Molecular Cell

The Nuclear-Retained Noncoding RNA MALAT1 Regulates Alternative Splicing by Modulating SR Splicing Factor Phosphorylation.

Publication Date: 2010 Aug 25 PMID: 20797886
Authors: Tripathi, V. - Ellis, J. D. - Shen, Z. - Song, D. Y. - Pan, Q. - Watt, A. T. - Freier, S. M. - Bennett, C. F. - Sharma, A. - Bubulya, P. A. - Blencowe, B. J. - Prasanth, S. G. - Prasanth, K. V.
Journal: Mol Cell

Alternative splicing (AS) of pre-mRNA is utilized by higher eukaryotes to achieve increased transcriptome and proteomic complexity. The serine/arginine (SR) splicing factors regulate tissue- or cell-type-specific AS in a concentration- and phosphorylation-dependent manner. However, the mechanisms that modulate the cellular levels of active SR proteins remain to be elucidated. In the present study, we provide evidence for a role for the long nuclear-retained regulatory RNA (nrRNA), MALAT1 in AS regulation. MALAT1 interacts with SR proteins and influences the distribution of these and other splicing factors in nuclear speckle domains. Depletion of MALAT1 or overexpression of an SR protein changes the AS of a similar set of endogenous pre-mRNAs. Furthermore, MALAT1 regulates cellular levels of phosphorylated forms of SR proteins. Taken together, our results suggest that MALAT1 regulates AS by modulating the levels of active SR proteins. Our results further highlight the role for an nrRNA in the regulation of gene expression.

post to: CiteULike

Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif.

Publication Date: 2010 Aug 27 PMID: 20797634
Authors: Matic, I. - Schimmel, J. - Hendriks, I. A. - van Santen, M. A. - van de Rijke, F. - van Dam, H. - Gnad, F. - Mann, M. - Vertegaal, A. C.
Journal: Mol Cell

Reversible protein modification by small ubiquitin-like modifiers (SUMOs) is critical for eukaryotic life. Mass spectrometry-based proteomics has proven effective at identifying hundreds of potential SUMO target proteins. However, direct identification of SUMO acceptor lysines in complex samples by mass spectrometry is still very challenging. We have developed a generic method for the identification of SUMO acceptor lysines in target proteins. We have identified 103 SUMO-2 acceptor lysines in endogenous target proteins. Of these acceptor lysines, 76 are situated in the SUMOylation consensus site [VILMFPC]KxE. Interestingly, eight sites fit the inverted SUMOylation consensus motif [ED]xK[VILFP]. In addition, we found direct mass spectrometric evidence for crosstalk between SUMOylation and phosphorylation with a preferred spacer between the SUMOylated lysine and the phosphorylated serine of four residues. In 16 proteins we identified a hydrophobic cluster SUMOylation motif (HCSM). SUMO conjugation of RanGAP1 and ZBTB1 via HCSMs is remarkably efficient.

post to: CiteULike

MMXD, a TFIIH-independent XPD-MMS19 protein complex involved in chromosome segregation.

Publication Date: 2010 Aug 27 PMID: 20797633
Authors: Ito, S. - Tan, L. J. - Andoh, D. - Narita, T. - Seki, M. - Hirano, Y. - Narita, K. - Kuraoka, I. - Hiraoka, Y. - Tanaka, K.
Journal: Mol Cell

Xeroderma pigmentosum group D (XPD) protein is one of the subunits of TFIIH that is required for nucleotide excision repair and transcription. We found a XPD protein complex containing MMS19 that was assumed to be a regulator of TFIIH. However, the MMS19-XPD complex did not contain any other subunits of TFIIH. Instead, it included FAM96B (now designated MIP18), Ciao1, and ANT2. MMS19, MIP18, and XPD localized to the mitotic spindle during mitosis. The siRNA-mediated knockdown of MMS19, MIP18, or XPD led to improper chromosome segregation and the accumulation of nuclei with abnormal shapes. In addition, the frequency of abnormal mitosis and nuclei was increased in XP-D and XP-D/CS patients' cells. These results indicate that the MMS19-XPD protein complex, now designated MMXD (MMS19-MIP18-XPD), is required for proper chromosome segregation, an abnormality of which could contribute to the pathogenesis in some cases of XP-D and XP-D/CS.

post to: CiteULike

A proteomic screen for nucleolar SUMO targets shows SUMOylation modulates the function of Nop5/Nop58.

