Achim Dickmanns

Conserved Stem II of the Box C/D Motif Is Essential for Nucleolar Localization and Is Required, Along with the 15.5K Protein, for the Hierarchical Assembly of the Box C/D snoRNP

ABSTRACT The 5′ stem-loop of the U4 snRNA and the box C/D motif of the box C/D snoRNAs can both be folded into a similar stem-internal loop-stem structure that binds the 15.5K protein. The homologous proteins NOP56 and NOP58 and 61K (hPrp31) associate with the box C/D snoRNPs and the U4/U6 snRNP, respectively. This raises the intriguing question of how the two homologous RNP complexes specifically assemble onto similar RNAs. Here we investigate the requirements for the specific binding of the individual snoRNP proteins to the U14 box C/D snoRNPs in vitro. This revealed that the binding of 15.5K to the box C/D motif is essential for the association of the remaining snoRNP-associated proteins, namely, NOP56, NOP58, fibrillarin, and the nucleoplasmic proteins TIP48 and TIP49. Stem II of the box C/D motif, in contrast to the U4 5′ stem-loop, is highly conserved, and we show that this sequence is responsible for the binding of NOP56, NOP58, fibrillarin, TIP48, and TIP49, but not of 15.5K, to the snoRNA. Indeed, the sequence of stem II was essential for nucleolar localization of U14 snoRNA microinjected into HeLa cells. Thus, the conserved sequence of stem II determines the specific assembly of the box C/D snoRNP.

Crystal Structure of the Nuclear Export Receptor CRM1 in Complex with Snurportin1 and RanGTP

Nuclear Import/Export Receptor Nuclear transport receptors constantly shuttle cargo between the nucleus and the cytoplasm through nuclear pore complexes. In the nucleus, RanGTP promotes the dissociation of cargo from importins, which import cargo into the nucleus (where RanGTP is guanosine 5′ triphosphate–bound Ran). Conversely, nuclear RanGTP promotes cargo-binding to exportins, which export cargo from the nucleus. Cargo is released from exportins in the cytoplasm upon hydrolysis of RanGTP. Cytoplasmically assembled RNA splicing components enter the nucleus together with an import adapter snurportin 1 (SPN1), but how then does the import adapter release its cargo and exit the nucleus to collect further cargo? The nuclear exportin CRM1 exports a broad range of substrates—including SPN1, ribosomes, and many regulatory proteins. Monecke et al. (p. 1087) describe the crystal structure of CRM1 bound to SPN1 and RanGTP. The structure shows that SPN1 cannot simultaneously bind its import cargo and the exportin CRM1, ensuring that only cargo-free SPN1 is returned to the cytoplasm. There are no direct contacts between Ran and SPN1 in the ternary complex, suggesting that RanGTP promotes cargo-binding through long-range conformational changes in CRM1. The structure of an exportin complex shows how nuclear transport complexes differentially recognize cargo. CRM1 mediates nuclear export of numerous unrelated cargoes, which may carry a short leucine-rich nuclear export signal or export signatures that include folded domains. How CRM1 recognizes such a variety of cargoes has been unknown up to this point. Here we present the crystal structure of the SPN1⋅CRM1⋅RanGTP export complex at 2.5 angstrom resolution (where SPN1 is snurportin1 and RanGTP is guanosine 5′ triphosphate–bound Ran). SPN1 is a nuclear import adapter for cytoplasmically assembled, m3G-capped spliceosomal U snRNPs (small nuclear ribonucleoproteins). The structure shows how CRM1 can specifically return the cargo-free form of SPN1 to the cytoplasm. The extensive contact area includes five hydrophobic residues at the SPN1 amino terminus that dock into a hydrophobic cleft of CRM1, as well as numerous hydrophilic contacts of CRM1 to m3G cap-binding domain and carboxyl-terminal residues of SPN1. The structure suggests that RanGTP promotes cargo-binding to CRM1 solely through long-range conformational changes in the exportin.

Crystal structure of the polysialic acid-degrading endosialidase of bacteriophage K1F.

Phages infecting the polysialic acid (polySia)-encapsulated human pathogen Escherichia coli K1 are equipped with capsule-degrading tailspikes known as endosialidases, which are the only identified enzymes that specifically degrade polySia. As polySia also promotes cellular plasticity and tumor metastasis in vertebrates, endosialidases are widely applied in polySia-related neurosciences and cancer research. Here we report the crystal structures of endosialidase NF and its complex with oligomeric sialic acid. The structure NF, which reveals three distinct domains, indicates that the unique polySia specificity evolved from a combination of structural elements characteristic of exosialidases and bacteriophage tailspike proteins. The endosialidase assembles into a catalytic trimer stabilized by a triple β-helix. Its active site differs markedly from that of exosialidases, indicating an endosialidase-specific substrate-binding mode and catalytic mechanism. Residues essential for endosialidase activity were identified by structure-based mutational analysis.

