Publications by authors named "Utz Fischer"

75 Publications

Identification and structural analysis of the Schizosaccharomyces pombe SMN complex.

Nucleic Acids Res 2021 Mar 23. Epub 2021 Mar 23.

Department of Biochemistry, Biocenter, University of Würzburg, Würzburg 97074, Germany.

The macromolecular SMN complex facilitates the formation of Sm-class ribonucleoproteins involved in mRNA processing (UsnRNPs). While biochemical studies have revealed key activities of the SMN complex, its structural investigation is lagging behind. Here we report on the identification and structural determination of the SMN complex from the lower eukaryote Schizosaccharomyces pombe, consisting of SMN, Gemin2, 6, 7, 8 and Sm proteins. The core of the SMN complex is formed by several copies of SMN tethered through its C-terminal alpha-helices arranged with alternating polarity. This creates a central platform onto which Gemin8 binds and recruits Gemins 6 and 7. The N-terminal parts of the SMN molecules extrude via flexible linkers from the core and enable binding of Gemin2 and Sm proteins. Our data identify the SMN complex as a multivalent hub where Sm proteins are collected in its periphery to allow their joining with UsnRNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkab158DOI Listing
March 2021

Interaction of 7SK with the Smn complex modulates snRNP production.

Nat Commun 2021 02 24;12(1):1278. Epub 2021 Feb 24.

Institute of Clinical Neurobiology, University Hospital Wuerzburg, Wuerzburg, Germany.

Gene expression requires tight coordination of the molecular machineries that mediate transcription and splicing. While the interplay between transcription kinetics and spliceosome fidelity has been investigated before, less is known about mechanisms regulating the assembly of the spliceosomal machinery in response to transcription changes. Here, we report an association of the Smn complex, which mediates spliceosomal snRNP biogenesis, with the 7SK complex involved in transcriptional regulation. We found that Smn interacts with the 7SK core components Larp7 and Mepce and specifically associates with 7SK subcomplexes containing hnRNP R. The association between Smn and 7SK complexes is enhanced upon transcriptional inhibition leading to reduced production of snRNPs. Taken together, our findings reveal a functional association of Smn and 7SK complexes that is governed by global changes in transcription. Thus, in addition to its canonical nuclear role in transcriptional regulation, 7SK has cytosolic functions in fine-tuning spliceosome production according to transcriptional demand.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-21529-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904863PMC
February 2021

The SARS-CoV-2 RNA-protein interactome in infected human cells.

Nat Microbiol 2021 03 21;6(3):339-353. Epub 2020 Dec 21.

Helmholtz Institute for RNA-based Infection Research, Helmholtz-Center for Infection Research, Würzburg, Germany.

Characterizing the interactions that SARS-CoV-2 viral RNAs make with host cell proteins during infection can improve our understanding of viral RNA functions and the host innate immune response. Using RNA antisense purification and mass spectrometry, we identified up to 104 human proteins that directly and specifically bind to SARS-CoV-2 RNAs in infected human cells. We integrated the SARS-CoV-2 RNA interactome with changes in proteome abundance induced by viral infection and linked interactome proteins to cellular pathways relevant to SARS-CoV-2 infections. We demonstrated by genetic perturbation that cellular nucleic acid-binding protein (CNBP) and La-related protein 1 (LARP1), two of the most strongly enriched viral RNA binders, restrict SARS-CoV-2 replication in infected cells and provide a global map of their direct RNA contact sites. Pharmacological inhibition of three other RNA interactome members, PPIA, ATP1A1, and the ARP2/3 complex, reduced viral replication in two human cell lines. The identification of host dependency factors and defence strategies as presented in this work will improve the design of targeted therapeutics against SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41564-020-00846-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7906908PMC
March 2021

Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1.

Biomolecules 2020 06 6;10(6). Epub 2020 Jun 6.

Department of Biochemistry, Theodor Boveri Institute, Biocenter of the University of Würzburg, 97070 Würzburg, Germany.

Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/biom10060872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356810PMC
June 2020

Stabilize and connect: the role of LARP7 in nuclear non-coding RNA metabolism.

RNA Biol 2021 Feb 3;18(2):290-303. Epub 2020 Jun 3.

Department of Biochemistry, Theodor Boveri-Institute, University of Würzburg, Würzburg, Germany.

