Matthias Wilm

University College Dublin | UCD · Conway Institute of Biomolecular & Biomedical Research

Membrane proteins carry out a wide variety of biological functions. The reproduction of specific properties that have evolved over millions of years of biological membranes in a technically controlled environment is of significant interest. Here a method is presented that allows the self-assembly of a macroscopically large, freely transportable mem...

Nanoelectrospray can be used to generate a layered structure consisting of bipolar lipids, detergent-solubilized membrane proteins, and glycerol that self-assembles upon detergent extraction into one extended layer of a protein containing membrane. This manuscript presents the first evidence that this method might allow membrane protein complexes t...

Polycomb group (PcG) protein complexes repress developmental regulator genes by modifying their chromatin. How different PcG proteins assemble into complexes and are recruited to their target genes is poorly understood. Here, we report the crystal structure of the core of the Drosophila PcG protein complex Pleiohomeotic (Pho)-repressive complex (Ph...

We report that low percentages of dimethylsulfoxide (DMSO) in liquid chromatography solvents lead to a strong enhancement of electrospray ionization of peptides, improving the sensitivity of protein identification in bottom-up proteomics by up to tenfold. The method can be easily implemented on any LC-MS/MS system without modification to hardware o...

Electrospray ionization is today the most widely used ionization technique in chemical and biochemical analysis. Interfaced with a mass spectrometer it allows the investigation of the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized. There is no limitation in mass which thus enables ev...

Electrospray ionization is today the most widely used ionization technique in chemical and bio-chemical analysis. Interfaced with a mass spectrometer it allows to investigate the molecular composition of liquid samples. With electrospray a large variety of chemical substances can be ionized. There is no limitation in mass which enables even the inv...

Thrombin is a key protease involved in blood coagulation, complement activation, inflammation, angiogenesis, and tumor invasion. Although induced in many (patho-)physiological conditions, the underlying mechanisms controlling prothrombin expression remained enigmatic. We have now discovered that prothrombin expression is regulated by a posttranscri...

Polycomb group (PcG) proteins are transcriptional repressors that control processes ranging from the maintenance of cell fate decisions and stem cell pluripotency in animals to the control of flowering time in plants. In Drosophila, genetic studies identified more than 15 different PcG proteins that are required to repress homeotic (HOX) and other...

Proteomics has enabled the direct investigation of biological material, at first through the analysis of individual proteins, then of lysates from cell cultures, and finally of extracts from tissues and biopsies from entire organisms. Its latest manifestation - quantitative proteomics - allows deeper insight into biological systems. This article re...

RBM5/Luca-15/H37 is a gene frequently inactivated in lung cancers and overexpressed in breast tumors. Its protein product has been detected in prespliceosomal complexes and modulates cell proliferation and Fas-mediated apoptosis. We report that RBM5 is a component of complexes involved in 3' splice site recognition and regulates alternative splicin...

Eukaryotic mRNAs possess a poly(A) tail that enhances translation via the (7)mGpppN cap structure or internal ribosome entry sequences (IRESs). Here we address the question of how cellular IRESs recruit the ribosome and how recruitment is augmented by the poly(A) tail. We show that the poly(A) tail enhances 48S complex assembly by the c-myc IRES. R...

Production of Ran-guanosine triphosphate (GTP) around chromosomes induces local nucleation and plus end stabilization of microtubules (MTs). The nuclear protein TPX2 is required for RanGTP-dependent MT nucleation. To find the MT stabilizer, we affinity purify nuclear localization signal (NLS)-containing proteins from Xenopus laevis egg extracts. Th...

Polyacrylamide gel electrophoresis (PAGE) is probably the most general and widespread protein separation technique. This simple method is used in almost every molecular biology and biochemistry laboratory to monitor protein experiments, including consecutive stages of protein purification. PAGE may even be the final step of a purification protocol....

