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Juri Rappsilber

Publications and source records attributed to Juri Rappsilber.

At least 19 recordsLinked to original sources
PubMed2026

Structural insights into Sld3-Sld7-dependent Cdc45 loading during replication initiation.

Regulated helicase activation by DDK kinase is central for genome stability. However, how DDK phosphorylation primes the MCM2-7 double hexamer (DH) for Sld3-Sld7 binding and Cdc45 loading remained unclear. We define this mechanism through cryo-EM structures of MCM2-7 DH-Sld3-Sld7 (MS) and MCM2-7 DH-Sld3-Sld7-Cdc45 (MSC). We reveal that the autoinhibitory Mcm4 tail engages not only Mcm4 but also Mcm6. Upon DDK-dependent phosphorylation, both of these sites become accessible. In the context of the MS structure, we identify that two short Sld3 motifs that contact Mcm4 and Mcm6 read out the DH phosphorylation state, while the Sld3 Treslin domain (STD) binds to Mcm2. In the MSC structure, Cdc45 dislodges the Sld3 STD from Mcm2, allowing Sld3 to position Cdc45 at the Mcm2/Mcm5 interface. Mutagenesis of the Sld3 STD-Cdc45 interface disrupts Cdc45 loading, validating this interaction. Together, our data reveal a phosphorylation-encoded mechanism coupling DDK-activated Mcm4/Mcm6 surfaces to distal Cdc45 placement, explaining how firing factors choreograph the DH-to-CMG transition.

Cell Cycle Proteins↗
PubMed2006

The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3.

Methylation of lysine and arginine residues on histone tails affects chromatin structure and gene transcription. Tri- and dimethylation of lysine 9 on histone H3 (H3K9me3/me2) is required for the binding of the repressive protein HP1 and is associated with heterochromatin formation and transcriptional repression in a variety of species. H3K9me3 has long been regarded as a 'permanent' epigenetic mark. In a search for proteins and complexes interacting with H3K9me3, we identified the protein GASC1 (gene amplified in squamous cell carcinoma 1), which belongs to the JMJD2 (jumonji domain containing 2) subfamily of the jumonji family, and is also known as JMJD2C. Here we show that three members of this subfamily of proteins demethylate H3K9me3/me2 in vitro through a hydroxylation reaction requiring iron and alpha-ketoglutarate as cofactors. Furthermore, we demonstrate that ectopic expression of GASC1 or other JMJD2 members markedly decreases H3K9me3/me2 levels, increases H3K9me1 levels, delocalizes HP1 and reduces heterochromatin in vivo. Previously, GASC1 was found to be amplified in several cell lines derived from oesophageal squamous carcinomas, and in agreement with a contribution of GASC1 to tumour development, inhibition of GASC1 expression decreases cell proliferation. Thus, in addition to identifying GASC1 as a histone trimethyl demethylase, we suggest a model for how this enzyme might be involved in cancer development, and propose it as a target for anti-cancer therapy.

Cell Proliferation↗
PubMed2006

Gemin8 is a novel component of the survival motor neuron complex and functions in small nuclear ribonucleoprotein assembly.

The survival motor neuron (SMN) protein is the product of the spinal muscular atrophy disease gene. SMN and Gemin2-7 proteins form a large macromolecular complex that localizes in the cytoplasm as well as in the nucleoplasm and in nuclear Gems. The SMN complex interacts with several additional proteins and likely functions in multiple cellular pathways. In the cytoplasm, a subset of SMN complexes containing unrip and Sm proteins mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs). Here, by mass spectrometry analysis of SMN complexes purified from HeLa cells, we identified a novel protein that is evolutionarily conserved in metazoans, and we named it Gemin8. Co-immunoprecipitation and immunolocalization experiments demonstrated that Gemin8 is associated with the SMN complex and is localized in the cytoplasm and in the nucleus, where it is highly concentrated in Gems. Gemin8 interacts directly with the Gemin6-Gemin7 heterodimer and, together with unrip, these proteins form a heteromeric subunit of the SMN complex. Gemin8 is also associated with Sm proteins, and Gemin8-containing SMN complexes are competent to carry out snRNP assembly. Importantly, RNA interference experiments indicate that Gemin8 knock-down impairs snRNP assembly, and Gemin8 expression is down-regulated in cells with low levels of SMN. These results demonstrate that Gemin8 is a novel integral component of the SMN complex and extend the repertoire of cellular proteins involved in the pathway of snRNP biogenesis.