La and La-related proteins (LARPs) are characterized by a common RNA interaction platform termed the La module. This structural hallmark allows LARPs to pervade various aspects of RNA biology. The metazoan LARP7 protein binds to the 7SK RNA as part of a 7SK small nuclear ribonucleoprotein (7SK snRNP), which inhibits the transcriptional activity of RNA polymerase II (Pol II). Additionally, recent findings revealed unanticipated roles of LARP7 in the assembly of other RNPs, as well as in the modification, processing and cellular transport of RNA molecules. Reduced levels of functional LARP7 have been linked to cancer and Alazami syndrome, two seemingly unrelated human diseases characterized either by hyperproliferation or growth retardation. Here, we review the intricate regulatory networks centered on LARP7 and assess how malfunction of these networks may relate to the etiology of LARP7-linked diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/15476286.2020.1767952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928028PMC
February 2021

The Alazami Syndrome-Associated Protein LARP7 Guides U6 Small Nuclear RNA Modification and Contributes to Splicing Robustness.

Mol Cell 2020 03 3;77(5):1014-1031.e13. Epub 2020 Feb 3.

Biochemistry Center Regensburg (BZR), Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany. Electronic address:

The La-related protein 7 (LARP7) forms a complex with the nuclear 7SK RNA to regulate RNA polymerase II transcription. It has been implicated in cancer and the Alazami syndrome, a severe developmental disorder. Here, we report a so far unknown role of this protein in RNA modification. We show that LARP7 physically connects the spliceosomal U6 small nuclear RNA (snRNA) with a distinct subset of box C/D small nucleolar RNAs (snoRNAs) guiding U6 2'-O-methylation. Consistently, these modifications are severely compromised in the absence of LARP7. Although general splicing remains largely unaffected, transcriptome-wide analysis revealed perturbations in alternative splicing in LARP7-depleted cells. Importantly, we identified defects in 2'-O-methylation of the U6 snRNA in Alazami syndrome siblings carrying a LARP7 mutation. Our data identify LARP7 as a bridging factor for snoRNA-guided modification of the U6 snRNA and suggest that alterations in splicing fidelity contribute to the etiology of the Alazami syndrome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.01.001DOI Listing
March 2020

LARP7-Mediated U6 snRNA Modification Ensures Splicing Fidelity and Spermatogenesis in Mice.

Mol Cell 2020 03 3;77(5):999-1013.e6. Epub 2020 Feb 3.

State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences - University of Chinese Academy of Sciences, Shanghai 200031, China; School of Life Science and Technology, Shanghai Tech University, Shanghai 201210, China. Electronic address:

U6 snRNA, as an essential component of the catalytic core of the pre-mRNA processing spliceosome, is heavily modified post-transcriptionally, with 2'-O-methylation being most common. The role of these modifications in pre-mRNA splicing as well as their physiological function in mammals have remained largely unclear. Here we report that the La-related protein LARP7 functions as a critical cofactor for 2'-O-methylation of U6 in mouse male germ cells. Mechanistically, LARP7 promotes U6 loading onto box C/D snoRNP, facilitating U6 2'-O-methylation by box C/D snoRNP. Importantly, ablation of LARP7 in the male germline causes defective U6 2'-O-methylation, massive alterations in pre-mRNA splicing, and spermatogenic failure in mice, which can be rescued by ectopic expression of wild-type LARP7 but not an U6-loading-deficient mutant LARP7. Our data uncover a novel role of LARP7 in regulating U6 2'-O-methylation and demonstrate the functional requirement of such modification for splicing fidelity and spermatogenesis in mice.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.01.002DOI Listing
March 2020

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes.

Cell 2019 12;179(7):1537-1550.e19

Department of Biochemistry and Cancer Therapy Research Center (CTRC), Theodor Boveri-Institute, University of Würzburg, Am Hubland, 97074 Würzburg, Germany; Genelux Corporation, 3030 Bunker Hill Street, San Diego, CA 92109, USA; Helmholtz Institute for RNA-based Infection Research (HIRI) and Helmholtz Centre for Infection Research (HZI), 97080 Würzburg, Germany. Electronic address:

Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNA. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen et al., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.11.024DOI Listing
December 2019

Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes.

Cell 2019 12;179(7):1525-1536.e12

Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany. Electronic address:

Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate mG-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of core and complete vRNAP complexes of the prototypic Vaccinia poxvirus (Grimm et al., 2019; in this issue of Cell). Here, we present the cryo-electron microscopy (cryo-EM) structures of Vaccinia vRNAP in the form of a transcribing elongation complex and in the form of a co-transcriptional capping complex that contains the viral capping enzyme (CE). The trifunctional CE forms two mobile modules that bind the polymerase surface around the RNA exit tunnel. RNA extends from the vRNAP active site through this tunnel and into the active site of the CE triphosphatase. Structural comparisons suggest that growing RNA triggers large-scale rearrangements on the surface of the transcription machinery during the transition from transcription initiation to RNA capping and elongation. Our structures unravel the basis for synthesis and co-transcriptional modification of poxvirus RNA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.11.023DOI Listing
December 2019

A missense mutation in SNRPE linked to non-syndromal microcephaly interferes with U snRNP assembly and pre-mRNA splicing.