Molecular basis for multiple sulfatase deficiency and mechanism for formylglycine generation of the human formylglycine-generating enzyme.

Sulfatases are enzymes essential for degradation and remodeling of sulfate esters. Formylglycine (FGly), the key catalytic residue in the active site, is unique to sulfatases. In higher eukaryotes, FGly is generated from a cysteine precursor by the FGly-generating enzyme (FGE). Inactivity of FGE results in multiple sulfatase deficiency (MSD), a fatal autosomal recessive syndrome. Based on the crystal structure, we report that FGE is a single-domain monomer with a surprising paucity of secondary structure and adopts a unique fold. The effect of all 18 missense mutations found in MSD patients is explained by the FGE structure, providing a molecular basis of MSD. The catalytic mechanism of FGly generation was elucidated by six high-resolution structures of FGE in different redox environments. The structures allow formulation of a novel oxygenase mechanism whereby FGE utilizes molecular oxygen to generate FGly via a cysteine sulfenic acid intermediate.

High intranuclear mobility and dynamic clustering of the splicing factor U1 snRNP observed by single particle tracking

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) are components of the splicing machinery that removes introns from precursor mRNA. Like other splicing factors, U snRNPs are diffusely distributed throughout the nucleus and, in addition, are concentrated in distinct nuclear substructures referred to as speckles. We have examined the intranuclear distribution and mobility of the splicing factor U1 snRNP on a single-molecule level. Isolated U1 snRNPs were fluorescently labeled and incubated with digitonin-permeabilized 3T3 cells in the presence of Xenopus egg extract. By confocal microscopy, U1 snRNPs were found to be imported into nuclei, yielding a speckled intranuclear distribution. Employing a laser video-microscope optimized for high sensitivity and high speed, single U1 snRNPs were visualized and tracked at a spatial precision of 35 nm and a time resolution of 30 ms. The single-particle data revealed that U1 snRNPs occurred in small clusters that colocalized with speckles. In the clusters, U1 snRNPs resided for a mean decay time of 84 ms before leaving the optical slice in the direction of the optical axis, which corresponded to a mean effective diffusion coefficient of 1 μm2/s. An analysis of the trajectories of single U1 snRNPs revealed that at least three kinetic classes of low, medium, and high mobility were present. Moreover, the mean square displacements of these fractions were virtually independent of time, suggesting arrays of binding sites. The results substantiate the view that nuclear speckles are not rigid structures but highly dynamic domains characterized by a rapid turnover of U1 snRNPs and other splicing factors.

The importin-beta binding domain of snurportin1 is responsible for the Ran- and energy-independent nuclear import of spliceosomal U snRNPs in vitro.

The nuclear localization signal (NLS) of spliceosomal U snRNPs is composed of the U snRNAs 2,2,7-trimethyl-guanosine (m3G)-cap and the Sm core domain. The m3G-cap is specifically bound by snurportin1, which contains an NH2-terminal importin-β binding (IBB) domain and a COOH-terminal m3G-cap–binding region that bears no structural similarity to known import adaptors like importin-α (impα). Here, we show that recombinant snurportin1 and importin-β (impβ) are not only necessary, but also sufficient for U1 snRNP transport to the nuclei of digitonin-permeabilized HeLa cells. In contrast to impα–dependent import, single rounds of U1 snRNP import, mediated by the nuclear import receptor complex snurportin1–impβ, did not require Ran and energy. The same Ran- and energy-independent import was even observed for U5 snRNP, which has a molecular weight of more than one million. Interestingly, in the presence of impβ and a snurportin1 mutant containing an impα IBB domain (IBBimpα), nuclear U1 snRNP import was Ran dependent. Furthermore, β-galactosidase (βGal) containing a snurportin1 IBB domain, but not IBBimpα-βGal, was imported into the nucleus in a Ran-independent manner. Our results suggest that the nature of the IBB domain modulates the strength and/or site of interaction of impβ with nucleoporins of the nuclear pore complex, and thus whether or not Ran is required to dissociate these interactions.

