@phdthesis{Huber2023, author = {Huber, Hannes}, title = {Biochemical and functional characterization of DHX30, an RNA helicase linked to neurodevelopmental disorder}, doi = {10.25972/OPUS-28050}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-280505}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2023}, abstract = {RNA helicases are key players in the regulation of gene expression. They act by remodeling local RNA secondary structures as well as RNA-protein interactions to enable the dynamic association of RNA binding proteins to their targets. The putative RNA helicase DHX30 is a member of the family of DEAH-box helicases with a putative role in the ATP-dependent unwinding of RNA secondary structures. Mutations in the DHX30 gene causes the autosomal dominant neuronal disease "Neurodevelopmental Disorder with severe Motor Impairment and Absent Language" (NEDMIAL;OMIM\#617804). In this thesis, a strategy was established that enabled the large-scale purification of enzymatically active DHX30. Through enzymatic studies performed in vitro, DHX30 was shown to act as an ATP-dependent 3' → 5' RNA helicase that catalyzes the unwinding of RNA:RNA and RNA:DNA substrates. Using recombinant DHX30, it could be shown that disease-causing missense mutations in the conserved helicase core caused the disruption of its ATPase and helicase activity. The protein interactome of DHX30 however, was unchanged indicating that the pathogenic missense-mutations do not cause misfolding of DHX30, but rather specifically affect its catalytic activity. DHX30 localizes predominantly in the cytoplasm where it forms a complex with ribosomes and polysomes. Using a cross-linking mass spectrometry approach, a direct interaction of the N-terminal double strand RNA binding domain of DHX30 with sites next to the ribosome's mRNA entry channel and the subunit interface was uncovered. RNA sequencing of DHX30 knockout cells revealed a strong de-regulation of mRNAs involved in neurogenesis and nervous system development, which is in line with the NEDMIAL disease phenotype. The knockdown of DHX30 results in a decreased 80S peak in polysome gradients, indicating that DHX30 has an effect on the translation machinery. Sequencing of the pool of active translating mRNAs revealed that upon DHX30 knockout mainly 5'TOP mRNAs are downregulated. These mRNAs are coding for proteins of the translational machinery and translation initiation factors. This study identified DHX30 as a factor of the translation machinery that selectively impacts the expression of a subset of proteins and provides insight on the etiology of NEDMIAL.}, language = {en} } @article{BenhalevyGuptaDananetal.2017, author = {Benhalevy, Daniel and Gupta, Sanjay K. and Danan, Charles H. and Ghosal, Suman and Sun, Hong-Wei and Kazemeier, Hinke G. and Paeschke, Katrin and Hafner, Markus and Juranek, Stefan A.}, title = {The Human CCHC-type Zinc Finger Nucleic Acid-Binding Protein Binds G-Rich Elements in Target mRNA Coding Sequences and Promotes Translation}, series = {Cell Reports}, volume = {18}, journal = {Cell Reports}, number = {12}, doi = {10.1016/j.celrep.2017.02.080}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171122}, pages = {2979-2990}, year = {2017}, abstract = {The CCHC-type zinc finger nucleic acid-binding protein (CNBP/ZNF9) is conserved in eukaryotes and is essential for embryonic development in mammals. It has been implicated in transcriptional, as well as post-transcriptional, gene regulation; however, its nucleic acid ligands and molecular function remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and function. We used photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to identify 8,420 CNBP binding sites on 4,178 mRNAs. CNBP preferentially bound G-rich elements in the target mRNA coding sequences, most of which were previously found to form G-quadruplex and other stable structures in vitro. Functional analyses, including RNA sequencing, ribosome profiling, and quantitative mass spectrometry, revealed that CNBP binding did not influence target mRNA abundance but rather increased their translational efficiency. Considering that CNBP binding prevented G-quadruplex structure formation in vitro, we hypothesize that CNBP is supporting translation by resolving stable structures on mRNAs.}, language = {en} } @article{PakniaChariStarketal.2016, author = {Paknia, Elham and Chari, Ashwin and Stark, Holger and Fischer, Utz}, title = {The Ribosome Cooperates with the Assembly Chaperone pICln to Initiate Formation of snRNPs}, series = {Cell Reports}, volume = {16}, journal = {Cell Reports}, number = {12}, doi = {10.1016/j.celrep.2016.08.047}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162420}, pages = {p3103-3112}, year = {2016}, abstract = {The formation of macromolecular complexes within the crowded environment of cells often requires aid from assembly chaperones. PRMT5 and SMN complexes mediate this task for the assembly of the common core of pre-mRNA processing small nuclear ribonucleoprotein particles (snRNPs). Core formation is initiated by the PRMT5-complex subunit pICln, which pre-arranges the core proteins into spatial positions occupied in the assembled snRNP. The SMN complex then accepts these pICln-bound proteins and unites them with small nuclear RNA (snRNA). Here, we have analyzed how newly synthesized snRNP proteins are channeled into the assembly pathway to evade mis-assembly. We show that they initially remain bound to the ribosome near the polypeptide exit tunnel and dissociate upon association with pICln. Coincident with its release activity, pICln ensures the formation of cognate heterooligomers and their chaperoned guidance into the assembly pathway. Our study identifies the ribosomal quality control hub as a site where chaperone-mediated assembly of macromolecular complexes can be initiated.