PLoS Genet 2019 10 31;15(10):e1008460. Epub 2019 Oct 31.

Department of Biology, Southern University of Science and Technology (SUSTech), Shenzhen, China.

Malfunction of pre-mRNA processing factors are linked to several human diseases including cancer and neurodegeneration. Here we report the identification of a de novo heterozygous missense mutation in the SNRPE gene (c.65T>C (p.Phe22Ser)) in a patient with non-syndromal primary (congenital) microcephaly and intellectual disability. SNRPE encodes SmE, a basal component of pre-mRNA processing U snRNPs. We show that the microcephaly-linked SmE variant is unable to interact with the SMN complex and as a consequence fails to assemble into U snRNPs. This results in widespread mRNA splicing alterations in fibroblast cells derived from this patient. Similar alterations were observed in HEK293 cells upon SmE depletion that could be rescued by the expression of wild type but not mutant SmE. Importantly, the depletion of SmE in zebrafish causes aberrant mRNA splicing alterations and reduced brain size, reminiscent of the patient microcephaly phenotype. We identify the EMX2 mRNA, which encodes a protein required for proper brain development, as a major mis-spliced down stream target. Together, our study links defects in the SNRPE gene to microcephaly and suggests that alterations of cellular splicing of specific mRNAs such as EMX2 results in the neurological phenotype of the disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1008460DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850558PMC
October 2019

Gene Knockdown in Zebrafish (Danio rerio) as a Tool to Model Photoreceptor Diseases.

Methods Mol Biol 2019 ;1834:209-219

Department of Biochemistry, Biocentre, University of Wuerzburg, Würzburg, Germany.

Disturbances in the general mRNA metabolism have been recognized as a major defect in a growing number of hereditary human diseases. One prominent example of this disease group is retinitis pigmentosa (RP), characterized by selective loss of photoreceptor cells. RP can be caused by dominant mutations in key factors of the pre-mRNA processing spliceosome. In these cases, the complex events leading to the RP phenotype can only insufficiently be analyzed in animal knockout models due to the essential functions of splice factors. Furthermore knockout animals frequently miss the specific phenotypes caused by knockdown of the respective genes. Here we introduce the zebrafish Danio rerio as a valuable vertebrate model system to study RP and related diseases in knockdown case scenarios.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-4939-8669-9_15DOI Listing
April 2019

Impaired Local Translation of β-actin mRNA in Ighmbp2-Deficient Motoneurons: Implications for Spinal Muscular Atrophy with respiratory Distress (SMARD1).

Neuroscience 2018 08 19;386:24-40. Epub 2018 Jun 19.

Institute of Clinical Neurobiology, University Hospital Würzburg, Würzburg, Germany. Electronic address:

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a fatal motoneuron disorder in children with unknown etiology. The disease is caused by mutations in the IGHMBP2 gene, encoding a Super Family 1 (SF1)-type RNA/DNA helicase. IGHMBP2 is a cytosolic protein that binds to ribosomes and polysomes, suggesting a role in mRNA metabolism. Here we performed morphological and functional analyses of isolated immunoglobulin μ-binding protein 2 (Ighmbp2)-deficient motoneurons to address the question whether the SMARD1 phenotype results from de-regulation of protein biosynthesis. Ighmbp2-deficient motoneurons exhibited only moderate morphological aberrations such as a slight increase of axonal branches. Consistent with the rather mild phenotypic aberrations, RNA sequencing of Ighmbp2-deficient motoneurons revealed only minor transcriptome alterations compared to controls. Likewise, we did not detect any global changes in protein synthesis using pulsed SILAC (Stable Isotope Labeling by Amino acids in Cell culture), FUNCAT (FlUorescent Non-Canonical Amino acid Tagging) and SUnSET (SUrface SEnsing of Translation) approaches. However, we observed reduced β-actin protein levels at the growth cone of Ighmbp2-deficient motoneurons which was accompanied by reduced level of IMP1/ZBP1, a known interactor of β-actin mRNA. Fluorescence Recovery after Photobleaching (FRAP) studies revealed translational down-regulation of an eGFP--β-actin 3'UTR mRNA in growth cones. Local translational regulation of β-actin mRNA was dependent on the 3' UTR but independent of direct Ighmbp2-binding to β-actin mRNA. Taken together, our data indicate that Ighmbp2 deficiency results in local but modest disruption of protein biosynthesis which might partially contribute to the motoneuron defects seen in SMARD1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuroscience.2018.06.019DOI Listing
August 2018

UsnRNP biogenesis: mechanisms and regulation.

