B1 Analyses of eukaryotic ribosome assembly | P. Milkereit, H. Tschochner, J. Griesenbeck

Production of ribosomes is one of the major energy consuming processes in proliferating cells which requires, among others, the correct folding of rRNA and the assembly of ribosomal proteins with rRNA. In the next funding period of the CRC we plan to continue our previous studies on how ribosome biogenesis factors and ribosomal proteins affect the assembly states and stability of ribosomal precursor particles in S. cerevisiae. Apart from this, we want to develop and apply biochemical methods to define the dynamics of the local RNA environment of specific ribosomal proteins and ribosome biogenesis factors in pre-ribosomal particles. We expect that these experiments will enable to correlate specific assembly states with pre-rRNA folding states and will give mechanistic insights into yeast pre-ribosome maturation.

B2 RNP complexes regulating the accessibility of higher order structures of chromatin | G. Längst
Chromatin is stably associated with RNA and RNP complexes can open higher order structures of chromatin. We study the functional role of the RNA and the mechanisms of chromatin opening. We perform ChIP-Seq and RNA IPs of DF31 and HMGN5 to identify the chromatin targeting signals and test them in interaction assays. We will functionally address the chromatin opening mechanism with an in vivo GFP-HMGN5 targeting system and mutants of the protein. To study RNA dependent chromatin opening in a genome wide scale, we established the differential MNase treatment of cells combined with high-throughput sequencing and advanced bioinformatic analysis. We will continue to work on the development of the bioinformatical and statistical analysis of the large scale data sets and combine the assay with the functional testing of our model protein.
B3 Post-translational regulation of small RNA-guided gene silencing pathways | G. Meister
Post-translational modifications are found on many proteins and contribute to functional diversifications of gene products. It is the aim of project B3 to catalogue and functional characterize post-translational modifications on the key-components of the miRNA pathway. In project B3, we will map phosphorylation sites on factors of the miRNA pathway (Drosha, Xpo-5, Lin28) and functionally characterize the identified phosphorylation sites on Ago and TNRC6 proteins. Signaling pathways and phosphorylation-dependent interaction partners will be investigated.

B4 Characterization of small RNA pathways in the nucleus of human somatic cells | G. Meister
Although miRNA-guided gene silencing is a cytoplasmic process, Ago proteins and other gene silencing factors are also found in the nucleus of human somatic cells. In project B4, nuclear import routes and nuclear functions of gene silencing factors will be investigated. We will investigate which proteins and RNAs Ago and TNRC6 proteins associate with in the nucleus. Furthermore, we will unravel which TNRC6-intrinsic structural features are important for the formation of P-bodies. In addition to TNRC6 proteins, we will further analyze implications of nuclear Ago localization on gene silencing processes in the cytoplasm and the nucleus.

B5 Gametogenesis-related small non-coding RNAs and Argonaute proteins in Arabidopsis | S. Sprunck
The composition and defined biological role(s) of Argonaute (AGO) RNP effector complexes in the female germline of flowering plants are almost unexplored. In the previous funding period we investigated AGO protein and gene expression in the female germline of Arabidopsis thaliana, performed functional studies, and identified more than hundred differential expressed miRNAs, siRNAs and new small RNA precursor sequences by small RNA-Seq. In the next funding period we aim to characterize the composition of AGO effector RNP complexes in the female germline, and to analyze affinity-purified AGOs for posttranslational protein modifications such as phosphorylation. Furthermore, we aim to characterize the population of small RNAs in isolated egg cells of Arabidopsis.

B6 Assembly of localized mRNPs and their function in regulating translation in Arabidopsis | T. Dresselhaus
The group of Thomas Dresselhaus (B6) aims to study the spatial and temporal control of protein biosynthesis during early seed development in Arabidopsis. The multi-step processes of initial mRNP assembly, transport and translational control will be studied using localized mRNAs and putative translational repressors identified in the first SFB funding period. They now aim to investigate transport dynamics, composition and translational activity of identified candidate mRNPs and RNA-binding proteins.

B9 Long noncoding RNAs in tissue homeostasis and disease | M. Kretz
The group of Markus Kretz analyzes the functional relevance and modes of action of long non-coding RNAs (lncRNAs) in human normal and neoplastic skin.
The human genome encodes several thousand long non-protein coding transcripts >200 nucleotides in length. Although recent studies have shown that lncRNAs play important roles in a variety of biological processes, their impact on controlling the transition of mature human tissue into a neoplastic state during cancer development remains largely unknown. Thus, characterization of lncRNAs that are miss-regulated in cancer will gain further insight into functions and mechanisms of lncRNAs in differentiation as well as neoplasia of mature human tissue, and may provide novel targets for prevention and treatment of disorders of epidermal homeostasis as well as skin cancer.
To approach this goal, we use human organotypic epidermis as a model system, to analyze the functional impacts of skin cancer-associated lncRNAs and interacting molecules on epidermal differentiation, neoplastic progression as well as tumor cell growth and invasion rate.

B10 Mechanism and regulation of the elF2-assembly factor Cdc123 and the link of cell cycle entry to mRNA translation | W. Seufert
The proposed work mostly involves molecular biological experiments in budding yeast. Towards the mechanism and physiological role of Cdc123, we will generate mutants guided by structural information and test their biological function and protein interaction by complementation, Y2H and coIP studies as well as in ATPase, kinase, and eIF2 assembly assays, and continue work on genetic interactions and phosphorylation of Cdc123. To understand how defects in mRNA translation prevent cell cycle entry, we will characterize the consequences of depleting various initiation factors, study in detail G1-cyclin CLN3 expression, and collaborate to analyze translational changes genome-wide by use of ribosome profiling.

B11 Regulation of alternative translation initiation | J. Medenbach
Alternative usage of translation initiation codons is employed to produce different protein isoforms with markedly different activities from a single mRNA species (e.g. FGF-2 mRNA). Despite the broad clinical importance, the underlying regulatory mechanisms are not yet understood. Recent technological advances now allow us to examine ribonucleoproteins (RNPs) in a highly parallel fashion, probing simultaneously for a multitude of protein-RNA interactions. By further developing, adapting and applying these high throughput techniques we plan to interrogate RNP dynamics under different cellular conditions in unprecedented detail to gain insight into the regulation of translation initiation and start codon choice on FGF2 mRNA.

B7 (finished): Functional implication of miRNA processing in malignant melanoma | A. Bosserhoff
Regulation of gene expression by miRNAs plays an important and still emerging role in physiological as well as pathophysiological processes. Details of miRNA function including the characterization of miRNPs still need to be examined to yield a complete understanding of the molecular processes. We have chosen to analyze changes in the miRNA processing machinery in melanoma. These will be determined in detail and effects of these variances will be studied. Preliminary data hinted to deregulation of Argonaute 2 (Ago2) expression in melanoma tumor cells. Therefore, regulation of Ago2 and effects of regulated Ago2 expression will be analyzed in detail. Additionally, we will concentrate on the miRNP and define the influence of protein methylation on miRNP formation and activity. Here, melanoma is an appealing model system as accumulation of a protein-methyltransferase inhibitor, MTA, was shown during cancer development. In summary, successful accomplishment of the aims will yield in a better understanding of the pathophysiological situation of miRNA processing in human disease but also to a broader knowledge on physiological processes.