Structural basis for activity switching in polymerases determining the fate of let-7 pre-miRNAs.

Tumor-suppressor let-7 pre-microRNAs (miRNAs) are regulated by terminal uridylyltransferases TUT7 and TUT4 that either promote let-7 maturation by adding a single uridine nucleotide to the pre-miRNA 3' end or mark them for degradation by the addition of multiple uridines. Oligo-uridylation is increased in cells by enhanced TUT7/4 expression and especially by the RNA-binding pluripotency factor LIN28A. Using cryogenic electron microscopy, we captured high-resolution structures of active forms of TUT7 alone, of TUT7 plus pre-miRNA and of both TUT7 and TUT4 bound with pre-miRNA and LIN28A.

Cyclin-dependent kinases: Masters of the eukaryotic universe.

A family of structurally related cyclin-dependent protein kinases (CDKs) drives many aspects of eukaryotic cell function. Much of the literature in this area has considered individual members of this family to act primarily either as regulators of the cell cycle, the context in which CDKs were first discovered, or as regulators of transcription. Until recently, CDK7 was the only clear example of a CDK that functions in both processes.

Koller and the dawn of cancer cytogenetics.

Shortly before the DNA era began, PC Koller described lagging chromosomes and chromosome numerical abnormalities in human carcinomas. While present-day cancer geneticists would question some of Koller's conclusions, this study ultimately contributed to the realisation that chromosomal instability is a widespread feature of solid tumours.

Effect of CFIm68 knockdown on RNA polymerase II transcription.

Objectives: Transcription of eukaryotic protein-coding genes by RNA polymerase II (pol II) is highly regulated at initiation, elongation and termination. Transcription is also coordinated with co-transcriptional processing of the emerging pre-mRNA by capping, splicing, and cleavage and polyadenylation. Polyadenylation (poly(A)) site recognition, which defines the end of the mRNA, relies on the cleavage and polyadenylation (CPA) complex.

Effect of CFIm25 knockout on RNA polymerase II transcription.

Objectives: Transcription of eukaryotic protein-coding genes by RNA polymerase II (pol II) is a highly regulated process. Most human genes have multiple poly(A) sites, which define different possible mRNA ends, suggesting the existence of mechanisms that regulate which poly(A) site is used. Poly(A) site selection may be mediated by cleavage factor I (CFIm), which is part of the cleavage and polyadenylation (CPA) complex.

Cleavage factor Im (CFIm) as a regulator of alternative polyadenylation.

Most mammalian protein coding genes are subject to alternative cleavage and polyadenylation (APA), which can generate distinct mRNA 3'UTRs with differing regulatory potential. Although this process has been intensely studied in recent years, it remains unclear how and to what extent cleavage site selection is regulated under different physiological conditions. The cleavage factor Im (CFIm) complex is a core component of the mammalian cleavage machinery, and the observation that its depletion causes transcriptome-wide changes in cleavage site use makes it a key candidate regulator of APA.

Improved crystallization and diffraction of caffeine-induced death suppressor protein 1 (Cid1).

The post-transcriptional addition of uridines to the 3'-end of RNAs is an important regulatory process that is critical for coding and noncoding RNA stability. In fission yeast and metazoans this untemplated 3'-uridylylation is catalysed by a single family of terminal uridylyltransferases (TUTs) whose members are adapted to specific RNA targets. In Schizosaccharomyces pombe the TUT Cid1 is responsible for the uridylylation of polyadenylated mRNAs, targeting them for destruction.

Structural plasticity of Cid1 provides a basis for its distributive RNA terminal uridylyl transferase activity.

Terminal uridylyl transferases (TUTs) are responsible for the post-transcriptional addition of uridyl residues to RNA 3' ends, leading in some cases to altered stability. The Schizosaccharomyces pombe TUT Cid1 is a model enzyme that has been characterized structurally at moderate resolution and provides insights into the larger and more complex mammalian TUTs, ZCCHC6 and ZCCHC11. Here, we report a higher resolution (1.

Cytoplasmic RNA: a case of the tail wagging the dog.

