SummaryTranslation and decay of eukaryotic mRNAs is controlled by shortening of the poly(A) tail and release of the poly(A)-binding protein Pab1/PABP. The Ccr4-Not complex contains two exonucleases—Ccr4 and Caf1/Pop2—that mediate mRNA deadenylation. Here, using a fully reconstituted biochemical system with proteins from the fission yeast Schizosaccharomyces pombe, we show that Pab1 interacts with Ccr4-Not, stimulates deadenylation, and differentiates the roles of the nuclease enzymes. Surprisingly, Pab1 release relies on Ccr4 activity. In agreement with this, in vivo experiments in budding yeast show that Ccr4 is a general deadenylase that acts on all mRNAs. In contrast, Caf1 only trims poly(A) not bound by Pab1. As a consequence, Caf1 is a specialized deadenylase required for the selective deadenylation of transcripts with lower rates of translation elongation and reduced Pab1 occupancy. These findings reveal a coupling between the rates of translation and deadenylation that is dependent on Pab1 and Ccr4-Not.

Prokaryotic messenger RNAs (mRNAs) are translated as they are transcribed. The pioneering ribosome potentially contacts RNA polymerase (RNAP), forming a supramolecular complex known as the expressome. The basis of expressome assembly and its consequences for transcription and translation are poorly understood. Here we present a series of structures representing uncoupled, coupled and collided expressome states determined by electron cryomicroscopy. A bridge between the ribosome and RNAP can be formed by the transcription factor NusG, stabilizing an otherwise variable interaction interface. Shortening of the intervening mRNA causes a substantial rearrangement that aligns the ribosome entrance-channel to the RNAP exit-channel. In this collided complex, NusG-linkage is no longer possible. These structures reveal mechanisms of coordination between transcription and translation and provide a framework for future study.

After the IECE 802.1 1 standardization group established the first ABSTRACT wireless LAN standard two years ago, 5everaI efforts were start^ ed to increilsc data rates and also to use other bands. This article dcicribes the new wire^ less LAN standards develolped by IEEE 802.1 1, ETSI BRAN, and MMAC. l~hc new stdndards are targeting data rates up lo 1 1 Mh/s in the 2.4 GHz band and up to 54 Mbis in the 5 GH2 band. L i k e t h e 1EEE 802.1 1 s l a i i d a r d , tlic El,ropcan TcIeeolnnliIIIieeti~,Ils St:ind;irils Inslitutc (ETSI) IIIPU11-I A N lvnc I siandard 131 soccilics hiith iiicc tlic hcgiiiiiing o l (lie I9Ulls, wirclcss lnciil xrcn & networks (W I A N s) lor ilic 900 Mllz, 2, 4, iiiiil 5 (itlz iiiduslriiil, scicntific, ;ind iiiccliciil (ISM) haiiils liiivc hccn available Ixiscil on a raugc oC proprietary produeis. I n. l u w 10'17, the Institute ol Elcciric:il ;ind I?lccironics Eiigiiiccrs ;ipprovctl iin inlcrnalioo;il intcropcr~i1,ility si;indard (IliliF, 802. I I) [I], 71ic sl;i~ulard specifics hoth mcdium ~C C C S S contnrl iiid thrcc dillcrcni plrysicxl kiycrs (PlHY). Tlicrc arc two I;idi~~-lxisccl FHYs using thc 2.4 GHI hand. T l~c iliirtl I'IIY nscs iiifnircd lighl. i\ll I'llYs support ii daia ralc of 1 Mh/s i i n d opiionally 2 Mbls. The 2.4 CiHz lrcqtmicy Lxind is iiviiiliililc fur liccnsc cxcnipi ~i s c in H u r q x , tlic llnilcd Stalcs, aiid J:ipiii. 'I'iiblc I list& ilic availa1,lc frcqiicncy hiinds i i l d lhc rcstriciirriis to dcviccs wliicli iisc this h;ind lin c(iiniiiiiiiiciitiiiiis. Uscs dcm;ind lor liiglicr hit rates uid intcriiaiioiid availahilitp of the 2.4 GHz band has spiirrcd thc dcvelopniciit of a higher-spccd cxtcnsiiio tu thc 802. I I siandiird. In .

SummaryCcr4-Not is a conserved protein complex that shortens the 3′ poly(A) tails of eukaryotic mRNAs to regulate transcript stability and translation into proteins. RNA-binding proteins are thought to facilitate recruitment of Ccr4-Not to certain mRNAs, but lack of an in-vitro-reconstituted system has slowed progress in understanding the mechanistic details of this specificity. Here, we generate a fully recombinant Ccr4-Not complex that removes poly(A) tails from RNA substrates. The intact complex is more active than the exonucleases alone and has an intrinsic preference for certain RNAs. The RNA-binding protein Mmi1 is highly abundant in preparations of native Ccr4-Not. We demonstrate a high-affinity interaction between recombinant Ccr4-Not and Mmi1. Using in vitro assays, we show that Mmi1 accelerates deadenylation of target RNAs. Together, our results support a model whereby both RNA-binding proteins and the sequence context of mRNAs influence deadenylation rate to regulate gene expression.

