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Part A: (r)RNA synthesizing machineries and chromatin

Project A1: H. Tschochner, J. Griesenbeck, P. Milkereit

Analyses of the RNA polymerase I (Pol I) machinery: specific transcriptional mechanisms, comparison with other RNA polymerases and structural determination of Pol I complexes

RNA polymerase I (Pol I) is a specialized enzyme which transcribes ribosomal RNA (rRNA) genes in all eukaryotes investigated so far. Pol I transcription is distinguished by specific DNA cis-elements and trans-acting factors as well as the usage of a specific chromatin template. The aim of this project is to unravel specific functional and structural features of the Pol I machinery on a molecular level and to get more insights into the mechanism of Pol I dependent RNA synthesis. Well-defined in vitro systems, which were established in the first funding period will now be used to investigate how certain subunits, domains and/or posttranslational modifications of the Pol I machinery are involved in specific steps of the transcription cycle. 

Project A4: G. Längst

Coordination of rRNA gene transcription initiation and termination in chromatin 

Project A4 focuses on the establishment and functional role of promoter-terminator DNA loops in rDNA regulation. In WP1 we will study the interplay between NoRC, pRNA and TTF-I in promoter recognition and nucleosome positioning. In WP2 we will functionally characterize the chromatin structure at the rRNA gene terminator and test whether this structure is a prerequisite for efficient transcription termination and rDNA looping. In WP3 we will study the dynamics of rDNA chromatin structure and activity during the cell cycle, monitoring the dynamics of chromatin structure and factor binding. We will establish genomically tagged TTF-I, UBF, NoRC and Chd proteins to reveal genomic locations, isolate complexes and to reveal the cell cycle dependent dynamics of these proteins.

Project A5: J. Griesenbeck, P. Milkereit, H. Tschochner

Compositional analysis of ribosomal RNA gene chromatin in distinct transcriptional states

The essential process of ribosomal RNA (rRNA) gene transcription in the nucleolus of eukaryotic cells occurs in the context of chromatin. Our project aims to define rRNA gene chromatin, in its composition, structure and function. In the last funding period we have further developed a technique for the native isolation of rRNA gene chromatin from S. cerevisiae, providing a detailed description of chromatin at different functional ribosomal DNA (rDNA) elements. Results obtained with the biochemically purified rDNA chromatin were verified by methods analyzing rDNA chromatin composition in vivo. In the next funding period we will use the combination of these approaches to obtain a detailed molecular description of dynamic rDNA chromatin transitions in different physiological situations. We expect to deepen our insights in the structure-function relationship between chromatin and transcription.

Project A6: K. Grasser

Transcript elongation and RNA processing/export in Arabidopsis  

The aim of this project is to elucidate possible interactions between ongoing transcription and mRNA processing and export in the Arabidopsis model. Mutants deficient in different transcript elongation factors (TEFs) will be examined for mRNA splicing defects. Affected genes are analysed in more detail to identify whether changes in chromatin structure correlate with splicing alterations.  In the second part, the interplay of transcript elongation and plant mRNA export will be studied to elucidate a possible link with the transcript elongation complex. Since in plants mRNA export proteins (i.e. export adaptors) are diversified, it will be analysed to which extent they are functionally redundant or have adopted specific roles in the export of mRNAs from the nucleus.

Project A7: D. Grohmann

Dynamics of transcriptional complexes and interplay of the archaeal RNA polymerase with RNA processing proteins

We will employ single-molecule fluorescence spectroscopy in conjunction with site-specific fluorophore engineering schemes and biochemical approaches to unravel the structure-function-dynamic relationship within archaeal and also eukaryotic RNAPI and RNAPIII transcription systems, which only recently became available in a recombinant form suitable for single-molecule interrogation. Here, we will especially focus on mechanisms of transcription initiation. Furthermore, we would like to characterise the influence of the archaeal RNA chaperone Hfq on RNA synthesis as these processes most likely occur simultaneously in vivo.