Amino Acid Sequence↗
PubMed2006

Modular stop and go extraction tips with stacked disks for parallel and multidimensional Peptide fractionation in proteomics.

Proteome complexity necessitates protein or peptide separation prior to analysis. We previously described a pipet-tip based peptide micropurification system named StageTips (STop and Go Extraction Tips), which consists of a very small disk of membrane-embedded separation material. Here, we extend this approach in several dimensions by stacking disks containing reversed phase (C(18)) and strong cation exchange (SCX) materials. Multidimensional fractionation as well as desalting, filtration, and concentration prior to mass spectrometry in single or tandem columns is described. C(18)-SCX-C(18) stacked disks significantly improved protein identification by LC-MS/MS for an E. coli protein digest and by MALDI-MS for a 12 standard protein digest. Sequential fractionation based on C(18)- followed by SCX material was also developed. This multidimensional fractionation approach was expanded to parallel sample preparation by incorporating C(18)-SCX-StageTips into a 96-well plate (StagePlate). Fractions were collected into other C(18)-StagePlates and desalted and eluted in parallel to sample well plates or MALDI targets. This approach is suitable for high throughput protein identification for moderately complex, low abundance samples using automated nanoelectrospray-MS/MS or MALDI-MS.

Bacterial Proteins↗
PubMed2005

Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.

To estimate absolute protein contents in complex mixtures, we previously defined a protein abundance index (PAI) as the number of observed peptides divided by the number of observable peptides per protein (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome. Res. 12, 1231-1245). Here we report that PAI values obtained at different concentrations of serum albumin show a linear relationship with the logarithm of protein concentration in LC-MS/MS experiments. This was also the case for 46 proteins in a mouse whole cell lysate. For absolute quantitation, PAI was converted to exponentially modified PAI (emPAI), equal to 10PAI minus one, which is proportional to protein content in a protein mixture. For the 46 proteins in the whole lysate, the deviation percentages of the emPAI-based abundances from the actual values were within 63% on average, similar or better than determination of abundance by protein staining. emPAI was applied to comprehensive protein expression analysis and to a comparison study between gene and protein expression in a human cancer cell line, HCT116. The values of emPAI are easily calculated and add important quantitation information to proteomic experiments; therefore we suggest that they should be reported in large scale proteomic identification projects.

Animals↗
PubMed2005

Unrip is a component of SMN complexes active in snRNP assembly.

A macromolecular complex containing survival of motor neurons (SMN), the spinal muscular atrophy protein, and Gemin2-7 interacts with Sm proteins and snRNAs to carry out the assembly of these components into spliceosomal small nuclear ribonucleoproteins (snRNPs). Here we report the characterization of unr-interacting protein (unrip), a GH-WD protein of unknown function, as a component of the SMN complex that interacts directly with Gemin6 and Gemin7. Unrip also binds a subset of Sm proteins, and unrip-containing SMN complexes are necessary and sufficient to mediate the assembly of spliceosomal snRNPs. These results demonstrate that unrip functions in the pathway of snRNP biogenesis and is a marker of cellular SMN complexes active in snRNP assembly.

Animals↗
PubMed2005

Sequence tag scanning: a new explorative strategy for recognition of unexpected protein alterations by nanoelectrospray ionization-tandem mass spectrometry.

