Hot off the presses! Jan 15 Mol Cell

The Jan 15 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:

• RNA Switches Out in the Cold
  - Mol Cell 37(1):1-2 (2010)
  In this issue of Molecular Cell, Giuliodori et al. (2010) describe a cold-induced genetic switch made of RNA. Could thermo-sensing RNAs be among the most common types of RNA genetic switches?
• Nuclear Poly(A)-Binding Proteins and Nuclear Degradation: Take the mRNA and Run?
  - Mol Cell 37(1):3-5 (2010)
  Recent work from Lemay et al. (2010) in this issue of Molecular Cell reveals a role for a nuclear poly(A)-binding protein in promoting degradation of small nucleolar RNAs (snoRNAs) by the nuclear exosome.
• Histone Acetylation by HBO1 Tightens Replication Licensing
  - Mol Cell 37(1):5-6 (2010)
  In this issue of Molecular Cell, Miotto and Struhl (2010) suggest that replication licensing, the loading of Mcm2-7 onto DNA, is promoted by HBO1 acetylating histone H4 at replication origins, providing a molecular view of how chromatin status influences origin usage.
• The CRISPR System: Small RNA-Guided Defense in Bacteria and Archaea
  - Mol Cell 37(1):7-19 (2010)
  All cellular systems evolve ways to combat predators and genomic parasites. In bacteria and archaea, numerous resistance mechanisms have developed against phage. Our understanding of this defensive repertoire has recently been expanded to include the CRISPR system of clustered, regularly interspaced short palindromic repeats. In this remarkable pathway, short sequence tags from invading genetic elements are actively incorporated into the host's CRISPR locus to be transcribed and processed into a set of small RNAs that guide the destruction of foreign genetic material. Here we review the inner workings of this adaptable and heritable immune system and draw comparisons to small RNA-guided defense mechanisms in eukaryotic cells.
• The cspA mRNA Is a Thermosensor that Modulates Translation of the Cold-Shock Protein CspA
  - Mol Cell 37(1):21-33 (2010)
  Cold induction of cspA, the paradigm Escherichia coli cold-shock gene, is mainly subject to posttranscriptional control, partly promoted by cis-acting elements of its transcript, whose secondary structure at 37°C and at cold-shock temperature has been elucidated here by enzymatic and chemical probing. The structures, which were also validated by mutagenesis, demonstrate that cspA mRNA undergoes a temperature-dependent structural rearrangement, likely resulting from stabilization in the cold of an otherwise thermodynamically unstable folding intermediate. At low temperature, the "cold-shock" structure is more efficiently translated and somewhat less susceptible to degradation than the 37°C structure. Overall, our data shed light on a molecular mechanism at the basis of the cold-shock response, indicating that cspA mRNA is able to sense temperature downshifts, adopting functionally distinct structures at different temperatures, even without the aid of trans-acting ! factors. Unlike with other previously studied RNA thermometers, these structural rearrangements do not result from melting of hairpin structures.
• The Nuclear Poly(A)-Binding Protein Interacts with the Exosome to Promote Synthesis of Noncoding Small Nucleolar RNAs
  - Mol Cell 37(1):34-45 (2010)
  Poly(A)-binding proteins (PABPs) are important to eukaryotic gene expression. In the nucleus, the PABP PABPN1 is thought to function in polyadenylation of pre-mRNAs. Deletion of fission yeast pab2, the homolog of mammalian PABPN1, results in transcripts with markedly longer poly(A) tails, but the nature of the hyperadenylated transcripts and the mechanism that leads to RNA hyperadenylation remain unclear. Here we report that Pab2 functions in the synthesis of noncoding RNAs, contrary to the notion that PABPs function exclusively on protein-coding mRNAs. Accordingly, the absence of Pab2 leads to the accumulation of polyadenylated small nucleolar RNAs (snoRNAs). Our findings suggest that Pab2 promotes poly(A) tail trimming from pre-snoRNAs by recruiting the nuclear exosome. This work unveils a function for the nuclear PABP in snoRNA synthesis and provides insights into exosome recruitment to polyadenylated RNAs.
