Hot off the presses! Apr 24 Mol Cell

The Apr 24 issue of the Mol Cell is now up on Pubget (About Mol Cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Targeting of mRNAs to Their Sites of Unconventional Splicing in the Unfolded Protein Response
  - Mol Cell 34(2):133-134 (2009)
  In this issue of Molecular Cell (Yanagitani et al., 2009) and recently published in Nature (Aragon et al., 2009) are reports on the importance of mRNA localization for unconventional splicing in the unfolded protein response, but the two papers suggests very different mechanisms in mammals and yeast.
• The APC/C: A Smörgåsbord for Proteolysis
  - Mol Cell 34(2):135-136 (2009)
  In a recent issue of Molecular Cell, Matyskiela and Morgan (2009) identify the sites on the APC/C that are required for activation and substrate binding, providing insights into how the APC/C works, with implications for the spindle assembly checkpoint that regulates it.
• Systems Biology Makes It Personal
  - Mol Cell 34(2):137-138 (2009)
  Systems biology promises to personalize medicine via network-based biomarkers that predict therapeutic effectiveness. Toward this goal, Chang et al. (2009) recently introduced a systems-based approach to break down oncogenic signaling networks into modules that predict the effectiveness of pathway-specific therapeutics.
• Protein Phosphorylation Goes Negative
  - Mol Cell 34(2):139-140 (2009)
  Analyzing global protein phosphorylation by mass spectrometry remains a challenging task. To this end, Old et al. (2009) recently described a strategy that improves the coverage of phosphorylated sites and employed it to identify B-Raf-dependent phosphorylation sites in melanoma cells.
• Hidesaburo Hanafusa 1929–2009
  - Mol Cell 34(2):141-143 (2009)
  
• Direct Binding of CoREST1 to SUMO-2/3 Contributes to Gene-Specific Repression by the LSD1/CoREST1/HDAC Complex
  - Mol Cell 34(2):145-154 (2009)
  Posttranslational modification of transcription factors by the small ubiquitin-related modifier SUMO is associated with transcriptional repression, but the underlying mechanisms remain incompletely described. We have identified binding of the LSD1/CoREST1/HDAC corepressor complex to SUMO-2. Here we show that CoREST1 binds directly and noncovalently to SUMO-2, but not SUMO-1, and CoREST1 bridges binding of the histone demethylase LSD1 to SUMO-2. Depletion of SUMO-2/3 conjugates led to transcriptional derepression, reduced occupancy of CoREST1 and LSD1, and changes in histone methylation and acetylation at some, but not all, LSD1/CoREST1/HDAC target genes. We have identified a nonconsensus SUMO-interaction motif (SIM) in CoREST1 required for SUMO-2 binding, and we show that mutation of the CoREST1 SIM disrupted SUMO-2 binding and transcriptional repression of some neuronal-specific genes in nonneuronal cells. Our results reveal that direct interactions between CoREST1 an! d SUMO-2 mediate SUMO-dependent changes in chromatin structure and transcription that are important for cell-type-specific gene expression.
• An Alpha Motif at Tas3 C Terminus Mediates RITS cis Spreading and Promotes Heterochromatic Gene Silencing
  - Mol Cell 34(2):155-167 (2009)
  RNA interference (RNAi) plays a pivotal role in the formation of heterochromatin at the fission yeast centromeres. The RNA-induced transcriptional silencing (RITS) complex, composed of heterochromatic small interfering RNAs (siRNAs), the siRNA-binding protein Ago1, the chromodomain protein Chp1, and the Ago1/Chp1-interacting protein Tas3, provides a physical tether between the RNAi and heterochromatin assembly pathways. Here, we report the structural and functional characterization of a C-terminal Tas3 α-helical motif (TAM), which self-associates into a helical polymer and is required for cis spreading of RITS in centromeric DNA regions. Site-directed mutations of key residues within the hydrophobic monomer-monomer interface disrupt Tas3-TAM polymeric self-association in vitro and result in loss of gene silencing, spreading of RITS, and a dramatic reduction in centromeric siRNAs in vivo. These results demonstrate that, in addition to the chromodomain of Chp1 and siRNA! -loaded Ago1, Tas3 self-association is required for RITS spreading and efficient heterochromatic gene silencing at centromeric repeat regions.