Protein analysis by database search engines using tandem mass spectra is limited by the presence of unexpected protein modifications, sequence isoforms which may not be in the protein databases, and poor quality tandem mass spectrometry (MS/MS) of low abundance proteins. The analysis of expected protein modifications can be efficiently addressed by precursor ion scanning. However, it is limited to modifications that show such a characteristic loss in a peptide independent manner. We observed that proline and aspartic acid induced backbone fragmentation is accompanied by a low intensity signal for loss of H3PO4 for several pSer- or pThr-phosphopeptides. We describe here the use of peptide-specific fragments that can be used after a protein was identified to allow in-depth characterization of modifications and isoforms. We consider high abundance fragments formed by cleavage at the C-terminal side of aspartic acid, at the N-terminal side of proline and low mass ions such as a2, b2, b3, y1, y2, and y3. The MS/MS dataset is filtered for each sequence tag of interest by an in silico precursor ion scan. The resulting extracted ion traces are then combined by multiplication to increase specificity. Since the strategy is based on common peptide segments which are shared by different isoforms of peptides it can be applied to the analysis of any post-translational modification or sequence variants of a protein. This is demonstrated for the cases of serine and threonine phosphorylation, histone H1 acetylation and the spotting of multiple H1 isoforms.

Acetylation↗
PubMed2005

Spot overlapping in two-dimensional maps: a serious problem ignored for much too long.

In the analysis of a neuroblastoma xenograft implanted in mice using two-dimensional maps, some 85 proteins were found to be up- or down-regulated (out of a total of 264 detected by a medium-sensitivity colloidal Coomassie stain). When these spots were eluted and analysed by mass spectrometry in a quadrupole time of flight mass spectrometer, a number of spots were found to be envelopes of different polypeptide chains. Out of a total of 74 proteins identified, 52 (71%) were found to be singlets, 14 (19%) were doublets, 6 (8%) were triplets, 1 was a quadruplet and 1 a quintuplet. Analysis of the DeltapI and DeltaMr of all species contained in a single gel segment eluted helped point out potential errors in protein identification. This was a unique case, in that very minute bioptic sample loads were applied to the gel. In normal cases, where sample loads of ca. 1 mg of total protein are applied and typically at least 1000 spots are visualised, the singlets will be the minority, rarely exceeding 30% of all spots analysed. The experimental data on the abundance of overlapping spots were in excellent agreement with theoretical data calculated on the basis of the statistical theory of spot overlapping, originally proposed by Davis and further developed by some of the authors. Ways and means for minimizing spot overlap and visualising a greater number of spots in a two-dimensional map are discussed.

Adrenal Gland Neoplasms↗
PubMed2005

Self-made frits for nanoscale columns in proteomics.

We report here the production of self-made frits for nano-columns. The frits introduce a minor dead volume and can be placed in capillaries with a wide range of diameters (20-250 microm tested) in an extremely simple and low-cost procedure. The obtained columns appear to be comparable to "no-frit" columns with near-ideal chromatographic characteristics. We expect that this frit will be useful for the spotting of gradients onto MALDI plates but also where special ESI set-ups do not allow for "no-frit" solutions.

Chromatography, Liquid↗
PubMed2004

eIF4A3 is a novel component of the exon junction complex.

The exon junction complex (EJC) is a protein complex that assembles near exon-exon junctions of mRNAs as a result of splicing. EJC proteins play important roles in postsplicing events including mRNA export, cytoplasmic localization, and nonsense-mediated decay. Recent evidence suggests that mRNA translation is also influenced by the splicing history of the transcript. Here we identify eIF4A3, a DEAD-box RNA helicase and a member of the eIF4A family of translation initiation factors, as a novel component of the EJC. We show that eIF4A3 associates preferentially with nuclear complexes containing the EJC proteins magoh and Y14. Furthermore, eIF4A3, but not the highly related eIF4A1 or eIF4A2, preferentially associates with spliced mRNA. In vitro splicing and mapping experiments demonstrate that eIF4A3 binds mRNAs at the position of the EJC. Using monoclonal antibodies, we show that eIF4A3 is found in the nucleus whereas eIF4A1 and eIF4A2 are found in the cytoplasm. Thus, eIF4A3 likely provides a splicing-dependent influence on the translation of mRNAs.

Amino Acid Sequence↗
PubMed2003

Detection of arginine dimethylated peptides by parallel precursor ion scanning mass spectrometry in positive ion mode.