The addition of poly(A) tails to eukaryotic nuclear mRNAs promotes their stability, export to the cytoplasm and translation. Subsequently, the balance between exonucleolytic deadenylation and selective re-establishment of translation-competent poly(A) tails by cytoplasmic poly(A) polymerases is essential for the appropriate regulation of gene expression from oocytes to neurons. In recent years, surprising roles for cytoplasmic poly(A) polymerase-related enzymes that add uridylyl, rather than adenylyl, residues to RNA 3' ends have also emerged.

The long and short of microRNA.

MicroRNAs (miRNAs) are versatile regulators of gene expression in higher eukaryotes. In order to silence many different mRNAs in a precise manner, miRNA stability and efficacy is controlled by highly developed regulatory pathways and fine-tuning mechanisms both affecting miRNA processing and altering mature miRNA target specificity.

RNA decay via 3' uridylation.

The post-transcriptional addition of non-templated nucleotides to the 3' ends of RNA molecules can have a profound impact on their stability and biological function. Evidence accumulated over the past few decades has identified roles for polyadenylation in RNA stabilisation, degradation and, in the case of eukaryotic mRNAs, translational competence. By contrast, the biological significance of RNA 3' modification by uridylation has only recently started to become apparent.

Structural basis for the activity of a cytoplasmic RNA terminal uridylyl transferase.

Cytoplasmic terminal uridylyl transferases comprise a conserved family of enzymes that negatively regulate the stability or biological activity of a variety of eukaryotic RNAs, including mRNAs and tumor-suppressor let-7 microRNAs. Here we describe crystal structures of the Schizosaccharomyces pombe cytoplasmic terminal uridylyl transferase Cid1 in two apo conformers and bound to UTP. We demonstrate that a single histidine residue, conserved in mammalian Cid1 orthologs, is responsible for discrimination between UTP and ATP.

Transcriptional and cellular responses to defective mitochondrial proteolysis in fission yeast.

Lon and m-AAA are the principal, regulated proteases required for protein maturation and turnover in the mitochondrial matrix of diverse species. To understand their roles in fission yeast (Schizosaccharomyces pombe) mitochondria, we generated deletion strains lacking Lon and m-AAA, individually (Δlon1 and Δm-AAA) or together, Δlon1Δm-AAA (Δ/Δ). All three strains were viable but incapable of respiratory growth on a non-fermentable carbon source due to mitochondrial dysfunction.

The human cytoplasmic RNA terminal U-transferase ZCCHC11 targets histone mRNAs for degradation.

Inhibition of eukaryotic DNA replication leads to the rapid suppression of histone synthesis, via 3' uridylation of cytoplasmic histone mRNAs followed by their Lsm1-7-mediated decapping and degradation. Here we show that the human cytoplasmic RNA terminal U-transferase ZCCHC11, recently implicated in microRNA metabolism, associates with replication-dependent histone mRNAs. Knockdown of ZCCHC11 selectively blocked histone mRNA degradation following inhibition of DNA replication, whereas knockdown of PAPD1 or PAPD5, previously proposed as candidate histone mRNA U-transferases, had no such effect.

3' Uridylation and the regulation of RNA function in the cytoplasm.

Degradation of cytoplasmic mRNAs is an important aspect of the regulation of gene function in eukaryotes. Much of what is currently known about the underlying pathways of mRNA decay is derived from studies of the budding yeast Saccharomyces cerevisiae, in which mRNA turnover is initiated by deadenylation, followed either by decapping and 5'-->3' degradation or by further 3'-->5' exonucleolysis. Our studies using RNA cRACE (circularization-based rapid amplification of cDNA ends) techniques indicate that mRNA decapping in the fission yeast Schizosaccharomyces pombe often does not require prior deadenylation.

Polyadenylation and beyond: emerging roles for noncanonical poly(A) polymerases.