Shortly after the introduction of electrospray as a viable ionization technique for large molecules (1), electrospray tandem mass spectrometry (ES MS/MS) techniques, such as HPLC-ES MS/MS, were used successfully for peptide sequencing at picomole and subpicomole levels (2–4). In LC-MS/MS, peptide sequence information is generated during the short t...

PHAX (phosphorylated adaptor for RNA export) is the key regulator of U snRNA nuclear export in metazoa. Our previous work revealed that PHAX is phosphorylated in the nucleus and is exported as a component of the U snRNA export complex to the cytoplasm, where it is dephosphorylated (M. Ohno, A. Segref, A. Bachi, M. Wilm, and I. W. Mattaj, Cell 101:1...

PRC2 is thought to be the histone methyltransferase (HMTase) responsible for H3-K27 trimethylation at Polycomb target genes. Here we report the biochemical purification and characterization of a distinct form of Drosophila PRC2 that contains the Polycomb group protein polycomblike (Pcl). Like PRC2, Pcl-PRC2 is an H3-K27-specific HMTase that mono-,...

Several label-free quantitation strategies have been introduced that obliterate the need for expensive isotopically labeled molecules. However label-free approaches have considerably higher demands in respect of repeatability of sample preparation and fractionation than multiplexing isotope labeling-based strategies. OFFGEL fractionation promises t...

Supplementary Figures

The prothrombin (F2) 3' end formation signal is highly susceptible to thrombophilia-associated gain-of-function mutations. In its unusual architecture, the F2 3' UTR contains an upstream sequence element (USE) that compensates for weak activities of the non-canonical cleavage site and the downstream U-rich element. Here, we address the mechanism of...

The sarcomere is the major structural and functional unit of striated muscle. Approximately 65 different proteins have been associated with the sarcomere, and their exact composition defines the speed, endurance, and biology of each individual muscle. Past analyses relied heavily on electrophoretic and immunohistochemical techniques, which only all...

Geminin has been proposed to coordinate cell cycle and differentiation events through balanced interactions with the cell cycle regulator Cdt1 and with homeobox transcription factors and chromatin remodeling activities implicated in cell fate decisions. Here we show that Geminin is cleaved in primary cells and cancer cell lines induced to undergo a...

Neurofibromin is the protein product of the tumor suppressor gene NF1, alterations of which are responsible for the pathogenesis of the common disorder Neurofibromatosis type I (NF1). The only well-characterized function of neurofibromin is its RasGAP activity, contained in the central GAP related domain (GRD). By solving the crystal structure of a...

The functional analysis of the common prothrombin (F2) 20210*A allele has recently revealed gain-of-function of 3′end processing as a novel genetic mechanism predisposing to human disease. The general susceptibility of the F2 mRNA for gain-of-function is further exemplified by F2 20209*T and F2 20221*T, and can be explained by an unusual architectu...

Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here th...

Polycomb response elements (PREs) are specific cis-regulatory sequences needed for transcriptional repression of HOX and other target genes by Polycomb group (PcG) proteins. Among the many PcG proteins known in Drosophila, Pho is the only sequence-specific DNA-binding protein. To gain insight into the function of Pho, we purified Pho protein comple...

GTP-loaded Ran induces the assembly of microtubules into aster-like and spindle-like structures in Xenopus egg extract. The microtubule-associated protein (MAP), TPX2, can mediate Ran's role in aster formation, but factors responsible for the transition from aster-like to spindle-like structures have not been described. Here we identify a complex t...

Dosage compensation in Drosophila is dependent on MSL proteins and involves hypertranscription of the male X chromosome, which ensures equal X-linked gene expression in both sexes. Here, we report the purification of enzymatically active MSL complexes from Drosophila embryos, Schneider cells, and human HeLa cells. We find a stable association of th...

MSL-2 (male-specific lethal 2) is the limiting component of the Drosophila dosage compensation complex (DCC) that specifically increases transcription from the male X chromosome. Ectopic expression of MSL-2 protein in females causes DCC assembly on both X chromosomes and lethality. Inhibition of MSL-2 synthesis requires the female-specific protein...