Project A2 (finished): P. Cramer

Structure and function of RNA polymerase I and initiation factor Rrn3

The first step in eukaryotic ribosome biogenesis is the production of the ribosomal RNA precursor by RNA polymerase I (Pol I). Pol I is a 14-subunit multi-protein complex with a molecular weight of 600 kDa. The regulation of Pol I and ribosome biogenesis controls cell growth. Despite its importance, the structure of Pol I and the mechanism of class I promoter-specific transcription initiation remain unknown. The long-term goal of this project is to provide the structural basis of Pol I transcription, and Pol I-specific transcription initiation, to understand key switches that control cell growth. In the past, we have determined crystal structures of Pol I subcomplexes A14/43 and A43/34.5 (Geiger et al., Mol. Cell 2010) and the hybrid structure of Pol I that used cryo-electron microscopy, modelling, and biochemical probing, to unravel the functional architecture of the complex (Kuhn, Cell 2007). During the first funding period of this SFB, we propose to determine the X-ray crystal structure of the complete yeast Pol I (Aim 1), and to determine the structure and function of the Pol I-specific initiation factor Rrn3, including its interaction with Pol I (Aim 2). In collaboration with other teams in the SFB, we will also improve crystals of the initiation complex of the archaeal Pyrococcus furiosus polymerase, provide assistance to teams who aim to crystallize factors involved in ribosome biogenesis, and provide help with structure-based prediction and interpretation of site-directed mutagenesis.

Project A3 (finished): M. Thomm

Structure function relationships in the archaeal transcription machinery

The archaeal transcription machinery is the evolutionary precursor and an excellent simplified model of the more complex eukaryotic transcription systems. The reconstituted RNA polymerase (RNAP) from Pyrococcus furiosus was a useful tool for the elucidation of major functions of key loops in the active centre of RNAP and the archaeal system was also helpful to unravel a function of the linker region of TFB and of the clamp coiled coil domain of RNAP in open complex formation. Eukaryotic RNAP subunits were shown to functionally replace subunits in the archaeal RNAP, and the archaeal subunit P in WT sequence and a mutated form of subunit H were able to complement deletion mutants of the general eukaryotic RNAP subunits Rpb12 and of the C-terminal domain of subunit Rpb5 in yeast. In this project we aim to analyze the architecture and dynamics of the preinitiation complex (PIC) in more detail in particular with respect to the location and function of different functional elements of TFB that are inserted in the RNA polymerase cleft in a TFIIB-RNA polymerase II cocrystal. We are specifically interested in the transitions from i) closed to open complexes and from ii) open to early elongating complexes. Furthermore, we continue to determine the location of TFE on the RNA-polymerase during initiation and elongation, both in vivo and in vitro. Another target of investigation is transcriptional proofreading of the archaeal RNA polymerase. Reconstitution of the archaeal RNA polymerases allows us to study deletion and point mutants in the trigger loop (TL) to define its contribution to internal cleavage activity of the RNA polymerase, transcriptional fidelity as well as a possible role of TL in interacting with elongation factor TFS, which is located in cocrystals of Pol II and TFIIS close to the TL. These studies will contribute to a deeper understanding of the mechanism of transcription and of the evolution of the three eukaryotic RNA polymerases from a single archaea-like precursor.


Part B: Assembly, function and regulation of RNPs

Project B1: P. Milkereit, H. Tschochner, J. Griesenbeck

Analyses of eukaryotic ribosome assembly

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.

Project B2: G. Längst

RNP complexes regulating the accessibility of higher order structures of chromatin

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.

Project B3: G. Meister

Post-translational regulation of small RNA-guided gene silencing pathways

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.

Project B4: G. Meister

Characterization of small RNA pathways in the nucleus of human somatic cells

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.

Project B5: S. Sprunck

Gametogenesis-related small non-coding RNAs and Argonaute proteins in Arabidopsis

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.

Project B6: T. Dresselhaus

Assembly of localized mRNPs and their function in regulating translation in Arabidopsis

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.

Project B9: M.Kretz

Long noncoding RNAs in tissue homeostasis and disease

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.

Project B10: W. Seufert

Mechanism and regulation of the eIF2-assembly factor Cdc123 and the link of cell cycle entry to mRNA translation

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.

Project B11: J. Medenbach

Regulation of alternative translation initiation

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.