Chromosoma 2017 10 1;126(5):577-593. Epub 2017 Aug 1.

Department of Biochemistry, University of Würzburg, Biozentrum, Am Hubland, D-97074, Würzburg, Germany.

Macromolecular complexes composed of proteins or proteins and nucleic acids rather than individual macromolecules mediate many cellular activities. Maintenance of these activities is essential for cell viability and requires the coordinated production of the individual complex components as well as their faithful incorporation into functional entities. Failure of complex assembly may have fatal consequences and can cause severe diseases. While many macromolecular complexes can form spontaneously in vitro, they often require aid from assembly factors including assembly chaperones in the crowded cellular environment. The assembly of RNA protein complexes implicated in the maturation of pre-mRNAs (termed UsnRNPs) has proven to be a paradigm to understand the action of assembly factors and chaperones. UsnRNPs are assembled by factors united in protein arginine methyltransferase 5 (PRMT5)- and survival motor neuron (SMN)-complexes, which act sequentially in the UsnRNP production line. While the PRMT5-complex pre-arranges specific sets of proteins into stable intermediates, the SMN complex displaces assembly factors from these intermediates and unites them with UsnRNA to form the assembled RNP. Despite advanced mechanistic understanding of UsnRNP assembly, our knowledge of regulatory features of this essential and ubiquitous cellular function remains remarkably incomplete. One may argue that the process operates as a default biosynthesis pathway and does not require sophisticated regulatory cues. Simple theoretical considerations and a number of experimental data, however, indicate that regulation of UsnRNP assembly most likely happens at multiple levels. This review will not only summarize how individual components of this assembly line act mechanistically but also why, how, and when the UsnRNP workflow might be regulated by means of posttranslational modification in response to cellular signaling cues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00412-017-0637-6DOI Listing
October 2017

Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation.

J Cell Biol 2017 08 21;216(8):2391-2407. Epub 2017 Jun 21.

Department of Biochemistry, Biocenter, University of Würzburg, Am Hubland, Würzburg, Germany

Specialized assembly factors facilitate the formation of many macromolecular complexes in vivo. The formation of Sm core structures of spliceosomal U-rich small nuclear ribonucleoprotein particles (UsnRNPs) requires assembly factors united in protein arginine methyltransferase 5 (PRMT5) and survival motor neuron (SMN) complexes. We demonstrate that perturbations of this assembly machinery trigger complex cellular responses that prevent aggregation of unassembled Sm proteins. Inactivation of the SMN complex results in the initial tailback of Sm proteins on the PRMT5 complex, followed by down-regulation of their encoding mRNAs. In contrast, reduction of pICln, a PRMT5 complex subunit, leads to the retention of newly synthesized Sm proteins on ribosomes and their subsequent lysosomal degradation. Overexpression of Sm proteins under these conditions results in a surplus of Sm proteins over pICln, promoting their aggregation. Our studies identify an elaborate safeguarding system that prevents individual Sm proteins from aggregating, contributing to cellular UsnRNP homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201611108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551706PMC
August 2017

Deciphering the mRNP Code: RNA-Bound Determinants of Post-Transcriptional Gene Regulation.

Trends Biochem Sci 2017 05 6;42(5):369-382. Epub 2017 Mar 6.

Theodor-Boveri Institute, University of Wuerzburg, Germany. Electronic address:

Eukaryotic cells determine the final protein output of their genetic program not only by controlling transcription but also by regulating the localization, translation and turnover rates of their mRNAs. Ultimately, the fate of any given mRNA is determined by the ensemble of all associated RNA-binding proteins (RBPs), non-coding RNAs and metabolites collectively known as the messenger ribonucleoprotein particle (mRNP). Although many mRNA-associated factors have been identified over the past years, little is known about the composition of individual mRNPs and the cooperation of their constituents. In this review we discuss recent progress that has been made on how this 'mRNP code' is established on individual transcripts and how it is interpreted during gene expression in eukaryotic cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tibs.2017.02.004DOI Listing
May 2017

Molding Photonic Boxes into Fluorescent Emitters by Direct Laser Writing.

Adv Mater 2017 Apr 16;29(16). Epub 2017 Feb 16.

Technische Physik, Universität Würzburg, Am Hubland, 97074, Würzburg, Germany.