}, language = {en} } @incollection{DasZografakisOeljeklausetal.2023, author = {Das, Hirakjyoti and Zografakis, Alexandros and Oeljeklaus, Silke and Warscheid, Bettina}, title = {Analysis of Yeast Peroxisomes via Spatial Proteomics}, series = {Peroxisomes}, booktitle = {Peroxisomes}, edition = {accepted version}, publisher = {Springer}, doi = {10.1007/978-1-0716-3048-8_2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-327532}, publisher = {Universit{\"a}t W{\"u}rzburg}, pages = {13-31}, year = {2023}, abstract = {Peroxisomes are ubiquitous organelles with essential functions in numerous cellular processes such as lipid metabolism, detoxification of reactive oxygen species and signaling. Knowledge of the peroxisomal proteome including multi-localized proteins and, most importantly, changes of its composition induced by altering cellular conditions or impaired peroxisome biogenesis and function is of paramount importance for a holistic view on peroxisomes and their diverse functions in a cellular context. In this chapter, we provide a spatial proteomics protocol specifically tailored to the analysis of the peroxisomal proteome of baker's yeast that enables the definition of the peroxisomal proteome under distinct conditions and to monitor dynamic changes of the proteome including the relocation of individual proteins to a different cellular compartment. The protocol comprises subcellular fractionation by differential centrifugation followed by Nycodenz density gradient centrifugation of a crude peroxisomal fraction, quantitative mass spectrometric measurements of subcellular and density gradient fractions and advanced computational data analysis, resulting in the establishment of organellar maps on a global scale.}, language = {en} } @article{SendellPriceTulenkoPetterssonetal.2023, author = {Sendell-Price, Ashley T. and Tulenko, Frank J. and Pettersson, Mats and Kang, Du and Montandon, Margo and Winkler, Sylke and Kulb, Kathleen and Naylor, Gavin P. and Phillippy, Adam and Fedrigo, Olivier and Mountcastle, Jacquelyn and Balacco, Jennifer R. and Dutra, Amalia and Dale, Rebecca E. and Haase, Bettina and Jarvis, Erich D. and Myers, Gene and Burgess, Shawn M. and Currie, Peter D. and Andersson, Leif and Schartl, Manfred}, title = {Low mutation rate in epaulette sharks is consistent with a slow rate of evolution in sharks}, series = {Nature Communications}, volume = {14}, journal = {Nature Communications}, doi = {10.1038/s41467-023-42238-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-357827}, year = {2023}, abstract = {Sharks occupy diverse ecological niches and play critical roles in marine ecosystems, often acting as apex predators. They are considered a slow-evolving lineage and have been suggested to exhibit exceptionally low cancer rates. These two features could be explained by a low nuclear mutation rate. Here, we provide a direct estimate of the nuclear mutation rate in the epaulette shark (Hemiscyllium ocellatum). We generate a high-quality reference genome, and resequence the whole genomes of parents and nine offspring to detect de novo mutations. Using stringent criteria, we estimate a mutation rate of 7×10\(^{-10}\) per base pair, per generation. This represents one of the lowest directly estimated mutation rates for any vertebrate clade, indicating that this basal vertebrate group is indeed a slowly evolving lineage whose ability to restore genetic diversity following a sustained population bottleneck may be hampered by a low mutation rate.}, language = {en} } @article{KoernerMeyerMarincolaetal.2023, author = {K{\"o}rner, Maria and Meyer, Susanne R. and Marincola, Gabriella and Kern, Maximilian J. and Grimm, Clemens and Schuelein-Voelk, Christina and Fischer, Utz and Hofmann, Kay and Buchberger, Alexander}, title = {The FAM104 proteins VCF1/2 promote the nuclear localization of p97/VCP}, series = {eLife}, volume = {12}, journal = {eLife}, doi = {10.7554/eLife.92409}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350222}, year = {2023}, abstract = {The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97-cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.