MS protein identification and quantitation are key proteomic techniques in biological research. Besides identification of proteins, MS is used increasingly to characterize secondary protein modifications. This often requires trimming the analytical strategy to a specific type of modification. Direct analysis of protein modifications in proteomic sa...

Nuclear envelope (NE) formation during cell division in multicellular organisms is a central yet poorly understood biological process. We report that the conserved nucleoporin Nup155 has an essential function in NE formation in Caenorhabditis elegans embryos and in Xenopus laevis egg extracts. In vivo depletion of Nup155 led to failure of nuclear l...

Asymmetric distribution of cellular components underlies many biological processes, and the localization of mRNAs within domains of the cytoplasm is one important mechanism of establishing and maintaining cellular asymmetry. mRNA localization often involves assembly of large ribonucleoproteins (RNPs) in the cytoplasm. Using an RNA affinity chromato...

Prothymosin alpha (ProTalpha) is a histone H1-binding protein that interacts with the transcription coactivator CREB-binding protein and potentiates transcription. Based on coimmunoprecipitation and mammalian two-hybrid assays, we show here that ProTalpha forms a complex with the oncoprotein SET. ProTalpha efficiently decondenses human sperm chroma...

The vacuolar (H+)-ATPase (or V-ATPase) is a membrane protein complex that is structurally related to F1 and F0 ATP synthases. The V-ATPase is composed of an integral domain (V0) and a peripheral domain (V1) connected by a central stalk and up to three peripheral stalks. The number of peripheral stalks and the proteins that comprise them remain cont...

Wortmannin Treatment Inhibits Fluid-Phase Uptake into Enlarged Rabankyrin-5 Structures NIH3T3 cells were pretreated for 20 min with wortmannin (100 nM) and incubated for 25 min with 0,5 μg/ml rhodamine-labelled transferrin and 2,5 mg/ml FITC-labelled dextran (MW, 70.000), fixed, processed for immunofluorescence, and analysed by confocal scanning mi...

Rabankyrin-5 Localizes to Macropinosomes Generated by Actin Dynamics Images were taken every 3 s over a time period of 15 min. RGB stacks were converted to QuickTime format using ImageJ (NIH). The movie is played at 15 frames per second. (11.1 MB MOV).

During catalytic activation of the spliceosome, snRNP remodeling events occur, leading to the formation of a 35S U5 snRNP that contains a large group of proteins, including Prp19 and CDC5, not found in 20S U5 snRNPs. To investigate the function of 35S U5 proteins, we immunoaffinity purified human spliceosomes that had not yet undergone catalytic ac...

In yeast cells, the THO complex has been implicated in mitotic recombination, transcription elongation and mRNA nuclear export. The stable core of THO consists of Tho2p, Hpr1p, Mft1p and Thp2p. Whether a complex with similar functions assembles in metazoa has not yet been established. Here we report that Drosophila melanogaster THO consists of THO2...

Importin alpha is well known as an adaptor that functions with Importin beta in the nuclear import of proteins containing specific nuclear localization signals (NLSs). We show here that either an excess or a lack of Importin alpha blocks nuclear envelope (NE) assembly in vitro, and our data suggest that soluble Importin alpha functions in NE assemb...

In metazoa, the nuclear envelope breaks down and reforms during each cell cycle. Nuclear pore complexes (NPCs), which serve as channels for transport between the nucleus and cytoplasm, assemble into the reforming nuclear envelope in a sequential process involving association of a subset of NPC proteins, nucleoporins, with chromatin followed by the...

mRNAs harbouring premature translation-termination codons are usually degraded by the nonsense-mediated mRNA decay (NMD) pathway. Human up-frameshift protein 1 (Hupf1) is an NMD factor that is conserved between yeast and mammals. To isolate cellular complexes that are formed with Hupf1 and to explore the role of cellular proteins in NMD, we generat...