Direct laser writing of photonic boxes into active layers of biologically produced recombinant fluorescent protein in optical microcavities is demonstrated. Irradiation with laser light above the photobleaching threshold induces photonic confinement potentials on the order of 40 meV. The technique provides high spatial selectivity and enables room-temperature lasing in protein rings, and circular and elliptical pillars with customized beam shapes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adma.201605236DOI Listing
April 2017

The right pick: structural basis of snRNA selection by Gemin5.

Genes Dev 2016 11;30(21):2341-2344

Department of Biochemistry, University of Würzburg, D-97074 Würzburg, Germany.

Macromolecular complexes, rather than individual biopolymers, perform many cellular activities. Faithful assembly of these complexes in vivo is therefore a vital challenge of all cells, and its failure can have fatal consequences. To form functional complexes, cells use elaborate measures to select the "right" components and combine them into working entities. How assembly is achieved at the molecular level is unclear in many cases. Three groups (Jin and colleagues, pp. 2391-2403; Xu and colleagues, pp. 2376-2390; and Tang and colleagues in Cell Research) have now provided insights into how an assembly factor specifically recognizes substrate RNA molecules and enables their usage for assembly of Sm-class uridine-rich small nuclear RNA-protein complexes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.293084.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131775PMC
November 2016

The Ribosome Cooperates with the Assembly Chaperone pICln to Initiate Formation of snRNPs.

Cell Rep 2016 09;16(12):3103-3112

Department of Biochemistry, University of Wuerzburg, 97074 Wuerzburg, Germany; Department of Radiation Medicine and Applied Sciences, University of California at San Diego, San Diego, CA 92037, USA. Electronic address:

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2016.08.047DOI Listing
September 2016

A critical examination of the recently reported crystal structures of the human SMN protein.

Hum Mol Genet 2016 08;25(21):4717–4725

Departement of Biochemistry, Biocenter of the University, University of Wuerzburg, Würzburg, Germany.

A recent publication by Seng et al. in this journal reports the crystallographic structure of refolded, full-length SMN protein and two disease-relevant derivatives thereof. Here, we would like to suggest that at least two of the structures reported in that study are incorrect. We present evidence that one of the associated crystallographic datasets is derived from a crystal of the bacterial Sm-like protein Hfq and that a second dataset is derived from a crystal of the bacterial Gab protein. Both proteins are frequent contaminants of bacterially overexpressed proteins which might have been co-purified during metal affinity chromatography. A third structure presented in the Seng et al. paper cannot be examined further because neither the atomic coordinates, nor the diffraction intensities were made publicly available. The Tudor domain protein SMN has been shown to be a component of the SMN complex, which mediates the assembly of RNA-protein complexes of uridine-rich small nuclear ribonucleoproteins (UsnRNPs). Importantly, this activity is reduced in SMA patients, raising the possibility that the aetiology of SMA is linked to RNA metabolism. Structural studies on diverse components of the SMN complex, including fragments of SMN itself have contributed greatly to our understanding of the cellular UsnRNP assembly machinery. Yet full-length SMN has so far evaded structural elucidation. The Seng et al. study claimed to have closed this gap, but based on the results presented here, the only conclusion that can be drawn is that the Seng et al. study is largely invalid and should be retracted from the literature.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/hmg/ddw298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418738PMC
August 2016

Accumulated common variants in the broader fragile X gene family modulate autistic phenotypes.

EMBO Mol Med 2015 Dec;7(12):1565-79

Clinical Neuroscience, Max Planck Institute of Experimental Medicine, Göttingen, Germany DFG Research Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen, Germany

Fragile X syndrome (FXS) is mostly caused by a CGG triplet expansion in the fragile X mental retardation 1 gene (FMR1). Up to 60% of affected males fulfill criteria for autism spectrum disorder (ASD), making FXS the most frequent monogenetic cause of syndromic ASD. It is unknown, however, whether normal variants (independent of mutations) in the fragile X gene family (FMR1, FXR1, FXR2) and in FMR2 modulate autistic features. Here, we report an accumulation model of 8 SNPs in these genes, associated with autistic traits in a discovery sample of male patients with schizophrenia (N = 692) and three independent replicate samples: patients with schizophrenia (N = 626), patients with other psychiatric diagnoses (N = 111) and a general population sample (N = 2005). For first mechanistic insight, we contrasted microRNA expression in peripheral blood mononuclear cells of selected extreme group subjects with high- versus low-risk constellation regarding the accumulation model. Thereby, the brain-expressed miR-181 species emerged as potential "umbrella regulator", with several seed matches across the fragile X gene family and FMR2. To conclude, normal variation in these genes contributes to the continuum of autistic phenotypes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/emmm.201505696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4693501PMC
December 2015

Crystallizing the 6S and 8S spliceosomal assembly intermediates: a complex project.