A jack of all trades: the multiple roles of the unique essential second messenger cyclic di-AMP

Second messengers are key components of many signal transduction pathways. In addition to cyclic AMP, ppGpp and cyclic di‐GMP, many bacteria use also cyclic di‐AMP as a second messenger. This molecule is synthesized by distinct classes of diadenylate cyclases and degraded by phosphodiesterases. The control of the intracellular c‐di‐AMP pool is very important since both a lack of this molecule and its accumulation can inhibit growth of the bacteria. In many firmicutes, c‐di‐AMP is essential, making it the only known essential second messenger. Cyclic di‐AMP is implicated in a variety of functions in the cell, including cell wall metabolism, potassium homeostasis, DNA repair and the control of gene expression. To understand the molecular mechanisms behind these functions, targets of c‐di‐AMP have been identified and characterized. Interestingly, c‐di‐AMP can bind both proteins and RNA molecules. Several proteins that interact with c‐di‐AMP are required to control the intracellular potassium concentration. In Bacillus subtilis, c‐di‐AMP also binds a riboswitch that controls the expression of a potassium transporter. Thus, c‐di‐AMP is the only known second messenger that controls a biological process by interacting with both a protein and the riboswitch that regulates its expression. Moreover, in Listeria monocytogenes c‐di‐AMP controls the activity of pyruvate carboxylase, an enzyme that is required to replenish the citric acid cycle. Here, we review the components of the c‐di‐AMP signaling system.

The Velvet Family of Fungal Regulators Contains a DNA-Binding Domain Structurally Similar to NF-κB

This study reveals an important family of fungal regulatory proteins to be transcription factors that contain a DNA-binding “velvet” domain structurally related to that of mammalian NFkB.

Structural basis for m3G-cap-mediated nuclear import of spliceosomal UsnRNPs by snurportin1

In higher eukaryotes the biogenesis of spliceosomal UsnRNPs involves a nucleocytoplasmic shuttling cycle. After the m7G‐cap‐dependent export of the snRNAs U1, U2, U4 and U5 to the cytoplasm, each of these snRNAs associates with seven Sm proteins. Subsequently, the m7G‐cap is hypermethylated to the 2,2,7‐trimethylguanosine (m3G)‐cap. The import adaptor snurportin1 recognises the m3G‐cap and facilitates the nuclear import of the UsnRNPs by binding to importin‐β. Here we report the crystal structure of the m3G‐cap‐binding domain of snurportin1 with bound m3GpppG at 2.4 Å resolution, revealing a structural similarity to the mRNA‐guanyly‐transferase. Snurportin1 binds both the hypermethylated cap and the first nucleotide of the RNA in a stacked conformation. This binding mode differs significantly from that of the m7G‐cap‐binding proteins Cap‐binding protein 20 (CBP20), eukaryotic initiation factor 4E (eIF4E) and viral protein 39 (VP39). The specificity of the m3G‐cap recognition by snurportin1 was evaluated by fluorescence spectroscopy, demonstrating the importance of a highly solvent exposed tryptophan for the discrimination of m7G‐capped RNAs. The critical role of this tryptophan and as well of a tryptophan continuing the RNA base stack was confirmed by nuclear import assays and cap‐binding activity tests using several snurportin1 mutants.

Cyclin E‐mediated elimination of p27 requires its interaction with the nuclear pore‐associated protein mNPAP60

The Cdk2 inhibitor, p27Kip1, is degraded in a phosphorylation‐ and ubiquitylation‐dependent manner at the G1–S transition of the cell cycle. Degradation of p27Kip1 requires import into the nucleus for phosphorylation by Cdk2. Phosphorylated p27Kip1 is thought to be subsequently re‐exported and degraded in the cytosol. Using two‐hybrid screens, we now show that p27Kip1 interacts with a nuclear pore‐associated protein, mNPAP60, map the interaction to the 310 helix of p27 and identify a point mutant in p27Kip1 that is deficient for interaction (R90G). In vivo and in vitro, the loss‐of‐interaction mutant is poorly transported into the nucleus, while ubiquitylation of p27R90G occurs normally. In vivo, co‐expression of cyclin E and Cdk2 rescues the import defect. However, mutant p27Kip1 accumulates in a phosphorylated form in the nucleus and is not efficiently degraded, arguing that at least one step in the degradation of phosphorylated p27Kip1 requires an interaction with the nuclear pore. Our results identify a novel component involved in p27Kip1 degradation and suggest that degradation of p27Kip1 is tightly linked to its intracellular transport.