In those cases where the information obtained by peptide mass fingerprinting or matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) is not sufficient for unambiguous protein identification, nano-electrospray ionization (nano-ESI) and/or electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis must be perf...

Liquid chromatography tandem mass spectrometry is a major tool for identifying proteins. The fragment spectra of peptides can be interpreted automatically in conjunction with a sequence database search. With the development of powerful automatic search engines, research now focuses on optimizing the result returned from database searches. We presen...

The sensitivity of protein identification by peptide sequencing using a nanoelectrospray ion source is limited by our ability to identify peptide ions in the mass spectrum. Their intensity must be higher than the chemical noise level to allow a rapid localization in the spectrum. Multiply-charged peptide ions on or below this level can only be foun...

Dosage compensation ensures equal expression of X-linked genes in males and females. In Drosophila, equalization is achieved by hypertranscription of the male X chromosome. This process requires an RNA/protein containing dosage compensation complex (DCC). Here we use RNA interference of individual DCC components to define the order of complex assem...

Nuclear pore complexes (NPCs) are large multiprotein assemblies that allow traffic between the cytoplasm and the nucleus. During mitosis in higher eukaryotes, the Nuclear Envelope (NE) breaks down and NPCs disassemble. How NPCs reassemble and incorporate into the NE upon mitotic exit is poorly understood. We demonstrate a function for the conserved...

A novel immobilized metal affinity chromatography (IMAC)-based protocol was successfully used for sample preparation prior to nanoelectrospray-based sequencing of phosphopeptides. In a first step, phosphorylated peptides are detected in an unseparated peptide mixture using precursor ion scanning in the negative ion mode on a triple-quadrupole mass...

The ensemble of expressed proteins in a given cell is organized in multiprotein complexes. The identification of the individual components of these complexes is essential for their functional characterization. The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide...

Genetic and molecular data have implicated theDrosophila gene female-lethal (2)d (fl (2)d) in alternative splicing regulation of genes involved in sexual determination. Sex-specific splicing is under the control of the female-specific regulatory protein sex-lethal (SXL). Co-immunoprecipitation and mass spectrometry results indicate that SXL and FL...

Detailed knowledge of the composition and structure of the spliceosome and its assembly intermediates is a prerequisite for understanding the complex process of pre-mRNA splicing. To this end, we have developed a tobramycin affinity-selection method that is generally applicable for the purification of native RNP complexes. By using this method, we...

Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in small nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of small nuclear ribonucleoprotein (snRNP) structures during spliceosome activation. Here, huma...

The SET domain proteins, SUV39 and G9a have recently been shown to be histone methyltransferases specific for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. We characterized the complex associated...

Mass spectrometry was used to identify novel proteins associated with the human 17S U2 snRNP and one of its stable subunits, SF3b. Several additional proteins were identified, demonstrating that 17S U2 snRNPs are significantly more complex than previously thought. Two of the newly identified proteins, namely the DEAD-box proteins SF3b125 and hPrp5...

Anopheles gambiae, the major vector of human malaria parasite, is an important insect model to study vector-parasite interactions. Here, we developed a simple in vivo double-stranded RNA (dsRNA) knockout approach to determine the function of the mosquito antimicrobial peptide gene Defensin. We injected dsRNA into adults and observed efficient and r...

Tandem affinity purification (TAP) and mass spectrometric peptide sequencing showed that the DEAD-box RNA helicase RHII/Gu is a functional interaction partner of c-Jun in human cells. The N-terminal transcription activation region of, c-Jun interacts with a C-terminal domain of RHII/Gu. This interaction is stimulated by anisomycin treatment in a ma...

Proteins separated by one-dimensional or two-dimensional gel electrophoresis can be digested in-gel with trypsin and the recovered peptides can be sequenced de novo using triple quadrupole or hybrid quadrupole time-of-flight instruments equipped with a nanoelectrospray ion source. The peptide sequences determined provide useful information for iden...

Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activitie...