Publication Date: 2010 Aug 27 PMID: 20797632
Authors: Westman, B. J. - Verheggen, C. - Hutten, S. - Lam, Y. W. - Bertrand, E. - Lamond, A. I.
Journal: Mol Cell

Posttranslational SUMO modification is an important mechanism of regulating protein function, especially in the cell nucleus. The nucleolus is the subnuclear organelle responsible for rRNA synthesis, processing, and assembly of the large and small ribosome subunits. Here, we have used SILAC-based quantitative proteomics to identify nucleolar SUMOylated proteins. This reveals a role for SUMOylation in the biogenesis and/or function of small nucleolar ribonucleoprotein complexes (snoRNPs) via the targeting of Nhp2 and Nop58. Using combined in vitro and in vivo approaches, both Nhp2 and Nop58 (also known as Nop5) are shown to be substrates for SUMOylation. Mutational analyses revealed the sites of modification on Nhp2 as K5, and on Nop58 as K467 and K497. Unlike Nop58 and Nhp2, the closely related Nop56 and 15.5K proteins appear not to be SUMO targets. SUMOylation is essential for high-affinity Nop58 binding to snoRNAs. This study provides direct evidence linking SUMO modification with snoRNP function.

post to: CiteULike

Replication termination at eukaryotic chromosomes is mediated by Top2 and occurs at genomic Loci containing pausing elements.

Publication Date: 2010 Aug 27 PMID: 20797631
Authors: Fachinetti, D. - Bermejo, R. - Cocito, A. - Minardi, S. - Katou, Y. - Kanoh, Y. - Shirahige, K. - Azvolinsky, A. - Zakian, V. A. - Foiani, M.
Journal: Mol Cell

Chromosome replication initiates at multiple replicons and terminates when forks converge. In E. coli, the Tus-TER complex mediates polar fork converging at the terminator region, and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes, termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies, we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs, counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S phase, and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs. We propose that in eukaryotes, replication fork barriers, Rrm3, and Top2 coordinate replication fork progression and fusion at TERs, thus counteracting abnormal genomic transitions.

post to: CiteULike

RNA polymerase I contains a TFIIF-related DNA-binding subcomplex.

Publication Date: 2010 Aug 27 PMID: 20797630
Authors: Geiger, S. R. - Lorenzen, K. - Schreieck, A. - Hanecker, P. - Kostrewa, D. - Heck, A. J. - Cramer, P.
Journal: Mol Cell

The eukaryotic RNA polymerases Pol I, II, and III use different promoters to transcribe different classes of genes. Promoter usage relies on initiation factors, including TFIIF and TFIIE, in the case of Pol II. Here, we show that the Pol I-specific subunits A49 and A34.5 form a subcomplex that binds DNA and is related to TFIIF and TFIIE. The N-terminal regions of A49 and A34.5 form a dimerization module that stimulates polymerase-intrinsic RNA cleavage and has a fold that resembles the TFIIF core. The C-terminal region of A49 forms a "tandem winged helix" (tWH) domain that binds DNA with a preference for the upstream promoter nontemplate strand and is predicted in TFIIE. Similar domains are predicted in Pol III-specific subunits. Thus, Pol I/III subunits that have no counterparts in Pol II are evolutionarily related to Pol II initiation factors and may have evolved to mediate promoter specificity and transcription processivity.

post to: CiteULike

Nuclear IKKbeta is an adaptor protein for IkappaBalpha ubiquitination and degradation in UV-induced NF-kappaB activation.

Publication Date: 2010 Aug 27 PMID: 20797629
Authors: Tsuchiya, Y. - Asano, T. - Nakayama, K. - Kato, T. Jr - Karin, M. - Kamata, H.
Journal: Mol Cell

Proinflammatory cytokines activate NF-kappaB using the IkappaB kinase (IKK) complex that phosphorylates inhibitory proteins (IkappaBs) at N-terminal sites resulting in their ubiquitination and degradation in the cytoplasm. Although ultraviolet (UV) irradiation does not lead to IKK activity, it activates NF-kappaB by an unknown mechanism through IkappaBalpha degradation without N-terminal phosphorylation. Here, we describe an adaptor function of nuclear IKKbeta in UV-induced IkappaBalpha degradation. UV irradiation induces the nuclear translocation of IkappaBalpha and association with IKKbeta, which constitutively interacts with beta-TrCP through heterogeneous ribonucleoprotein-U (hnRNP-U) leading to IkappaBalpha ubiquitination and degradation. Furthermore, casein kinase 2 (CK2) and p38 associate with IKKbeta and promote IkappaBalpha degradation by phosphorylation at C-terminal sites. Thus, nuclear IKKbeta acts as an adaptor protein for IkappaBalpha degradation in UV-induced NF-kappaB activation. NF-kappaB activated by the nuclear IKKbeta adaptor protein suppresses anti-apoptotic gene expression and promotes UV-induced cell death.

post to: CiteULike

The three-dimensional organization of polyribosomes in intact human cells.