}, language = {en} } @article{FetivaLissGertzmannetal.2023, author = {Fetiva, Maria Camila and Liss, Franziska and Gertzmann, D{\"o}rthe and Thomas, Julius and Gantert, Benedikt and Vogl, Magdalena and Sira, Nataliia and Weinstock, Grit and Kneitz, Susanne and Ade, Carsten P. and Gaubatz, Stefan}, title = {Oncogenic YAP mediates changes in chromatin accessibility and activity that drive cell cycle gene expression and cell migration}, series = {Nucleic Acids Research}, volume = {51}, journal = {Nucleic Acids Research}, number = {9}, doi = {10.1093/nar/gkad107}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-350218}, pages = {4266-4283}, year = {2023}, abstract = {YAP, the key protein effector of the Hippo pathway, is a transcriptional co-activator that controls the expression of cell cycle genes, promotes cell growth and proliferation and regulates organ size. YAP modulates gene transcription by binding to distal enhancers, but the mechanisms of gene regulation by YAP-bound enhancers remain poorly understood. Here we show that constitutive active YAP5SA leads to widespread changes in chromatin accessibility in untransformed MCF10A cells. Newly accessible regions include YAP-bound enhancers that mediate activation of cycle genes regulated by the Myb-MuvB (MMB) complex. By CRISPR-interference we identify a role for YAP-bound enhancers in phosphorylation of Pol II at Ser5 at MMB-regulated promoters, extending previously published studies that suggested YAP primarily regulates the pause-release step and transcriptional elongation. YAP5SA also leads to less accessible 'closed' chromatin regions, which are not directly YAP-bound but which contain binding motifs for the p53 family of transcription factors. Diminished accessibility at these regions is, at least in part, a consequence of reduced expression and chromatin-binding of the p53 family member ΔNp63 resulting in downregulation of ΔNp63-target genes and promoting YAP-mediated cell migration. In summary, our studies uncover changes in chromatin accessibility and activity that contribute to the oncogenic activities of YAP.}, language = {en} } @unpublished{HennigPrustyKauferetal.2022, author = {Hennig, Thomas and Prusty, Archana B. and Kaufer, Benedikt and Whisnant, Adam W. and Lodha, Manivel and Enders, Antje and Thomas, Julius and Kasimir, Francesca and Grothey, Arnhild and Herb, Stefanie and J{\"u}rges, Christopher and Meister, Gunter and Erhard, Florian and D{\"o}lken, Lars and Prusty, Bhupesh K.}, title = {Selective inhibition of miRNA 1 processing by a herpesvirus encoded miRNA}, edition = {accepted version}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-267862}, year = {2022}, abstract = {Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation thereof 1,2. A long appreciated, yet elusively defined relationship exists between the lytic-latent switch and viral non-coding RNAs 3,4. Here, we identify miRNA-mediated inhibition of miRNA processing as a thus far unknown cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defense and drive the lytic-latent switch. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective pri-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30/p53/Drp1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily drugable master regulator of the herpesvirus lytic-latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 provides exciting therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.}, language = {en} } @article{DenkSchmidtSchurretal.2021, author = {Denk, S. and Schmidt, S. and Schurr, Y. and Schwarz, G. and Schote, F. and Diefenbacher, M. and Armendariz, C. and Dejure, F. and Eilers, M. and Wiegering, Armin}, title = {CIP2A regulates MYC translation (via its 5′UTR) in colorectal cancer}, series = {International Journal of Colorectal Disease}, volume = {36}, journal = {International Journal of Colorectal Disease}, number = {5}, doi = {10.1007/s00384-020-03772-y}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-280092}, pages = {911-918}, year = {2021}, abstract = {Background Deregulated expression of MYC is a driver of colorectal carcinogenesis, suggesting that decreasing MYC expression may have significant therapeutic value. CIP2A is an oncogenic factor that regulates MYC expression. CIP2A is overexpressed in colorectal cancer (CRC), and its expression levels are an independent marker for long-term outcome of CRC. Previous studies suggested that CIP2A controls MYC protein expression on a post-transcriptional level. Methods To determine the mechanism by which CIP2A regulates MYC in CRC, we dissected MYC translation and stability dependent on CIP2A in CRC cell lines. Results Knockdown of CIP2A reduced MYC protein levels without influencing MYC stability in CRC cell lines. Interfering with proteasomal degradation of MYC by usage of FBXW7-deficient cells or treatment with the proteasome inhibitor MG132 did not rescue the effect of CIP2A depletion on MYC protein levels. Whereas CIP2A knockdown had marginal influence on global protein synthesis, we could demonstrate that, by using different reporter constructs and cells expressing MYC mRNA with or without flanking UTR, CIP2A regulates MYC translation. This interaction is mainly conducted by the MYC 5′UTR. Conclusions Thus, instead of targeting MYC protein stability as reported for other tissue types before, CIP2A specifically regulates MYC mRNA translation in CRC but has only slight effects on global mRNA translation. In conclusion, we propose as novel mechanism that CIP2A regulates MYC on a translational level rather than affecting MYC protein stability in CRC.}, language = {en} }