Acta Crystallogr D Biol Crystallogr 2015 Oct 26;71(Pt 10):2040-53. Epub 2015 Sep 26.

Department of Biochemistry, Theodor Boveri Institute, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.

The small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/6 and U5 are major constituents of the pre-mRNA processing spliceosome. They contain a common RNP core that is formed by the ordered binding of Sm proteins onto the single-stranded Sm site of the snRNA. Although spontaneous in vitro, assembly of the Sm core requires assistance from the PRMT5 and SMN complexes in vivo. To gain insight into the key steps of the assembly process, the crystal structures of two assembly intermediates of U snRNPs termed the 6S and 8S complexes have recently been reported. These multimeric protein complexes could only be crystallized after the application of various rescue strategies. The developed strategy leading to the crystallization and solution of the 8S crystal structure was subsequently used to guide a combination of rational crystal-contact optimization with surface-entropy reduction of crystals of the related 6S complex. Conversely, the resulting high-resolution 6S crystal structure was used during the restrained refinement of the 8S crystal structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1399004715014832DOI Listing
October 2015

Drug-Encoded Biomarkers for Monitoring Biological Therapies.

PLoS One 2015 8;10(9):e0137573. Epub 2015 Sep 8.

Department of Biochemistry, Biocenter, University of Würzburg, Würzburg, Germany; Department of Radiation Oncology, Moores Cancer Center, University of California San Diego, San Diego, CA, United States of America.

Blood tests are necessary, easy-to-perform and low-cost alternatives for monitoring of oncolytic virotherapy and other biological therapies in translational research. Here we assessed three candidate proteins with the potential to be used as biomarkers in biological fluids: two glucuronidases from E. coli (GusA) and Staphylococcus sp. RLH1 (GusPlus), and the luciferase from Gaussia princeps (GLuc). The three genes encoding these proteins were inserted individually into vaccinia virus GLV-1h68 genome under the control of an identical promoter. The three resulting recombinant viruses were used to infect tumor cells in cultures and human tumor xenografts in nude mice. In contrast to the actively secreted GLuc, the cytoplasmic glucuronidases GusA and GusPlus were released into the supernatants only as a result of virus-mediated oncolysis. GusPlus resulted in the most sensitive detection of enzyme activity under controlled assay conditions in samples containing as little as 1 pg/ml of GusPlus, followed by GusA (25 pg/ml) and GLuc (≥375 pg/ml). Unexpectedly, even though GusA had a lower specific activity compared to GusPlus, the substrate conversion in the serum of tumor-bearing mice injected with the GusA-encoding virus strains was substantially higher than that of GusPlus. This was attributed to a 3.2 fold and 16.2 fold longer half-life of GusA in the blood stream compared to GusPlus and GLuc respectively, thus a more sensitive monitor of virus replication than the other two enzymes. Due to the good correlation between enzymatic activity of expressed marker gene and virus titer, we conclude that the amount of the biomarker protein in the body fluid semiquantitatively represents the amount of virus in the infected tumors which was confirmed by low light imaging. We found GusA to be the most reliable biomarker for monitoring oncolytic virotherapy among the three tested markers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137573PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562523PMC
June 2016

ProteoPlex: stability optimization of macromolecular complexes by sparse-matrix screening of chemical space.

Nat Methods 2015 Sep 3;12(9):859-65. Epub 2015 Aug 3.

Research Group of 3D Electron Cryomicroscopy, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules with a variety of functions. Structure determination of these complexes in a purified state is often tedious owing to their compositional complexity and the associated relative structural instability. To improve the stability of macromolecular complexes in vitro, we present a generic method that optimizes the stability, homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behavior in the presence of various buffers and small molecules. The method includes the automated analysis of thermal unfolding curves based on a biophysical unfolding model for complexes. We found that under stabilizing conditions, even large multicomponent complexes reveal an almost ideal two-state unfolding behavior. We envisage an improved biochemical understanding of purified macromolecules as well as a substantial boost in successful macromolecular complex structure determination by both X-ray crystallography and cryo-electron microscopy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.3493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5136620PMC
September 2015

Reconstitution of the human U snRNP assembly machinery reveals stepwise Sm protein organization.

EMBO J 2015 Jul 11;34(14):1925-41. Epub 2015 Jun 11.