Dimethylation at arginine residues has been shown to be central in cellular processes such as signal transduction, transcription activation, and protein sorting. The two methyl groups are either placed symmetric or asymmetric on the zeta standing nitrogen atoms of the arginine side chain. Here, we introduce a novel method that enables the localization of dimethylarginine (DMA) residues in gel-separated proteins at a level of sensitivity of better than 1 pmol and that allows one to distinguish between the isomeric symmetric and asymmetric position of the methyl groups. The method utilizes two side-chain fragments of DMA, the dimethylammonium ion (m/z 46.06) and the dimethylcarbodiimidium ion (m/z 71.06), for positive ion mode precursor ion scanning. Dimethylcarbodiimidium ions (m/z 71.06) are produced by symmetric as well as asymmetric dimethylarginine but are observed more strongly for symmetric DMA. It is utilized here in the precursor of m/z 71 scan to indicate the presence of DMA in a peptide. The dimethylammonium ion (m/z 46.06) is specific for asymmetric DMA and is utilized here in the precursor of m/z 46 scan. The positive ion mode allows for the identification of the protein by peptide sequencing and simultaneous detection and localization of the modified residues. The analysis can be conducted on any mass spectrometer capable of precursor ion scanning. However, the high resolution of a quadrupole TOF instrument is beneficial to assign the accurate charge state of the often highly charged precursors. Using the precursor of m/z 71 scan, we found FUS/TLS and Sam68 to be DMA-containing proteins. We discovered at least 20 DMA sites in FUS/TLS. In MS/MS, we observed neutral loss of dimethylamine (m/z 45.05) from which it follows that the dimethylation in FUS/TLS is asymmetric. Monitoring in parallel the fragments m/z 46.06 and 71.06 in precursor ion scans and peptide sequencing, we identified at least nine asymmetric DMA modifications in Sam68. The parallel monitoring of fragments in precursor ion scans is a versatile tool to specify the nature of protein modifications in cases where a single fragment is not conclusive.

Amino Acid Sequence↗
PubMed2003

Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics.

Proteomics is critically dependent on optimal sample preparation. Particularly, the interface between protein digestion and mass spectrometric analysis has a large influence on the overall quality and sensitivity of the analysis. We here describe a novel procedure in which a very small disk of beads embedded in a Teflon meshwork is placed as a microcolumn into pipet tips. Termed Stage, for STop And Go Extraction, the procedure has been implemented with commercially available material (C18 Empore Disks (3M, Minneapolis, MN)) as frit and separation material. The disk is introduced in a simple and fast process yielding a convenient and completely reliable procedure for the production of self-packed microcolumns in pipet tips. It is held in place free of obstacles solely by the narrowing tip, ensuring optimized loading and elution of analytes. Five disks are conveniently placed in 1 min, adding <0.1 cent in material costs to the price of each tip. The system allows fast loading with low backpressure (>300 micro/min for the packed column using manual force) while eliminating the possibility of blocking. The loading capacity of C18-StageTips (column bed: 0.4 mm diameter, 0.5 mm length) is 2-4 microg of protein digest, which can be increased by using larger diameter or stacked disks. Five femtomole of tryptic BSA digest could be recovered quantitatively. We have found that the Stage system is well-suited as a universal sample preparation system for proteomics.

Animals↗
PubMed2002

Microcolumns with self-assembled particle frits for proteomics.

LC-MS-MS experiments in proteomics are usually performed with packed microcolumns employing frits or outlets smaller than the particle diameter to retain the packing material. We have developed packed microcolumns using self-assembled particles (SAPs) as frits that are smaller than the size of the outlet. A five to one ratio of outlet size to particle diameter appears to be the upper maximum. In these situations the particles assembled into an arch over the outlet like the stones in a stone bridge. When 3 microm particles were packed into a tapered column with an 8 microm outlet, two particles bridged the outlet with 0.3 pl dead volume and perfect success rate. In peptide analysis by LC-MS, the peak width at half height was normally less than 6 s, compared to 12 s without SAPs. The LC-MS-MS system provided 37% sequence coverage (21 matched peptides) for a tryptically-digested sample of 10 fmol bovine serum albumin. We also describe application of the SAP principle to make disposable pipette tip columns with short pieces of fused-silica capillary as the outlet.