• A Subset of the Histone H3 Lysine 9 Methyltransferases Suv39h1, G9a, GLP, and SETDB1 Participate in a Multimeric Complex
  - Mol Cell 37(1):46-56 (2010)
  Lysine 9 of histone 3 (H3K9) can be mono-, di-, or trimethylated, inducing distinct effects on gene expression and chromatin compaction. H3K9 methylation can be mediated by several histone methyltransferases (HKMTs) that possess mono-, di-, or trimethylation activities. Here we provide evidence that a subset of each of the main H3K9 HKMTs, G9a/KMT1C, GLP/KMT1D, SETDB1/KMT1E, and Suv39h1/KMT1A, coexist in the same megacomplex. Moreover, in Suv39h or G9a null cells, the remaining HKMTs are destabilized at the protein level, indicating that the integrity of these HKMTs is interdependent. The four HKMTs are recruited to major satellite repeats, a known Suv39h1 genomic target, but also to multiple G9a target genes. Moreover, we report a functional cooperation between the four H3K9 HKMTs in the regulation of known G9a target genes. Altogether, our data identify a H3K9 methylation multimeric complex.
• HBO1 Histone Acetylase Activity Is Essential for DNA Replication Licensing and Inhibited by Geminin
  - Mol Cell 37(1):57-66 (2010)
  HBO1, an H4-specific histone acetylase, is a coactivator of the DNA replication licensing factor Cdt1. HBO1 acetylase activity is required for licensing, because a histone acetylase (HAT)-defective mutant of HBO1 bound at origins is unable to load the MCM complex. H4 acetylation at origins is cell-cycle regulated, with maximal activity at the G1/S transition, and coexpression of HBO1 and Jade-1 increases histone acetylation and MCM complex loading. Overexpression of the Set8 histone H4 tail-binding domain specifically inhibits MCM loading, suggesting that histones are a physiologically relevant target for licensing. Lastly, Geminin inhibits HBO1 acetylase activity in the context of a Cdt1-HBO1 complex, and it associates with origins and inhibits H4 acetylation and licensing in vivo. Thus, H4 acetylation at origins by HBO1 is critical for replication licensing by Cdt1, and negative regulation of licensing by Geminin is likely to involve inhibition of HBO1 histone acetyl! ase activity.
• Mitochondrial RNA Polymerase Is Needed for Activation of the Origin of Light-Strand DNA Replication
  - Mol Cell 37(1):67-78 (2010)
  Mitochondrial DNA is replicated by a unique enzymatic machinery, which is distinct from the replication apparatus used for copying the nuclear genome. We examine here the mechanisms of origin-specific initiation of lagging-strand DNA synthesis in human mitochondria. We demonstrate that the mitochondrial RNA polymerase (POLRMT) is the primase required for initiation of DNA synthesis from the light-strand origin of DNA replication (OriL). Using only purified POLRMT and DNA replication factors, we can faithfully reconstitute OriL-dependent initiation in vitro. Leading-strand DNA synthesis is initiated from the heavy-strand origin of DNA replication and passes OriL. The single-stranded OriL is exposed and adopts a stem-loop structure. At this stage, POLRMT initiates primer synthesis from a poly-dT stretch in the single-stranded loop region. After about 25 nt, POLRMT is replaced by DNA polymerase γ, and DNA synthesis commences. Our findings demonstrate that POLRMT can func! tion as an origin-specific primase in mammalian mitochondria.
• The Molecular Chaperone Hsp90 Regulates Accumulation of DNA Polymerase η at Replication Stalling Sites in UV-Irradiated Cells
  - Mol Cell 37(1):79-89 (2010)
  DNA polymerase η (Pol η) is a member of the mammalian Y family polymerases and performs error-free translesion synthesis across UV-damaged DNA. For this function, Pol η accumulates in nuclear foci at replication stalling sites via its interaction with monoubiquitinated PCNA. However, little is known about the posttranslational control mechanisms of Pol η, which regulate its accumulation in replication foci. Here, we report that the molecular chaperone Hsp90 promotes UV irradiation-induced nuclear focus formation of Pol η through control of its stability and binding to monoubiquitinated PCNA. Our data indicate that Hsp90 facilitates the folding of Pol η into an active form in which PCNA- and ubiquitin-binding regions are functional. Furthermore, Hsp90 inhibition potentiates UV-induced cytotoxicity and mutagenesis in a Pol η-dependent manner. Our studies identify Hsp90 as an essential regulator of Pol η-mediated translesion synthesis.
• Primase Directs the Release of DnaC from DnaB
  - Mol Cell 37(1):90-101 (2010)
  An AAA+ ATPase, DnaC, delivers DnaB helicase at the E. coli chromosomal origin by a poorly understood process. This report shows that mutant proteins bearing alanine substitutions for two conserved arginines in a motif named box VII are defective in DNA replication, but this deficiency does not arise from impaired interactions with ATP, DnaB, or single-stranded DNA. Despite their ability to deliver DnaB to the chromosomal origin to form the prepriming complex, this intermediate is inactive. Quantitative analysis of the prepriming complex suggests that the DnaB-DnaC complex contains three DnaC monomers per DnaB hexamer and that the interaction of primase with DnaB and primer formation triggers the release of DnaC, but not the mutants, from DnaB. The interaction of primase with DnaB and the release of DnaC mark discrete events in the transition from initiation to the elongation stage of DNA replication.