Previous UV cross-linking studies demonstrated that, upon integration of the U2 snRNP into the spliceosome, a 14 kDa protein (p14) interacts directly with the branch adenosine, the nucleophile for the first transesterification step of splicing. We have identified the cDNA encoding this protein by microsequencing a 14 kDa protein isolated from U2-ty...

Identification of components present in biological complexes requires their purification to near homogeneity. Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases. We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purificati...

The ability to identify proteins with mass spectrometry has a profound impact on biological research. Its long term effects can only be compared with the significance PCR enzyme had for the advancement of biological science. Suddenly proteins can be identified having only minute quantities available. The efforts invested in to the technology lead t...

Despite the progress in genomic DNA sequencing de novo sequencing of peptides is still required in a biological research environment since many experiments are done in organisms whose genomes are not sequenced. A way to unambiguously retrieve a peptide sequence from a tandem mass spectrum is to assign the correct ion type to the fragments. Here we...

The small GTPase Ran, bound to GTP, is required for the induction of spindle formation by chromosomes in M phase. High concentrations of Ran.GTP are proposed to surround M phase chromatin. We show that the action of Ran.GTP in spindle formation requires TPX2, a microtubule-associated protein previously known to target a motor protein, Xklp2, to mic...

TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673-685). Here, we report the cloning and functional charac...

Vertebrate TAP and its yeast ortholog Mex67p are involved in the export of messenger RNAs from the nucleus. TAP has also been implicated in the export of simian type D viral RNAs bearing the constitutive transport element (CTE). Although TAP directly interacts with CTE-bearing RNAs, the mode of interaction of TAP/Mex67p with cellular mRNAs is diffe...

Recent developments in technology and instrumentation have made mass spectrometry the method of choice for the identification of gel-separated proteins using rapidly growing sequence databases (1). Proteins with a full-length sequence present in a database can be identified with high certainty and high throughput using the accurate masses obtained...

Protein identification in complete sequence databases or partial sequence databases is done preferentially by mass spectrometric techniques. However, protein de novo sequencing with mass spectrometry is more difficult to achieve. Generally, it is done by C-terminal labeling of the peptide. C-terminal fragment ions are identified in the tandem mass...

ilter is asymmetric having a sharply rising flank towards the selected m/z value and a slower falling tail to the higher m/z range. This allows to generate the two spectra with the full transmission of a quadrupole set to sub-isotopic resolution. This high transmission makes the method applicable to low level de novo sequencing. Since only the C-te...

The actin cytoskeleton is regulated by GTP-hydrolysing proteins, the Rho GTPases, which act as molecular switches in diverse signal-transduction processes. Various bacterial toxins can inactivate Rho GTPases by ADP-ribosylation or glucosylation. Previous research has identified Rho proteins as putative targets for Escherichia coli cytotoxic necroti...

Although LOX mRNA accumulates early during differentiation, a differentiation control element in its 3′ untranslated region confers translational silencing until late stage erythropoiesis. We have purified two proteins from rabbit reticulocytes that specifically mediate LOX silencing and identified them as hnRNPs K and E1. Transfection of hnRNP K a...

It is now possible to obtain sequence information from gel-separated proteins by mass spectrometry at levels too low for conventional approaches. Usually this tandem mass spectrometric data are used for database searches with the aim of identifying the corresponding gene. Recently it has been shown that long and accurate amino acid sequences can be...

The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that ma...

Peptide sequencing remains one of the most important analytical problems in molecular biology. Automated Edman degradation is established as a routine technique in most protein bio-chemical core facilities, and is a straightforward method to obtain enough sequence information to clone a gene [1, 2]. At sufficiently high sample amounts data interpre...

Mass spectrometry is a venerable analytical tool that has been used for some time in biochemistry for the analysis of small molecules, such as steroids. More recently, physicists have solved the problems associated with vaporizing and ionizing proteins and peptides, thereby allowing mass spectrometry to take on new roles in investigating protein se...