Project B7: A. Bosserhoff (finished)

Functional implication of miRNA processing in malignant melanoma

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.


Z - Service Projects

Project Z1: A. Bruckmann / R. Deutzmann

Mass spectrometry of proteins

The aim of service project Z1 is to provide mass spectrometry based tools and techniques that are required for the various research projects of the CRC, and to implement new, state-of the art methods. The most important applications are qualitative and quantitative analysis of protein complexes and their regulation by phosphorylation. Quantification is done by SRM-methods and iTRAQ. Recently SILAC has been added and we plan to establish further methods, such as label free quantification, and determination of the topology of constituent proteins in a complex by cross-linking.

Project Z2a: J. Engelmann

Second generation sequencing and sequence bioinformatics

Project Z2a will provide high-throughput sequencing with the Illumina HiSeq 1000 platform and bioinformatic analyses of short read sequence data. In collaboration with projects A6, B4, B5, B6, B9 and B10, we will characterize gene expression, alternative splicing and in particular retained intron events, as well as small RNA expression and RNAs bound to specific proteins with functions in RNA processing and translation. Experimental protocols and computational algorithms will be customized to the needs of the individual research project.

Project Z2b: R. Merkl

Computational analysis of sequence and 3D-structure for a deeper understanding of chromatin structure and gene silencing pathways

To support projects A4 and B2, we will improve statistical tests that allow us to study alterations of chromatin density due to different experimental conditions. Moreover, we will adapt these tests to make possible a comparison of different regions of the chromosomes in order to predict structural changes related to multicopy genes. To support projects A6, B3, B4 and B6, we will characterize proteins based on multiple sequence alignments, homology models, and molecular dynamics simulations and try to detail their function.

Project Z3: Central Tasks


Graduate Research Academy "RNA Biology"

Graduate Research Academy "RNA Biology"

The majority of scientific personnel to be hired by the SFB “Ribosome formation: principles of RNP-biogenesis and control of their function” represent PhD students (24 students). All PhD students accepted by the faculty of Biology and Preclinical Medicine at the University of Regensburg are integrated in the Regensburg International Graduate School Life Sciences (RIGeL) that offers a structured PhD program to prepare students both for an academic and non-academic career. RIGeL is subdivided into four key activities, Cellular Biochemistry & Biophysics, Molecular & Chemical Ecology, Neurosciences and Biomedicine, and offers summer schools, lab courses and quality control instruments (www.biologie.uni-regensburg.de/RIGeL). The Graduate Research Academy "RNA Biology" shall represent a branch of excellence within the RIGeL activity Cellular Biochemistry & Biophysics. Its major goal is to provide a scientifically highly attractive doctorate accompanying education program for the PhD students of the SFB as well as for PhD students working in their doctoral research projects on topics related to the SFB, but which are funded by other sources. In summary the Graduate Research Academy will encourage creative and highly motivated graduate students to become an active and executive part of the Graduate Research Academy, to develop innovative but critical scientific thinking, responsibility, self-organisation and management of research projects at an early stage in their (scientific) career. The program of the Graduate Research Academy will be attractive to provide the SFB with the possibility to compete with other excellent graduate schools for the most ambitious and outstanding graduate students. Key elements of the Graduate Research Academy are (i) a competitive recruiting system allowing the Graduate Research Academy to invite and evaluate external students from national and international institutions for a period of up to three months, (ii) to provide each SFB PhD student with the possibility to participate at one international method course and (iii) to organize an own RNA biology course for external PhD students and (iv) to participate at generic skill courses shaped according to the demands of the Graduate Research Academy students. Moreover, the Graduate Research Academy will (v) provide support for the PhD students of the school to attend at least one international conference, (vi) to support PhD students to organize a lecture series on RNA biology and invite several outside speakers, (vii) to organize an annual summer academy and (viii) to organize one session including a round table discussion at the biennial international conference organized by the SFB. Finally, (ix) support will be provided for each SFB PhD student to spend short-time periods (one to three months) in a foreign top research lab to extent their scientific knowledge and methodological repertoire as well as to consolidate international collaborations and for making contacts for postdoc positions.