• Rtr1 Is a CTD Phosphatase that Regulates RNA Polymerase II during the Transition from Serine 5 to Serine 2 Phosphorylation
  - Mol Cell 34(2):168-178 (2009)
  Messenger RNA processing is coupled to RNA polymerase II (RNAPII) transcription through coordinated recruitment of accessory proteins to the Rpb1 C-terminal domain (CTD). Dynamic changes in CTD phosphorylation during transcription elongation are responsible for their recruitment, with serine 5 phosphorylation (S5-P) occurring toward the 5′ end of genes and serine 2 phosphorylation (S2-P) occurring toward the 3′ end. The proteins responsible for regulation of the transition state between S5-P and S2-P CTD remain elusive. We show that a conserved protein of unknown function, Rtr1, localizes within coding regions, with maximum levels of enrichment occurring between the peaks of S5-P and S2-P RNAPII. Upon deletion of Rtr1, the S5-P form of RNAPII accumulates in both whole-cell extracts and throughout coding regions; additionally, RNAPII transcription is decreased, and termination defects are observed. Functional characterization of Rtr1 reveals its role as a CTD phosph! atase essential for the S5-to-S2-P transition.
• SR Protein Family Members Display Diverse Activities in the Formation of Nascent and Mature mRNPs In Vivo
  - Mol Cell 34(2):179-190 (2009)
  The SR proteins are a family of pre-mRNA splicing factors with additional roles in gene regulation. To investigate individual family members in vivo, we generated a comprehensive panel of stable cell lines expressing GFP-tagged SR proteins under endogenous promoter control. Recruitment of SR proteins to nascent FOS RNA was transcription dependent and RNase sensitive, with unique patterns of accumulation along the gene specified by the RNA recognition motifs (RRMs). In addition, all SR protein interactions with Pol II were RNA dependent, indicating that SR proteins are not preassembled with Pol II. SR protein interactions with RNA were confirmed in situ by FRET/FLIM. Interestingly, SC35-GFP also exhibited FRET with DNA and failed to associate with cytoplasmic mRNAs, whereas all other SR proteins underwent nucleocytoplasmic shuttling and associated with specific nuclear and cytoplasmic mRNAs. Because different constellations of SR proteins bound nascent, nuclear, and cyt! oplasmic mRNAs, mRNP remodeling must occur throughout an mRNA's lifetime.
• Cotranslational Targeting of XBP1 Protein to the Membrane Promotes Cytoplasmic Splicing of Its Own mRNA
  - Mol Cell 34(2):191-200 (2009)
  Endoplasmic reticulum (ER) stress triggers the cytoplasmic splicing of XBP1 mRNA by the transmembrane endoribonuclease IRE1α, resulting in activation of the unfolded protein response, which maintains ER homeostasis. We show that the unspliced XBP1 (XBP1u) mRNA is localized to the membrane, although its product is neither a secretory nor a membrane protein and is released to the cytosol after splicing. Biochemical and mutagenic analyses demonstrated that membrane localization of XBP1u mRNA required its in-frame translation. An insertional frame-shift mutation greatly diminished both membrane localization and splicing of the XBP1u mRNA. Furthermore, membrane localization was compromised by puromycin treatment and required a hydrophobic region within XBP1u. These data demonstrate that the nascent XBP1u polypeptide recruits its own mRNA to the membrane. This system serves to enhance cytoplasmic splicing and could facilitate a more rapid response to ER stress, and represen! ts a unique way of cotranslational protein targeting coupled to mRNA maturation.
• The Plasticity of a Translation Arrest Motif Yields Insights into Nascent Polypeptide Recognition inside the Ribosome Tunnel
  - Mol Cell 34(2):201-211 (2009)
  The recognition of a C-terminal motif in E. coli SecM (150FXXXXWIXXXXGIRAGP166) inside the ribosome tunnel causes translation arrest, but the mechanism of recognition is unknown. Whereas single mutations in this motif impair recognition, we demonstrate that new arrest-inducing peptides can be created through remodeling of the SecM C terminus. We found that R163 is indispensable but that flanking residues that vary in number and position play an important secondary role in translation arrest. The observation that individual SecM variants showed a distinct pattern of crosslinking to ribosomal proteins suggests that each peptide adopts a unique conformation inside the tunnel. Based on the results, we propose that translation arrest occurs when the peptide conformation specified by flanking residues moves R163 into a precise intratunnel location. Our data indicate that translation arrest results from extensive communication between SecM and the tunnel and help to explain t! he striking diversity of arrest-inducing peptides found throughout nature.