Publication Date: 2010 Aug 27 PMID: 20797628
Authors: Brandt, F. - Carlson, L. A. - Hartl, F. U. - Baumeister, W. - Grunewald, K.
Journal: Mol Cell

Structural studies have provided detailed insights into different functional states of the ribosome and its interaction with factors involved in nascent peptide folding, processing, and targeting. However, how the translational machinery is organized spatially in native cellular environments is not yet well understood. Here we have mapped individual ribosomes in electron tomograms of intact human cells by template matching and determined the average structure of the ribosome in situ. Characteristic features of active ribosomes in the cellular environment were assigned to the tRNA channel, elongation factors, and additional densities near the peptide tunnel. Importantly, the relative spatial configuration of neighboring ribosomes in the cell is clearly nonrandom. The preferred configurations are specific for active polysomes and were largely abrogated in puromycin-treated control cells. The distinct neighbor orientations found in situ resemble configurations of bacterial polysomes in vitro, indicating a conserved supramolecular organization with implications for nascent polypeptide folding.

post to: CiteULike

Catalysis of lysine 48-specific ubiquitin chain assembly by residues in E2 and ubiquitin.

Publication Date: 2010 Aug 27 PMID: 20797627
Authors: Rodrigo-Brenni, M. C. - Foster, S. A. - Morgan, D. O.
Journal: Mol Cell

Protein ubiquitination is catalyzed by ubiquitin-conjugating enzymes (E2s) in collaboration with ubiquitin-protein ligases (E3s). This process depends on nucleophilic attack by a substrate lysine on a thioester bond linking the C terminus of ubiquitin to a cysteine in the E2 active site. Different E2 family members display specificity for lysines in distinct contexts. We addressed the mechanistic basis for this lysine selectivity in Ubc1, an E2 that catalyzes the ubiquitination of lysine 48 (K48) in ubiquitin, leading to the formation of K48-linked polyubiquitin chains. We identified a cluster of polar residues near the Ubc1 active site, as well as a residue in ubiquitin itself, that are required for catalysis of K48-specific ubiquitin ligation, but not for general activity toward other lysines. Our results suggest that the active site of Ubc1, as well as the surface of ubiquitin, contains specificity determinants that channel specific lysines to the central residues involved directly in catalysis.

post to: CiteULike

Identification of regulators of chaperone-mediated autophagy.

Publication Date: 2010 Aug 27 PMID: 20797626
Authors: Bandyopadhyay, U. - Sridhar, S. - Kaushik, S. - Kiffin, R. - Cuervo, A. M.
Journal: Mol Cell

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of cytosolic proteins in lysosomes that contributes to cellular quality control and becomes an additional source of amino acids when nutrients are scarce. A chaperone complex delivers CMA substrates to a receptor protein at the lysosomal membrane that assembles into multimeric translocation complexes. However, the mechanisms regulating this process remain, for the most part, unknown. In this work, we have identified two regulatory proteins, GFAP and EF1alpha, that mediate a previously unknown inhibitory effect of GTP on CMA. GFAP stabilizes the multimeric translocation complex against chaperone-mediated disassembly, whereas GTP-mediated release of EF1alpha from the lysosomal membrane promotes self-association of GFAP, disassembly of the CMA translocation complex, and the consequent decrease in CMA. The dynamic interactions of these two proteins at the lysosomal membrane unveil now a role for GTP as a negative regulator of CMA.

post to: CiteULike

Combined functional genomic and proteomic approaches identify a PP2A complex as a negative regulator of Hippo signaling.

Publication Date: 2010 Aug 27 PMID: 20797625
Authors: Ribeiro, P. S. - Josue, F. - Wepf, A. - Wehr, M. C. - Rinner, O. - Kelly, G. - Tapon, N. - Gstaiger, M.
Journal: Mol Cell

The Hippo (Hpo) pathway is a central determinant of tissue size in both Drosophila and higher organisms. The core of the pathway is a kinase cascade composed of an upstream kinase Hpo (MST1/2 in mammals) and a downstream kinase Warts (Wts, Lats1/2 in mammals), as well as several scaffold proteins, Sav, dRASSF, and Mats. Activation of the core kinase cassette results in phosphorylation and inactivation of the progrowth transcriptional coactivator Yki, leading to increased apoptosis and reduced tissue growth. The mechanisms that prevent inappropriate Hpo activation remain unclear, and in particular, the identity of the phosphatase that antagonizes Hpo is unknown. Using combined proteomic and RNAi screening approaches, we identify the dSTRIPAK PP2A complex as a major regulator of Hpo signaling. dSTRIPAK depletion leads to increased Hpo activatory phosphorylation and repression of Yki target genes in vivo, suggesting this phosphatase complex prevents Hpo activation during development.

post to: CiteULike

Hsp12 is an intrinsically unstructured stress protein that folds upon membrane association and modulates membrane function.