Department of Biochemistry, Biocenter, University of Würzburg, Würzburg, Germany Department of Radiation Medicine and Applied Sciences, University of California, San Diego, San Diego, CA, USA

The assembly of spliceosomal U snRNPs depends on the coordinated action of PRMT5 and SMN complexes in vivo. These trans-acting factors enable the faithful delivery of seven Sm proteins onto snRNA and the formation of the common core of snRNPs. To gain mechanistic insight into their mode of action, we reconstituted the assembly machinery from recombinant sources. We uncover a stepwise and ordered formation of distinct Sm protein complexes on the PRMT5 complex, which is facilitated by the assembly chaperone pICln. Upon completion, the formed pICln-Sm units are displaced by new pICln-Sm protein substrates and transferred onto the SMN complex. The latter acts as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to prevent mis-assembly and to ensure the transfer of Sm proteins to cognate RNA. Investigation of mutant SMN complexes provided insight into the contribution of individual proteins to these activities. The biochemical reconstitution presented here provides a basis for a detailed molecular dissection of the U snRNP assembly reaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.15252/embj.201490350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547896PMC
July 2015

LARP4B is an AU-rich sequence associated factor that promotes mRNA accumulation and translation.

RNA 2015 Jul 22;21(7):1294-305. Epub 2015 May 22.

Biozentrum Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany Rudolf-Virchow-Zentrum für Experimentelle Biomedizin, Universität Würzburg, D-97080 Würzburg, Germany Department of Radiation Medicine and Applied Sciences, University of California at San Diego, San Diego, California 92037, USA.

mRNAs are key molecules in gene expression and subject to diverse regulatory events. Regulation is accomplished by distinct sets of trans-acting factors that interact with mRNAs and form defined mRNA-protein complexes (mRNPs). The resulting "mRNP code" determines the fate of any given mRNA and thus controlling gene expression at the post-transcriptional level. The La-related protein 4B (LARP4B) belongs to an evolutionarily conserved family of RNA-binding proteins characterized by the presence of a La-module implicated in direct RNA binding. Biochemical experiments have shown previously direct interactions of LARP4B with factors of the translation machinery. This finding along with the observation of an association with actively translating ribosomes suggested that LARP4B is a factor contributing to the mRNP code. To gain insight into the function of LARP4B in vivo we tested its mRNA association at the transcriptome level and its impact on the proteome. PAR-CLIP analyses allowed us to identify the in vivo RNA targets of LARP4B. We show that LARP4B binds to a distinct set of cellular mRNAs by contacting their 3' UTRs. Biocomputational analysis combined with in vitro binding assays identified the LARP4B-binding motif on mRNA targets. The reduction of cellular LARP4B levels leads to a marked destabilization of its mRNA targets and consequently their reduced translation. Our data identify LARP4B as a component of the mRNP code that influences the expression of its mRNA targets by affecting their stability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.051441.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4478348PMC
July 2015

mRNA metabolism and neuronal disease.

FEBS Lett 2015 Jun 13;589(14):1598-606. Epub 2015 May 13.

Institute for Genetics, University of Cologne, Zülpicher Str. 47a, 50674 Köln, Germany.

To serve as templates for translation eukaryotic mRNAs undergo an elaborate processing and maturation pathway. In eukaryotes this process comprises the synthesis of mRNA precursors, their processing and transport to the site of translation and eventually their decay. During the entire life cycle, mRNAs interact with distinct sets of trans-acting factors that determine their fate at any given phase of gene expression. Recent studies have shown that mutations in components acting in trans on mRNAs are frequent causes of a large variety of different human disorders. The etiology of most of these diseases is, however, only poorly understood, mostly because the consequences for mRNA-metabolism are unclear. Here we discuss three prominent genetic diseases that fall into this category, namely spinal muscular atrophy (SMA), retinitis pigmentosa (RP) and X-linked syndromic mental retardation (XLMR). Whereas SMA and RP can be directly linked to mRNA processing, XLMR results from mutations in the mRNA surveillance system. We discuss how defects in mRNA maturation and turnover might lead to the tissue specific defects seen in these diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.febslet.2015.04.052DOI Listing
June 2015

Assembly of RNPs: help needed.

RNA 2015 Apr;21(4):613-4

Max-Planck-Institute for Biophysical Chemistry, D-37077 Goettingen, Germany.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1261/rna.049791.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371304PMC
April 2015

The structure of apo ArnA features an unexpected central binding pocket and provides an explanation for enzymatic cooperativity.

Acta Crystallogr D Biol Crystallogr 2015 Mar 26;71(Pt 3):687-96. Epub 2015 Feb 26.