The addition of nontemplated nucleotides, particularly adenylyl and uridylyl residues, to the 3' ends of RNA substrates has been the focus of much attention in recent years, and these studies have generated some intriguing surprises. In addition to the well-known canonical poly(A) polymerase (PAP) that polyadenylates mRNAs prior to export from the nucleus to the cytoplasm, a separate class of noncanonical poly(A) polymerases has emerged over the past decade. Studies on various organisms have led to the realization that these noncanonical PAPs, which are conserved from yeast to mammals, play crucial and diverse roles in the regulation of gene expression.

Vgl1, a multi-KH domain protein, is a novel component of the fission yeast stress granules required for cell survival under thermal stress.

Multiple KH-domain proteins, collectively known as vigilins, are evolutionarily highly conserved proteins that are present in eukaryotic organisms from yeast to metazoa. Proposed roles for vigilins include chromosome segregation, messenger RNA (mRNA) metabolism, translation and tRNA transport. As a step toward understanding its biological function, we have identified the fission yeast vigilin, designated Vgl1, and have investigated its role in cellular response to environmental stress.

Decapping is preceded by 3' uridylation in a novel pathway of bulk mRNA turnover.

Both end structures of eukaryotic mRNAs, namely the 5' cap and 3' poly(A) tail, are necessary for transcript stability, and loss of either is sufficient to stimulate decay. mRNA turnover is classically thought to be initiated by deadenylation, as has been particularly well described in Saccharomyces cerevisiae. Here we describe two additional, parallel decay pathways in the fission yeast Schizosaccharomyces pombe.

Telomere maintenance: all's well that ends well.

The nucleoprotein structures termed telomeres serve to prevent the mis-identification of eukaryotic chromosome ends as sites of DNA damage, but are also among the genomic regions that pose the most problems during DNA replication. Here, we summarize some of the apparent difficulties encountered by the DNA replication machinery when it approaches the chromosome ends. Eukaryotic cells have evolved diverse mechanisms to overcome these problems, underlining the importance of telomere maintenance for a number of aspects of chromosome function.

The Cid1 poly(U) polymerase.

The Schizosaccharomyces pombe cytoplasmic protein Cid1 acts as a poly(U) polymerase (PUP). Polyadenylated actin mRNA, a target of this activity, is uridylated upon arrest in S phase and is likely to be one of many such Cid1 targets. This RNA uridylation pathway appears to be conserved, as Cid1 orthologs in Arabidopsis thaliana, Caenorhabditis elegans and humans display PUP activity either in vitro or in Xenopus laevis oocytes.

Non-invasive prenatal diagnosis of single gene disorders: how close are we?

Analysis of cell free fetal DNA (cffDNA) in maternal plasma provides the opportunity for reliable, timely, safe and cost-effective diagnosis of single gene disorders. The detection of certain fetal loci using cffDNA and conventional molecular analytic approaches is possible from 4 weeks gestation. To date, non-invasive first-trimester analysis for single gene disorders has been limited by assay sensitivity and specificity, due to the background maternal DNA.

The INT6 cancer gene and MEK signaling pathways converge during zebrafish development.

Background: Int-6 (integration site 6) was identified as an oncogene in a screen of tumorigenic mouse mammary tumor virus (MMTV) insertions. INT6 expression is altered in human cancers, but the precise role of disrupted INT6 in tumorigenesis remains unclear, and an animal model to study Int-6 physiological function has been lacking.

Principal Findings: Here, we create an in vivo model of Int6 function in zebrafish, and through genetic and chemical-genetic approaches implicate Int6 as a tissue-specific modulator of MEK-ERK signaling.

A randomized phase II pharmacokinetic and pharmacodynamic study of indisulam as second-line therapy in patients with advanced non-small cell lung cancer.

Purpose: The primary aim of this study was to measure the objective tumor response rate following treatment with indisulam [E7070; N-(3-chloro-7-indolyl)-1,4-benzenedisulfonamide] as second-line therapy in patients with advanced non-small cell lung cancer. The secondary aims were to determine progression-free survival, to assess the safety and tolerability of indisulam, and to study its pharmacokinetic and pharmacodynamic profile.

Experimental Design: Patients were randomized to receive indisulam every 3 weeks either as a single i.