Publication Date: 2010 Aug 27 PMID: 20797624
Authors: Welker, S. - Rudolph, B. - Frenzel, E. - Hagn, F. - Liebisch, G. - Schmitz, G. - Scheuring, J. - Kerth, A. - Blume, A. - Weinkauf, S. - Haslbeck, M. - Kessler, H. - Buchner, J.
Journal: Mol Cell

Hsp12 of S. cerevisiae is upregulated several 100-fold in response to stress. Our phenotypic analysis showed that this protein is important for survival of a variety of stress conditions, including high temperature. In the absence of Hsp12, we observed changes in cell morphology under stress conditions. Surprisingly, in the cell, Hsp12 exists both as a soluble cytosolic protein and associated to the plasma membrane. The in vitro analysis revealed that Hsp12, unlike all other Hsps studied so far, is completely unfolded; however, in the presence of certain lipids, it adopts a helical structure. The presence of Hsp12 does not alter the overall lipid composition of the plasma membrane but increases membrane stability.

post to: CiteULike

STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking inflammation to cancer.

Publication Date: 2010 Aug 27 PMID: 20797623
Authors: Iliopoulos, D. - Jaeger, S. A. - Hirsch, H. A. - Bulyk, M. L. - Struhl, K.
Journal: Mol Cell

A transient inflammatory signal can initiate an epigenetic switch from nontransformed to cancer cells via a positive feedback loop involving NF-kappaB, Lin28, let-7, and IL-6. We identify differentially regulated microRNAs important for this switch and putative transcription factor binding sites in their promoters. STAT3, a transcription factor activated by IL-6, directly activates miR-21 and miR-181b-1. Remarkably, transient expression of either microRNA induces the epigenetic switch. MiR-21 and miR-181b-1, respectively, inhibit PTEN and CYLD tumor suppressors, leading to increased NF-kappaB activity required to maintain the transformed state. These STAT3-mediated regulatory circuits are required for the transformed state in diverse cell lines and tumor growth in xenografts, and their transcriptional signatures are observed in colon adenocarcinomas. Thus, STAT3 is not only a downstream target of IL-6 but, with miR-21, miR-181b-1, PTEN, and CYLD, is part of the positive feedback loop that underlies the epigenetic switch that links inflammation to cancer.

post to: CiteULike

Apollo-taking the lead in telomere protection.

Publication Date: 2010 Aug 27 PMID: 20797622
Authors: Sarthy, J. F. - Baumann, P.
Journal: Mol Cell

The single-stranded overhangs at the ends of telomeres are thought to be critical for telomere maintenance, but how they are generated has been largely unclear. Two studies (one in this issue of Molecular Cell, Wu et al., 2010) have now implicated the Apollo nuclease in maintaining the overhang specifically at those telomeres generated by leading-strand DNA synthesis.

post to: CiteULike

Termination at sTop2.

Publication Date: 2010 Aug 27 PMID: 20797621
Authors: Alver, R. C. - Bielinsky, A. K.
Journal: Mol Cell

In this issue of Molecular Cell, Fachinetti et al. provide the first comprehensive map of replication termination sites (TERs) in Saccharomyces cerevisiae (Fachinetti et al., 2010). Strikingly, the majority of TERs are occupied by topoisomerase 2, which shields these regions against genomic instability.

post to: CiteULike

GTP: gatekeeper for autophagy.

Publication Date: 2010 Aug 27 PMID: 20797620
Authors: Beau, I. - Codogno, P.
Journal: Mol Cell

Chaperone-mediated autophagy (CMA) is involved in cellular quality control, and in the response to stress, deterioration in CMA contributes to the aging phenotype and to various disorders. The paper by Bandyopadhyay et al. in this issue of Molecular Cell demonstrates the role of GTP binding by elongation factor 1 alpha (EF1alpha) in regulating the lysosomal uptake of CMA substrates.

post to: CiteULike

"Depupylation" of Prokaryotic Ubiquitin-like Protein from Mycobacterial Proteasome Substrates.

Publication Date: 2010 Aug 11 PMID: 20705495
Authors: Burns, K. E. - Cerda-Maira, F. A. - Wang, T. - Li, H. - Bishai, W. R. - Darwin, K. H.
Journal: Mol Cell

Ubiquitin (Ub) provides the recognition and specificity required to deliver proteins to the eukaryotic proteasome for destruction. Prokaryotic ubiquitin-like protein (Pup) is functionally analogous to Ub in Mycobacterium tuberculosis (Mtb), as it dooms proteins to the Mtb proteasome. Studies suggest that Pup and Ub do not share similar mechanisms of activation and conjugation to target proteins. Dop (deamidase of Pup; Mtb Rv2112c/MT2172) deamidates the C-terminal glutamine of Pup to glutamate, preparing it for ligation to target proteins by proteasome accessory factor A (PafA). While studies have shed light on the conjugation of Pup to proteins, it was not known if Pup could be removed from substrates in a manner analogous to the deconjugation of Ub from eukaryotic proteins. Here, we show that Mycobacteria have a "depupylase" activity provided by Dop. The discovery of a depupylase strengthens the parallels between the Pup- and Ub-tagging systems of prokaryotes and eukaryotes, respectively.

post to: CiteULike

S-nitrosylation of beta-catenin by eNOS-derived NO promotes VEGF-induced endothelial cell permeability.