Chromatography, Liquid↗
PubMed2002

Identification and characterization of Gemin7, a novel component of the survival of motor neuron complex.

The survival of motor neurons (SMN) protein is the product of the gene mutated or deleted in the neurodegenerative disease, spinal muscular atrophy. SMN is part of a large macromolecular complex that also contains Gemin2, Gemin3, Gemin4, Gemin5, and Gemin6. The SMN complex functions in the assembly of spliceosomal small nuclear ribonucleoproteins and probably other ribonucleoprotein particles. We have identified a novel protein component of the SMN complex termed Gemin7 using native purified SMN complexes and peptide sequencing by mass spectrometry. Coimmunoprecipitation and immunolocalization experiments demonstrate that Gemin7 is a component of the SMN complex and colocalizes with SMN in the cytoplasm and in gems. Binding experiments show that Gemin7 interacts directly with SMN and Gemin6 and mediates the association of Gemin6 with the SMN complex. The amino acid sequence of Gemin7 does not contain any recognizable motifs with the exception of several arginine and glycine repeats that are necessary for its interaction with SMN. Moreover, Gemin7 interacts with several Sm proteins of spliceosomal small nuclear ribonucleoproteins, in particular, with SmE. With the identification of Gemin7, the inventory of the core components of the SMN complex appears essentially complete.

Amino Acid Sequence↗
PubMed2002

miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs.

Gemin3 is a DEAD-box RNA helicase that binds to the Survival of Motor Neurons (SMN) protein and is a component of the SMN complex, which also comprises SMN, Gemin2, Gemin4, Gemin5, and Gemin6. Reduction in SMN protein results in Spinal muscular atrophy (SMA), a common neurodegenerative disease. The SMN complex has critical functions in the assembly/restructuring of diverse ribonucleoprotein (RNP) complexes. Here we report that Gemin3 and Gemin4 are also in a separate complex that contains eIF2C2, a member of the Argonaute protein family. This novel complex is a large approximately 15S RNP that contains numerous microRNAs (miRNAs). We describe 40 miRNAs, a few of which are identical to recently described human miRNAs, a class of small endogenous RNAs. The genomic sequences predict that miRNAs are likely to be derived from larger precursors that have the capacity to form stem-loop structures.

Animals↗
PubMed2002

What does it mean to identify a protein in proteomics?

The annotation of the human genome indicates the surprisingly low number of approximately 40,000 genes. However, the estimated number of proteins encoded by these genes is two to three orders of magnitude higher. The ability to unambiguously identify the proteins is a prerequisite for their functional investigation. As proteins derived from the same gene can be largely identical, and might differ only in small but functionally relevant details, protein identification tools must not only identify a large number of proteins but also be able to differentiate between close relatives. This information can be generated by mass spectrometry, an approach that identifies proteins by partial analysis of their digestion-derived peptides. Information gleaned from databases fills in the missing sequence information. Because both sequence databases and experimental data are limited, a certain ambiguity often remains concerning which sequence variant(s) and modification(s) are present. As the common denominator of all the isoforms is a gene, in our opinion, it would be more accurate to state that a product of this particular gene rather than a certain protein has been identified by mass spectrometry.

Gene Expression Profiling↗
PubMed2002

Large-scale proteomic analysis of the human spliceosome.

In a previous proteomic study of the human spliceosome, we identified 42 spliceosome-associated factors, including 19 novel ones. Using enhanced mass spectrometric tools and improved databases, we now report identification of 311 proteins that copurify with splicing complexes assembled on two separate pre-mRNAs. All known essential human splicing factors were found, and 96 novel proteins were identified, of which 55 contain domains directly linking them to functions in splicing/RNA processing. We also detected 20 proteins related to transcription, which indicates a direct connection between this process and splicing. This investigation provides the most detailed inventory of human spliceosome-associated factors to date, and the data indicate a number of interesting links coordinating splicing with other steps in the gene expression pathway.

Amino Acid Sequence↗