multiscale approach probing chromatin domain conformation and dynamics of rapidly inducible estrogen target genes in mammary tumor cells. Live cell imaging will be combined with promoter capture Hi-C and polymer modeling to ask how structural reorganization regulates transcriptional output, and how this may be perturbed. Insights into chromosome biology and technological developments will foster new applications for diagnostics and drug discovery.