Publication Date: 2010 Aug 13 PMID: 20705246
Authors: Thibeault, S. - Rautureau, Y. - Oubaha, M. - Faubert, D. - Wilkes, B. C. - Delisle, C. - Gratton, J. P.
Journal: Mol Cell

Disruption of adherens junctions between endothelial cells results in compromised endothelial barrier function and in altered angiogenesis. Nitric oxide (NO) produced by endothelial NO synthase (eNOS) is essential for increased vascular permeability induced by vascular endothelial growth factor (VEGF). However, the molecular mechanisms by which NO modulates endothelial permeability remain elusive. Here, we show that, within adherens junctions, beta-catenin is a substrate for S-nitrosylation by NO. Stimulation of endothelial cells with VEGF induces S-nitrosylation of beta-catenin, which is dependent on expression and activity of eNOS. Furthermore, VEGF-induced S-nitrosylation of beta-catenin is inhibited in eNOS(-/-) mice. We identify Cys619, located within the VE-cadherin interaction site, as the major S-nitrosylation locus in response to VEGF. Inhibition of S-nitrosylation at Cys619 prevents NO-dependent dissociation of beta-catenin from VE-cadherin and disassembly of adherens junction complexes and inhibits VEGF-stimulated endothelial permeability. Thus, we identify S-nitrosylation of beta-catenin as a modulator of intercellular contacts between endothelial cells.

post to: CiteULike

Spindle position is coordinated with cell-cycle progression through establishment of mitotic exit-activating and -inhibitory zones.

Publication Date: 2010 Aug 13 PMID: 20705245
Authors: Chan, L. Y. - Amon, A.
Journal: Mol Cell

How spatial information is translated into a chemical signal is a fundamental problem in all organisms. The spindle position checkpoint is a prime example of this problem. This checkpoint senses spindle position and, in budding yeast, inhibits the mitotic exit network (MEN), a signaling pathway that promotes exit from mitosis. We find that spindle position is sensed by a system composed of MEN-inhibitory and -activating zones and a sensor that moves between them. The MEN inhibitory zone is located in the mother cell, the MEN-activating zone in the bud, and the spindle pole body (SPB), where the components of the MEN reside, functions as the sensor. Only when an SPB escapes the MEN inhibitor Kin4 in the mother cell and moves into the bud where the MEN activator Lte1 resides can exit from mitosis occur. In this manner, spatial information is sensed and translated into a chemical signal.

post to: CiteULike

Sin1-mTORC2 suppresses rag and il7r gene expression through Akt2 in B cells.

Publication Date: 2010 Aug 13 PMID: 20705244
Authors: Lazorchak, A. S. - Liu, D. - Facchinetti, V. - Di Lorenzo, A. - Sessa, W. C. - Schatz, D. G. - Su, B.
Journal: Mol Cell

Mammalian target of rapamycin (mTOR) is an important mediator of phosphoinositol-3-kinase (PI3K) signaling. PI3K signaling regulates B cell development, homeostasis, and immune responses. However, the function and molecular mechanism of mTOR-mediated PI3K signaling in B cells has not been fully elucidated. Here we show that Sin1, an essential component of mTOR complex 2 (mTORC2), regulates B cell development. Sin1 deficiency results in increased IL-7 receptor (il7r) and RAG recombinase (rag1 and rag2) gene expression, leading to enhanced pro-B cell survival and augmented V(D)J recombinase activity. We further show that Akt2 specifically mediates the Sin1-mTORC2 dependent suppression of il7r and rag gene expression in B cells by regulating FoxO1 phosphorylation. Finally, we demonstrate that the mTOR inhibitor rapamycin induces rag expression and promotes V(D)J recombination in B cells. Our study reveals that the Sin1/mTORC2-Akt2 signaling axis is a key regulator of FoxO1 transcriptional activity in B cells.

post to: CiteULike

Phospholipase D2-dependent inhibition of the nuclear hormone receptor PPARgamma by cyclic phosphatidic acid.

Publication Date: 2010 Aug 13 PMID: 20705243
Authors: Tsukahara, T. - Tsukahara, R. - Fujiwara, Y. - Yue, J. - Cheng, Y. - Guo, H. - Bolen, A. - Zhang, C. - Balazs, L. - Re, F. - Du, G. - Frohman, M. A. - Baker, D. L. - Parrill, A. L. - Uchiyama, A. - Kobayashi, T. - Murakami-Murofushi, K. - Tigyi, G.
Journal: Mol Cell

Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists.

post to: CiteULike

Structural basis for the major role of O-phosphoseryl-tRNA kinase in the UGA-specific encoding of selenocysteine.