• Substrate-Assisted Inhibition of Ubiquitin-like Protein-Activating Enzymes: The NEDD8 E1 Inhibitor MLN4924 Forms a NEDD8-AMP Mimetic In Situ
  - Mol Cell 37(1):102-111 (2010)
  The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strat! egy for selective inhibition of UBL conjugation pathways.
• Pin1At Encoding a Peptidyl-Prolyl cis/trans Isomerase Regulates Flowering Time in Arabidopsis
  - Mol Cell 37(1):112-122 (2010)
  Floral transition in plants is regulated by an integrated network of flowering genetic pathways. We show that an Arabidopsis PIN1-type parvulin 1, Pin1At, controls floral transition by accelerating cis/trans isomerization of the phosphorylated Ser/Thr-Pro motifs in two MADS-domain transcription factors, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and AGAMOUS-LIKE 24 (AGL24). Pin1At regulates flowering, which is genetically mediated by AGL24 and SOC1. Pin1At interacts with the phosphorylated AGL24 and SOC1 in vitro and with AGL24 and SOC1 in vivo and accelerates the cis/trans conformational change of phosphorylated Ser/Thr-Pro motifs of AGL24 and SOC1. We further demonstrate that these Ser/Thr-Pro motifs are important for Pin1At function in promoting flowering through AGL24 and SOC1 and that the interaction between Pin1At and AGL24 mediates the AGL24 stability in the nucleus. Taken together, we propose that phosphorylation-dependent prolyl cis/trans isomerization of key! transcription factors is an important flowering regulatory mechanism.
• TMEPAI, a Transmembrane TGF-β-Inducible Protein, Sequesters Smad Proteins from Active Participation in TGF-β Signaling
  - Mol Cell 37(1):123-134 (2010)
  Transforming growth factor-β (TGF-β) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-β signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-β signaling, antagonizes TGF-β signaling by interfering with TGF-β type I receptor (TβRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TβRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-β-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-β-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-β. Consisten! tly, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-β/Smad signaling.
• A Mammalian Herpesvirus Uses Noncanonical Expression and Processing Mechanisms to Generate Viral MicroRNAs
  - Mol Cell 37(1):135-142 (2010)
  Canonical primary microRNA (pri-miRNA) precursors are transcribed by RNA polymerase II and then processed by the Drosha endonuclease to generate 60 nt pre-miRNA hairpins. Pre-miRNAs in turn are cleaved by Dicer to generate mature miRNAs. Previously, some short introns, called miRtrons, were reported to fold into pre-miRNA hairpins after splicing and debranching, and miRNAs can also be excised by Dicer cleavage of rare endogenous short hairpin RNAs. Here we report that the miRNAs encoded by murine γ-herpesvirus 68 (MHV68) are also generated via atypical mechanisms. Specifically, MHV68 miRNAs are transcribed from RNA polymerase III promoters located within adjacent viral tRNA-like sequences. The resultant pri-miRNAs, which bear a 5′ tRNA moiety, are not processed by Drosha but instead by cellular tRNase Z, which cleaves 3′ to the tRNA to liberate pre-miRNA hairpins that are then processed by Dicer to yield the mature viral miRNAs.
• CRL4Cdt2 E3 Ubiquitin Ligase Monoubiquitinates PCNA to Promote Translesion DNA Synthesis
  - Mol Cell 37(1):143-149 (2010)
  Monoubiquitination of proliferating cell nuclear antigen (PCNA) is a critical posttranslational modification essential for DNA repair by translesion DNA synthesis (TLS). The Rad18 E3 ubiquitin ligase cooperates with the E2 Rad6 to monoubiquitinate PCNA in response to DNA damage. How PCNA is monoubiquitinated in unperturbed cells and whether this plays a role in the repair of DNA associated with replication is not known. We show that the CRL4Cdt2 E3 ubiquitin ligase complex promotes PCNA monoubiqutination in proliferating cells in the absence of external DNA damage independent of Rad18. PCNA monoubiquitination via CRL4Cdt2 is constitutively antagonized by the action of the ubiquitin-specific protease 1 (USP1). In vitro, CRL4Cdt2 monoubiquitinates PCNA at Lys164, the same residue that is monoubiquitinated by Rad18. Significantly, CRL4Cdt2 is required for TLS in nondamaged cells via a mechanism that is dependent on PCNA monoubiquitination. We propose that CRL4Cdt2 regulat! es PCNA-dependent TLS associated with stresses accompanying DNA replication.