Associated Projects

AP 1: S. Ferreira-Cerca

Ribosome biogenesis in Archaea

Ribosomes are evolutionary conserved macromolecular machines involved in protein synthesis. Given their pivotal role in gene expression, cellular growth and proliferation, the detailed molecular understanding of ribosomes function and how they are biosynthesized are fundamental questions in biology.

Eubacterial and eukaryotic ribosome biogenesis have been extensively analyzed during the last decades. However our understanding of the archaeal ribosome biogenesis pathway remains relatively scarce. In order to fill this gap and shed light on this fundamental process, we aim to systematically analyze the archaeal in vivo ribosome biogenesis pathway.

To this end we are using a combination of functional genomic and proteomic approaches in order to discover the archaeal ribosome biogenesis pathway using the advantage of two genetically tractable organisms: the halophile euryarchaeon Haloferax volcannii and the thermo-acidophile crenoarchaeon Sulfolobus acidocaldarius as model organisms.

As a proof of principle we have already generated mutants of putative archaeal ribosome biogenesis factors conserved either in bacterial and/ or in eukaryotic cells and are currently analyzing their putative role(s) in the archaeal ribosome biogenesis pathway in vivo. Our preliminary work already suggest that the archaeal ribosome biogenesis assembly pathway is characterized by the usage of a mixture of bacterial- and eukaryotic-like features that are complemented by additional Archaea specific features.

Overall our work will help to better characterize the in vivo principles of RNP assembly in Archaea. Thereby providing among others; a conceptual framework for the understanding of key evolutionary conserved principles of ribosome assembly, and a molecular understanding of the gain of functions and/ specialization of the archaeal ribosome biogenesis pathway.

AP2: J. Perez-Fernandez

Structural and functional analysis of early acting ribosome biogenesis factors

Ribosome biogenesis is an energetic expensive process which requires the action of more than 150 ribosome biogenesis factors (hereafter called auxiliary factors) conserved between yeast and humans. Some auxiliary factors form protein subcomplexes (called UTP) which are also conserved through evolution. The inefficient production of fully active ribosomes seems to be the specific cause of a new set of diseases grouped as ribosomopathies. Among the mutations found in those diseases, some are located in genes coding for auxiliary factors but the molecular explanation of their effects remains elusive. In this regard, the advanced knowledge of the yeast ribosome biogenesis process is a clear advantage to understand this process in higher eukaryotes. Recently, we have described the in vitro reconstitution of yeast UTPs which is a valuable tool for functional studies and high resolution structural analysis. Moreover, the results suggest that assembly of UTPs occurs independently of ongoing ribosomal production. The aim of this project is to characterize: i) the in vivo formation of UTPs, ii) The structure and function of the different UTP components and iii) The molecular effects of mutations found on genes coding for UTP components.

AP 3: W. Hausner

Identification and characterisation of components associated with the archaeal transcription machinery

The archaeal transcription system is a more simplified version of the eukaryotic one and therefore provides a kind of facilitated access for studying some details of the transcription process. Our model organism is Pyrococcus furiosus, which belongs to the hyperthermophilic euryarchaeota. This organism is genetically tractable and in addition the archaeal RNA polymerase can be completely reconstituted from recombinant components. We have also established the ChIP-Seq technique for this organism and a genome size below 2 Mbp provides excellent conditions for global approaches.

The main focus of the prosposal will be on the identification of additional components which interact with the basic transcription machinery during initiation or elongation. One of these components is the replication protein A, a single stranded DNA binding protein. In cooperation with the group of Didier Flament (University of Brest, France) we could demonstrate that this protein stimulates archaeal transcription in vitro. Recent in vivo data also indicate a role of this protein in eukaryotic transcription. As the functional details of this protein are not known, we would like to analyse the role of RPA during transcription. We have also evidence that Rad25, a helicase which is in Eukarya a part of TFIIH, is in contact with the archaeal RNA polymerase. The function in archaeal transcription is not known. Furthermore, we are interested to investigate the composition of transcription complexes transcribing protein or ribosomal genes. Due to the prokaryotic nature it is most likely that the archaeal Spt4/Spt5 complex combines transcription with translation, but if Spt4/5 is also involved in rRNA transcription is still an open question. To answer this question we would like to establish a procedure which allows the isolation of gene-specific complexes from formaldehyde fixed cells.