Publication Date: 2010 Aug 13 PMID: 20705242
Authors: Chiba, S. - Itoh, Y. - Sekine, S. - Yokoyama, S.
Journal: Mol Cell

The 21(st) amino acid, selenocysteine (Sec), is assigned to the codon UGA and is biosynthesized on the selenocysteine-specific tRNA (tRNA(Sec)) with the corresponding anticodon. In archaea/eukarya, tRNA(Sec) is ligated with serine by seryl-tRNA synthetase (SerRS), the seryl moiety is phosphorylated by O-phosphoseryl-tRNA kinase (PSTK), and the phosphate group is replaced with selenol by Sep-tRNA:Sec-tRNA synthase. PSTK selectively phosphorylates seryl-tRNA(Sec), while SerRS serylates both tRNA(Ser) and tRNA(Sec). In this study, we determined the crystal structures of the archaeal tRNA(Sec).PSTK complex. PSTK consists of two independent linker-connected domains, the N-terminal catalytic domain (NTD) and the C-terminal domain (CTD). The D-arm.CTD binding occurs independently of and much more strongly than the acceptor-arm.NTD binding. PSTK thereby distinguishes the characteristic D arm with the maximal stem and the minimal loop of tRNA(Sec) from the canonical D arm of tRNA(Ser), without interacting with the anticodon. This mechanism is essential for the UGA-specific encoding of selenocysteine.

post to: CiteULike

The DEAH box ATPases Prp16 and Prp43 cooperate to proofread 5' splice site cleavage during pre-mRNA splicing.

Publication Date: 2010 Aug 13 PMID: 20705241
Authors: Koodathingal, P. - Novak, T. - Piccirilli, J. A. - Staley, J. P.
Journal: Mol Cell

To investigate the mechanisms underlying accurate pre-mRNA splicing, we developed an in vitro assay sensitive to proofreading of 5' splice site cleavage. We inactivated spliceosomes by disrupting a metal-ligand interaction at the catalytic center and discovered that, when the DEAH box ATPase Prp16 was disabled, these spliceosomes catalyzed 5' splice site cleavage but at a reduced rate. Although Prp16 does not promote splicing of a genuine substrate until after 5' splice site cleavage, we found that Prp16 can associate with spliceosomes before 5' splice site cleavage, consistent with a role for Prp16 in proofreading 5' splice site cleavage. We established that Prp16-mediated rejection is reversible, necessitating a downstream discard pathway that we found requires the DEAH box ATPase Prp43, a spliceosome disassembly factor. These data indicate that spliceosomes distinguish slow substrates and that the mechanisms for establishing the fidelity of 5' splice site cleavage and exon ligation share a common ATP-dependent framework.

post to: CiteULike

Smad proteins bind a conserved RNA sequence to promote microRNA maturation by Drosha.

Publication Date: 2010 Aug 13 PMID: 20705240
Authors: Davis, B. N. - Hilyard, A. C. - Nguyen, P. H. - Lagna, G. - Hata, A.
Journal: Mol Cell

The signal transducers of the transforming growth factor beta (TGFbeta)/bone morphogenetic protein (BMP), the Smads, promote the expression of a subset of miRNAs by facilitating the cleavage reaction by Drosha. The mechanism that limits Smad-mediated processing to a selective group of miRNAs remained hitherto unexplored. In this study, we expand the number of TGFbeta/BMP-regulated miRNAs (T/B-miRs) to 20. Of interest, a majority of T/B-miRs contain a consensus sequence (R-SBE) within the stem region of the primary transcripts of T/B-miRs (pri-T/B-miRs). Here, we demonstrate that Smads directly bind the R-SBE. Mutation of the R-SBE abrogates TGFbeta/BMP-induced recruitment of Smads, Drosha, and DGCR8 to pri-T/B-miRs and impairs their processing, whereas introduction of R-SBE to unregulated pri-miRNAs is sufficient to recruit Smads and to allow regulation by TGFbeta/BMP. Thus, Smads are multifunctional proteins that modulate gene expression transcriptionally through DNA binding and posttranscriptionally through pri-miRNA binding and regulation of miRNA processing.

post to: CiteULike

The methyltransferase activity of Clr4Suv39h triggers RNAi independently of histone H3K9 methylation.