We report the first experimental observation in the optical domain of a dramatic width-dependent lateral leakage loss behavior for the TM-like mode of tight vertical confinement ridge waveguides formed in silicon-on-insulator. The lateral leakage loss displays a series of sharp cyclic minima at precise waveguide widths, and appears to be inherent to waveguide geometries of central importance to a wide variety of active devices in silicon photonics requiring lateral electrical access. This behavior is not predicted by the often-used effective-index-based methods, but is understood phenomenologically and also compared to prior numerical analysis and predictions of leaky mode behavior. It is shown that TM-like mode operation, critical to the operation of some active component designs, will require precision control of waveguide dimensions to achieve high performance.

Approximate Bayesian Computation (ABC) is a popular computational method for likelihood-free Bayesian inference. The term "likelihoodfree" refers to problems where the likelihood is intractable to compute or estimate directly, but where it is possible to generate simulated data X relatively easily given a candidate set of parameters θ simulated from a prior distribution. Parameters which generate simulated data within some tolerance δ of the observed data x * are regarded as plausible, and a collection of such θ is used to estimate the posterior distribution θ | X = x * . Suitable choice of δ is vital for ABC methods to return good approximations to θ in reasonable computational time.While ABC methods are widely used in practice, particularly in population genetics, rigorous study of the mathematical properties of ABC estimators lags behind practical developments of the method. We prove that ABC estimates converge to the exact solution under very weak assumptions and, under slightly stronger assumptions, quantify the rate of this convergence. In particular, we show that the bias of the ABC estimate is asymptotically proportional to δ 2 as δ ↓ 0. At the same time, the computational cost for generating one ABC sample increases like δ −q where q is the dimension of the observations. Rates of convergence are obtained by optimally balancing the mean squared error against the computational cost. Our results can be used to guide the choice of the tolerance parameter δ. keywords: Approximate Bayesian Computation, likelihood-free inference, Monte Carlo methods, convergence of estimators, rate of convergence MSC2010 classes: 62F12 (Asymptotic properties of estimators), 62F15 (Bayesian inference), 65C05 (Monte Carlo methods)

The Ccr4-Not complex removes mRNA poly(A) tails to regulate eukaryotic mRNA stability and translation. RNA-binding proteins contribute to specificity by interacting with both Ccr4-Not and target mRNAs, but this is not fully understood. Here, we reconstitute accelerated and selective deadenylation of RNAs containing AU-rich elements (AREs) and Pumilio-response elements (PREs). We find that the fission yeast homologues of Tristetraprolin/TTP and Pumilio/Puf (Zfs1 and Puf3) interact with Ccr4-Not via multiple regions within low-complexity sequences, suggestive of a multipartite interface that extends beyond previously defined interactions. Using a two-color assay to simultaneously monitor poly(A) tail removal from different RNAs, we demonstrate that Puf3 can distinguish between RNAs of very similar sequence. Analysis of binding kinetics reveals that this is primarily due to differences in dissociation rate constants. Consequently, motif quality is a major determinant of mRNA stability for Puf3 targets in vivo and can be used for the prediction of mRNA targets.

Mutations in the Plasmodium falciparum ‘chloroquine resistance transporter’ (PfCRT) confer resistance to chloroquine (CQ) and related antimalarials by enabling the protein to transport these drugs away from their targets within the parasite’s digestive vacuole (DV). However, CQ resistance-conferring isoforms of PfCRT (PfCRTCQR) also render the parasite hypersensitive to a subset of structurally-diverse pharmacons. Moreover, mutations in PfCRTCQR that suppress the parasite’s hypersensitivity to these molecules simultaneously reinstate its sensitivity to CQ and related drugs. We sought to understand these phenomena by characterizing the functions of PfCRTCQR isoforms that cause the parasite to become hypersensitive to the antimalarial quinine or the antiviral amantadine. We achieved this by measuring the abilities of these proteins to transport CQ, quinine, and amantadine when expressed in Xenopus oocytes and complemented this work with assays that detect the drug transport activity of PfCRT in its native environment within the parasite. Here we describe two mechanistic explanations for PfCRT-induced drug hypersensitivity. First, we show that quinine, which normally accumulates inside the DV and therewithin exerts its antimalarial effect, binds extremely tightly to the substrate-binding site of certain isoforms of PfCRTCQR. By doing so it likely blocks the normal physiological function of the protein, which is essential for the parasite’s survival, and the drug thereby gains an additional killing effect. In the second scenario, we show that although amantadine also sequesters within the DV, the parasite’s hypersensitivity to this drug arises from the PfCRTCQR-mediated transport of amantadine from the DV into the cytosol, where it can better access its antimalarial target. In both cases, the mutations that suppress hypersensitivity also abrogate the ability of PfCRTCQR to transport CQ, thus explaining why rescue from hypersensitivity restores the parasite’s sensitivity to this antimalarial. These insights provide a foundation for understanding clinically-relevant observations of inverse drug susceptibilities in the malaria parasite.