• Misfolded Membrane Proteins Are Specifically Recognized by the Transmembrane Domain of the Hrd1p Ubiquitin Ligase
  - Mol Cell 34(2):212-222 (2009)
  Quality control pathways such as ER-associated degradation (ERAD) employ a small number of factors to specifically recognize a wide variety of protein substrates. Delineating the mechanisms of substrate selection is a principle goal in studying quality control. The Hrd1p ubiquitin ligase mediates ERAD of numerous misfolded proteins including soluble, lumenal ERAD-L and membrane-anchored ERAD-M substrates. We tested if the Hrd1p multispanning membrane domain was involved in ERAD-M specificity. In this work, we have identified site-directed membrane domain mutants of Hrd1p impaired only for ERAD-M and normal for ERAD-L. Furthermore, other Hrd1p variants were specifically deficient for degradation of individual ERAD-M substrates. Thus, the Hrd1p transmembrane region bears determinants of high specificity in the ERAD-M pathway. From in vitro and interaction studies, we suggest a model in which the Hrd1p membrane domain employs intramembrane residues to evaluate substrate m! isfolding, leading to selective ubiquitination of appropriate ERAD-M clients.
• Structural Insights into Formation of an Active Signaling Complex between Rac and Phospholipase C Gamma 2
  - Mol Cell 34(2):223-233 (2009)
  Rho family GTPases are important cellular switches and control a number of physiological functions. Understanding the molecular basis of interaction of these GTPases with their effectors is crucial in understanding their functions in the cell. Here we present the crystal structure of the complex of Rac2 bound to the split pleckstrin homology (spPH) domain of phospholipase C-γ2 (PLCγ2). Based on this structure, we illustrate distinct requirements for PLCγ2 activation by Rac and EGF and generate Rac effector mutants that specifically block activation of PLCγ2, but not the related PLCβ2 isoform. Furthermore, in addition to the complex, we report the crystal structures of free spPH and Rac2 bound to GDP and GTPγS. These structures illustrate a mechanism of conformational switches that accompany formation of signaling active complexes and highlight the role of effector binding as a common feature of Rac and Cdc42 interactions with a variety of effectors.
• The Structure of a Receptor with Two Associating Transmembrane Domains on the Cell Surface: Integrin αIIbβ3
  - Mol Cell 34(2):234-249 (2009)
  Structures of intact receptors with single-pass transmembrane domains are essential to understand how extracellular and cytoplasmic domains regulate association and signaling through transmembrane domains. A chemical and computational method to determine structures of the membrane regions of such receptors on the cell surface is developed here and validated with glycophorin A. An integrin heterodimer structure reveals association over most of the lengths of the α and β transmembrane domains and shows that the principles governing association of hetero and homo transmembrane dimers differ. A turn at the Gly of the juxtamembrane GFFKR motif caps the α TM helix and brings the two Phe of GFFKR into the α/β interface. A juxtamembrane Lys residue in β also has an important role in the interface. The structure shows how transmembrane association/dissociation regulates integrin signaling. A joint ectodomain and membrane structure shows that substantial flexibility betwee! n the extracellular and TM domains is compatible with TM signaling.
• Global Regulators Orchestrate Group II Intron Retromobility
  - Mol Cell 34(2):250-256 (2009)
  Group II introns are hypothesized to share common ancestry with both nuclear spliceosomal introns and retrotransposons, which collectively occupy the majority of genome space in higher eukaryotes. These phylogenetically diverse introns are mobile retroelements that move through an RNA intermediate. Disruption of Escherichia coli genes encoding enzymes that catalyze synthesis of global regulators cAMP and ppGpp inhibits group II intron retromobility. These small molecules program genetic transitions between nutrient excess and starvation. Accordingly, we demonstrated that glucose depletion of wild-type cells and cAMP supplementation of mutants stimulated retromobility. Likewise, amino acid starvation, which induces the alarmone ppGpp, activated retromobility. In both cases, retrotransposition to ectopic sites was favored over retrohoming. Interestingly, these stimulatory effects are mediated at the level of the DNA target, rather than of expression of the retroelement. ! Thereby, during metabolic stress, cAMP and ppGpp control group II intron movement in concert with the cell's global genetic circuitry, stimulating genetic diversity.