Publication Date: 2010 Aug 13 PMID: 20705239
Authors: Gerace, E. L. - Halic, M. - Moazed, D.
Journal: Mol Cell

In fission yeast, the pericentromeric dg and dh repeats are transcribed and give rise to small interfering RNAs (siRNAs) by a mechanism that depends on the Clr4(suv39h) histone H3 lysine 9 (H3K9) methyltransferase. Here, we show that Clr4 activity promotes the assembly of a tripartite complex composed of the Clr4-containing CLRC complex and complexes involved in siRNA generation. However, unlike dh siRNAs, dg siRNAs accumulate to near wild-type levels in cells with H3K9 substitutions that cannot be methylated. Thus, Clr4 activity controls siRNA amplification from the different repeat regions by different mechanisms, H3K9 methylation dependent versus independent. Furthermore, artificial tethering of Rik1, a core subunit of the CLRC complex, to a euchromatic RNA mediates RNAi-dependent silencing that partially bypasses the requirement for other CLRC subunits. These findings establish Rik1 as a key link between CLRC and RNAi and reveal distinct centromeric siRNA amplification mechanisms that depend on the Clr4 methyltransferase activity.

post to: CiteULike

Homologous recombination restarts blocked replication forks at the expense of genome rearrangements by template exchange.

Publication Date: 2010 Aug 13 PMID: 20705238
Authors: Lambert, S. - Mizuno, K. - Blaisonneau, J. - Martineau, S. - Chanet, R. - Freon, K. - Murray, J. M. - Carr, A. M. - Baldacci, G.
Journal: Mol Cell

Template switching induced by stalled replication forks has recently been proposed to underlie complex genomic rearrangements. However, the resulting models are not supported by robust physical evidence. Here, we analyzed replication and recombination intermediates in a well-defined fission yeast system that blocks replication forks. We show that, in response to fork arrest, chromosomal rearrangements result from Rad52-dependent nascent strand template exchange occurring during fork restart. This template exchange occurs by both Rad51-dependent and -independent mechanisms. We demonstrate that Rqh1, the BLM homolog, limits Rad51-dependent template exchange without affecting fork restart. In contrast, we report that the Srs2 helicase promotes both fork restart and template exchange. Our data demonstrate that template exchange occurs during recombination-dependent fork restart at the expense of genome rearrangements.

post to: CiteULike

Regulation of DNA repair through deSUMOylation and SUMOylation of replication protein A complex.

Publication Date: 2010 Aug 13 PMID: 20705237
Authors: Dou, H. - Huang, C. - Singh, M. - Carpenter, P. B. - Yeh, E. T.
Journal: Mol Cell

The replication protein A complex (RPA) plays a crucial role in DNA replication and damage response. However, it is not known whether this complex is regulated by the SUMOylation pathway. Here, we show that the 70 kDa subunit of RPA (RPA70) associates with a Sentrin/SUMO-specific protease, SENP6, in the nucleus to maintain RPA70 in a hypoSUMOylated state during S phase. Campothecin (CPT), an inducer of replication stress, dissociates SENP6 from RPA70, allowing RPA70 to be modified by a small ubiquitin-like modifier 2/3 (SUMO-2/3). RPA70 SUMOylation facilitates recruitment of Rad51 to the DNA damage foci to initiate DNA repair through homologous recombination (HR). Cell lines that expressed a RPA70 mutant that cannot be SUMOylated are defective in HR and have a marked increase in sensitivity to CPT. These results demonstrate that SUMOylation status of RPA70 plays a critical role in the regulation of DNA repair through homologous recombination.

post to: CiteULike

Gymnastics of molecular chaperones.

Publication Date: 2010 Aug 13 PMID: 20705236
Authors: Mayer, M. P.
Journal: Mol Cell

Molecular chaperones assist folding processes and conformational changes in many proteins. In order to do so, they progress through complex conformational cycles themselves. In this review, I discuss the diverse conformational dynamics of the ATP-dependent chaperones of the Hsp60, Hsp70, Hsp90, and Hsp100 families.

post to: CiteULike

A novel effector protein modulates response regulator activity without altering phosphorylation.

Publication Date: 2010 Aug 13 PMID: 20705235
Authors: Curtis, P. D. - Brun, Y. V.
Journal: Mol Cell

Genes positively regulated by the global transcriptional response regulator CtrA are not expressed during a life cycle stage of Caulobacter crescentus when the regulator is activated by phosphorylation. Gora et al. (2010), in this issue of Molecular Cell, have discovered a novel effector protein that prevents activation but not repression by the regulator without altering its phosphorylation.

post to: CiteULike

CBP80 choreographs the NMD two-step.

Publication Date: 2010 Aug 13 PMID: 20705234
Authors: Ivanov, P. - Anderson, P.
Journal: Mol Cell

In this issue of Molecular Cell, Hwang et al. (2010) show that the cap-binding protein CBP80 promotes nonsense-mediated decay (NMD) at two steps. In this dual capacity, CBP80 may facilitate essential communication between the premature termination codon (PTC) and the exon-junction complex (EJC) to trigger NMD.

post to: CiteULike

SMADs stimulate miRNA processing.

Publication Date: 2010 Aug 13 PMID: 20705233
Authors: Treiber, T. - Meister, G.
Journal: Mol Cell

MicroRNAs (miRNAs) are potent regulators of gene expression. Consequently, miRNA expression is tightly regulated itself. In this issue, Hata and coworkers provide mechanistic insights into how SMAD proteins regulated the biogenesis of a specific subset of human miRNAs.

post to: CiteULike

UPF1 association with the cap-binding protein, CBP80, promotes nonsense-mediated mRNA decay at two distinct steps.