AP 4: A. Németh

Nucleolar control of genome organization and function

The nucleolus is the site of ribosomal RNA transcription and ribosome biogenesis, and facilitates a number of other cellular processes like cell cycle progression, stress sensing and RNA modification. Although it is the largest compartment of the cell’s nucleus, its role in genome organization and function is poorly understood.

Nucleolus-associated chromosome domains were identified genome-wide in human cells (Németh et al., 2010, van Koningsbruggen et al., 2010) providing a snapshot of genome organization around the nucleolus. Yet, questions about the dynamics of the spatial arrangement of this part of the genome remained to be answered. Since the nuclear and nucleolar architecture get largely remodeled during cellular aging, we aim to systematically analyze nucleolus-associated chromatin reorganization in this fundamental biological process.

To this end, we will make use a combination of functional genomics and DNA-combing-based single-molecule analyses approaches, which were recently developed in our laboratory. These investigations will be complemented with conventional immunofluorescence analyses, 3D-immuno-FISH experiments, quantitative RT-qPCR and northern blot analyses to reveal the exact sequence of alterations in chromatin structure, DNA modification patterns, transcriptional activity and nuclear organization of specific nucleolus-associated chromosome domains during cellular senescence.

In the initial phase of the project nucleoli of IMR90 human lung embryonic fibroblasts were isolated from a young, proliferating cell population, as well as from senescent cells. Nucleolus-associated DNA was analyzed on high-resolution microarrays by comparative genomic hybridization. Additionally, RNA was extracted from the same cell populations and subjected to global gene expression analysis. The results indicate the role of the nucleolus-associated chromatin in genome reorganization and gene regulation during senescence. Furthermore, based on the results of the high-throughput analyses we could select specific chromosomal domains and chromatin regulator proteins, which we aim to analyze in detail in subsequent experiments. In summary, our investigations will provide novel insights into the spatial and transcriptional dynamics of the genome during cellular aging and shed light on the role of the nucleolus, the largest RNP-producing compartment of the nucleus, in nuclear organization and function.

AP5: C. Ziegler

Structure determination of the dsRNA-transporting channel SID-1 involved in RNAi in C. elegans by single particle cryo-EM

Single particle (SP) cryo-EM has achieved lately near-atomic resolution for asymmetric and dynamic protein assemblies. This breakthrough towards atomic resolution was accomplished by improvements in microscope design and fast digital cameras detecting electrons directly. In addition new image processing algorithm compensate for resolution-limiting movements of proteins caused by the electron beam. Recently also atomic structures of membrane proteins could be solved by SP cryo-EM, e.g., the TRPV1 ion channel in distinct conformations. We will use state-of-the-art SP cryo-EM to determine the structure of the highly conserved dsRNA-gated and dsRNA-transporting channel SID-1 from C. elegans. This channel is required for the import of RNAi triggers in non-neuronal tissues in C. elegans where RNAi is remarkably potent. Recent SAXS studies on the extracellular domain of SID-1 have revealed a tetrameric assembly with a central pore in the dimensions of dsRNA. We will express SID-1 as well as structural and functional mutants in the baculovirus system and reconstitute the channel in amphiphilic detergents or nanodiscs. Moreover, we will study its regulatory interaction with SID-2, a small single-pass membrane protein that is expressed in gut cells where it interacts most likely with ingested dsRNA. The mechanisms and regulation of dsRNA import mediated by SID-1/SID-2 and its role in RNAi in C. elegans will provide new perspectives for optimizing RNAi in other species. Furthermore, we will exploit x-ray crystallography and NMR to investigate structure and dynamics of the extracellular domains that are suggested to control the access of dsRNA. In addition, we will provide our knowledge on state-of-the-art cryo-EM also to other groups, e.g., we will in collaboration with Dr. Philip Milkereit (University Regensburg, Biochemistry III) apply SP cryo-EM to investigate the structure of different ribosome assembly intermediates from Saccharomyces cerevisiae in complex with their respective assembly factors.

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