Department of Biochemistry, Biocenter of the University of Würzburg, Am Hubland, 97074 Würzburg, Germany.

The bacterial protein ArnA is an essential enzyme in the pathway leading to the modification of lipid A with the pentose sugar 4-amino-4-deoxy-L-arabinose. This modification confers resistance to polymyxins, which are antibiotics that are used as a last resort to treat infections with multiple drug-resistant Gram-negative bacteria. ArnA contains two domains with distinct catalytic functions: a dehydrogenase domain and a transformylase domain. The protein forms homohexamers organized as a dimer of trimers. Here, the crystal structure of apo ArnA is presented and compared with its ATP- and UDP-glucuronic acid-bound counterparts. The comparison reveals major structural rearrangements in the dehydrogenase domain that lead to the formation of a previously unobserved binding pocket at the centre of each ArnA trimer in its apo state. In the crystal structure, this pocket is occupied by a DTT molecule. It is shown that formation of the pocket is linked to a cascade of structural rearrangements that emerge from the NAD(+)-binding site. Based on these findings, a small effector molecule is postulated that binds to the central pocket and modulates the catalytic properties of ArnA. Furthermore, the discovered conformational changes provide a mechanistic explanation for the strong cooperative effect recently reported for the ArnA dehydrogenase function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S1399004714026686DOI Listing
March 2015

The catalytically inactive tyrosine phosphatase HD-PTP/PTPN23 is a novel regulator of SMN complex localization.

Mol Biol Cell 2015 Jan 12;26(2):161-71. Epub 2014 Nov 12.

Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Universität Heidelberg, D-69120 Heidelberg, Germany

The survival motor neuron (SMN) complex fulfils essential functions in the assembly of snRNPs, which are key components in the splicing of pre-mRNAs. Little is known about the regulation of SMN complex activity by posttranslational modification despite its complicated phosphorylation pattern. Several phosphatases had been implicated in the regulation of SMN, including the nuclear phosphatases PPM1G and PP1γ. Here we systematically screened all human phosphatase gene products for a regulatory role in the SMN complex. We used the accumulation of SMN in Cajal bodies of intact proliferating cells, which actively assemble snRNPs, as a readout for unperturbed SMN complex function. Knockdown of 29 protein phosphatases interfered with SMN accumulation in Cajal bodies, suggesting impaired SMN complex function, among those the catalytically inactive, non-receptor-type tyrosine phosphatase PTPN23/HD-PTP. Knockdown of PTPN23 also led to changes in the phosphorylation pattern of SMN without affecting the assembly of the SMN complex. We further show interaction between SMN and PTPN23 and document that PTPN23, like SMN, shuttles between nucleus and cytoplasm. Our data provide the first comprehensive screen for SMN complex regulators and establish a novel regulatory function of PTPN23 in maintaining a highly phosphorylated state of SMN, which is important for its proper function in snRNP assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E14-06-1151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4294665PMC
January 2015

Identification of a PRPF4 loss-of-function variant that abrogates U4/U6.U5 tri-snRNP integration and is associated with retinitis pigmentosa.

PLoS One 2014 10;9(11):e111754. Epub 2014 Nov 10.

Department of Biochemistry, University of Würzburg, Würzburg, Germany.

Pre-mRNA splicing by the spliceosome is an essential step in the maturation of nearly all human mRNAs. Mutations in six spliceosomal proteins, PRPF3, PRPF4, PRPF6, PRPF8, PRPF31 and SNRNP200, cause retinitis pigmentosa (RP), a disease characterized by progressive photoreceptor degeneration. All splicing factors linked to RP are constituents of the U4/U6.U5 tri-snRNP subunit of the spliceosome, suggesting that the compromised function of this particle may lead to RP. Here, we report the identification of the p.R192H variant of the tri-snRNP factor PRPF4 in a patient with RP. The mutation affects a highly conserved arginine residue that is crucial for PRPF4 function. Introduction of a corresponding mutation into the zebrafish homolog of PRPF4 resulted in a complete loss of function in vivo. A series of biochemical experiments suggested that p.R192H disrupts the binding interface between PRPF4 and its interactor PRPF3. This interferes with the ability of PRPF4 to integrate into the tri-snRNP, as shown in a human cell line and in zebrafish embryos. These data suggest that the p.R192H variant of PRPF4 represents a functional null allele. The resulting haploinsufficiency of PRPF4 compromises the function of the tri-snRNP, reinforcing the notion that this spliceosomal particle is of crucial importance in the physiology of the retina.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111754PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4226509PMC
July 2015