Publication Date: 2010 Aug 13 PMID: 20691628
Authors: Hwang, J. - Sato, H. - Tang, Y. - Matsuda, D. - Maquat, L. E.
Journal: Mol Cell

Nonsense-mediated mRNA decay (NMD) is an mRNA surveillance mechanism that in mammals generally occurs upon recognition of a premature termination codon (PTC) during a pioneer round of translation. This round involves newly synthesized mRNA that is bound at its 5' end by the cap-binding protein (CBP) heterodimer CBP80-CBP20. Here we show that precluding the binding of the NMD factor UPF1 to CBP80 inhibits NMD at two steps: the association of SMG1 and UPF1 with the two eukaryotic release factors (eRFs) during SURF complex formation at a PTC, and the subsequent association of SMG1 and UPF1 with an exon-junction complex. We also demonstrate that UPF1 binds PTC-containing mRNA more efficiently than the corresponding PTC-free mRNA in a way that is promoted by the UPF1-CBP80 interaction. A unifying model proposes a choreographed series of protein-protein interactions occurring on an NMD target.

post to: CiteULike

K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody.

Publication Date: 2010 Aug 13 PMID: 20655260
Authors: Matsumoto, M. L. - Wickliffe, K. E. - Dong, K. C. - Yu, C. - Bosanac, I. - Bustos, D. - Phu, L. - Kirkpatrick, D. S. - Hymowitz, S. G. - Rape, M. - Kelley, R. F. - Dixit, V. M.
Journal: Mol Cell

Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin. We engineered a K11 linkage-specific antibody and use it to demonstrate that K11 chains are highly upregulated in mitotic human cells precisely when substrates of the ubiquitin ligase anaphase-promoting complex (APC/C) are degraded. These chains increased with proteasomal inhibition, suggesting they act as degradation signals in vivo. Inhibition of the APC/C strongly impeded the formation of K11-linked chains, suggesting that a single ubiquitin ligase is the major source of mitotic K11-linked chains. Our results underscore the importance of K11-linked ubiquitin chains as critical regulators of mitotic protein degradation.

post to: CiteULike

Apollo contributes to G overhang maintenance and protects leading-end telomeres.

Publication Date: 2010 Aug 27 PMID: 20619712
Authors: Wu, P. - van Overbeek, M. - Rooney, S. - de Lange, T.
Journal: Mol Cell

Mammalian telomeres contain a single-stranded 3' overhang that is thought to mediate telomere protection. Here we identify the TRF2-interacting factor Apollo as a nuclease that contributes to the generation/maintenance of this overhang. The function of mouse Apollo was determined using Cre-mediated gene deletion, complementation with Apollo mutants, and the TRF2-F120A mutant that cannot bind Apollo. Cells lacking Apollo activated the ATM kinase at their telomeres in S phase and showed leading-end telomere fusions. These telomere dysfunction phenotypes were accompanied by a reduction in the telomeric overhang signal. The telomeric functions of Apollo required its TRF2-interaction and nuclease motifs. Thus, TRF2 recruits the Apollo nuclease to process telomere ends synthesized by leading-strand DNA synthesis, thereby creating a terminal structure that avoids ATM activation and resists end-joining. These data establish that the telomeric overhang is required for the protection of telomeres from the DNA damage response.

post to: CiteULike

A cell-type-specific protein-protein interaction modulates transcriptional activity of a master regulator in Caulobacter crescentus.

Publication Date: 2010 Aug 13 PMID: 20598601
Authors: Gora, K. G. - Tsokos, C. G. - Chen, Y. E. - Srinivasan, B. S. - Perchuk, B. S. - Laub, M. T.
Journal: Mol Cell

Progression through the Caulobacter cell cycle is driven by the master regulator CtrA, an essential two-component signaling protein that regulates the expression of nearly 100 genes. CtrA is abundant throughout the cell cycle except immediately prior to DNA replication. However, the expression of CtrA-activated genes is generally restricted to S phase. We identify the conserved protein SciP (small CtrA inhibitory protein) and show that it accumulates during G1, where it inhibits CtrA from activating target genes. The depletion of SciP from G1 cells leads to the inappropriate induction of CtrA-activated genes and, consequently, a disruption of the cell cycle. Conversely, the ectopic synthesis of SciP is sufficient to inhibit CtrA-dependent transcription, also disrupting the cell cycle. SciP binds directly to CtrA without affecting stability or phosphorylation; instead, SciP likely prevents CtrA from recruiting RNA polymerase. CtrA is thus tightly regulated by a protein-protein interaction which is critical to cell-cycle progression.

post to: CiteULike