A genetics screen highlights emerging roles for CPL3, RST1 and URT1 in RNA metabolism and silencing.

Post-transcriptional gene silencing (PTGS) is a major mechanism regulating gene expression in higher eukaryotes. To identify novel players in PTGS, a forward genetics screen was performed on an Arabidopsis thaliana line overexpressing a strong growth-repressive gene, ETHYLENE RESPONSE FACTOR6 (ERF6). We identified six independent ethyl-methanesulfonate mutants rescuing the dwarfism of ERF6-overexpressing plants as a result of transgene silencing. Among the causative genes, ETHYLENE-INSENSITIVE5, SUPERKILLER2 and HASTY1 have previously been reported to inhibit PTGS. Notably, the three other causative genes have not, to date, been related to PTGS: UTP:RNA-URIDYLYLTRANSFERASE1 (URT1), C-TERMINAL DOMAIN PHOSPHATASE-LIKE3 (CPL3) and RESURRECTION1 (RST1). We show that these genes may participate in protecting the 3' end of transgene transcripts. We present a model in which URT1, CPL3 and RST1 are classified as PTGS suppressors, as compromisation of these genes provokes the accumulation of aberrant transcripts which, in turn, trigger the production of small interfering RNAs, initiating RNA silencing.

PAPD5/7 Are Host Factors That Are Required for Hepatitis B Virus RNA Stabilization.

RG7834 is a potent, orally bioavailable small-molecule inhibitor of hepatitis B virus (HBV) gene expression that belongs to the dihydroquinolizinone (DHQ) chemical class and uniquely blocks production of both viral DNA and antigens. In this study, we used DHQ compounds as tools in a compound-based adaptation version of the yeast three-hybrid screen to identify the cognate cellular protein targets, the non-canonical poly(A) RNA polymerase associated domain containing proteins 5 and 7 (PAPD5 and PAPD7). Interaction with RG7834 was mapped to the catalytic domains of the two cellular enzymes. The role of PAPD5 and PAPD7 in HBV replication was confirmed by oligonucleotide-mediated knockdown studies that phenocopied the result seen with RG7834-treated HBV-infected hepatocytes. The greatest effect on HBV gene expression was seen when PAPD5 and PAPD7 mRNAs were simultaneously knocked down, suggesting that the two cellular proteins play a redundant role in maintaining HBV mRNA levels. In addition, as seen previously with RG7834 treatment, PAPD5 and PAPD7 knockdown led to destabilization and degradation of HBV mRNA without impacting production of viral RNA transcripts. Conclusion: We identify PAPD5 and PAPD7 as cellular host factors required for HBV RNA stabilization and as therapeutic targets for the HBV cure.

The RNA uridyltransferase Zcchc6 is expressed in macrophages and impacts innate immune responses.

Alveolar macrophages orchestrate pulmonary innate immunity and are essential for early immune surveillance and clearance of microorganisms in the airways. Inflammatory signaling must be sufficiently robust to promote host defense but limited enough to prevent excessive tissue injury. Macrophages in the lungs utilize multiple transcriptional and post-transcriptional mechanisms of inflammatory gene expression to delicately balance the elaboration of immune mediators. RNA terminal uridyltransferases (TUTs), including the closely homologous family members Zcchc6 (TUT7) and Zcchc11 (TUT4), have been implicated in the post-transcriptional regulation of inflammation from studies conducted in vitro. In vivo, we observed that Zcchc6 is expressed in mouse and human primary macrophages. Zcchc6-deficient mice are viable and born in Mendelian ratios and do not exhibit an observable spontaneous phenotype under basal conditions. Following an intratracheal challenge with S. pneumoniae, Zcchc6 deficiency led to a modest but significant increase in the expression of select cytokines including IL-6, CXCL1, and CXCL5. These findings were recapitulated in vitro whereby Zcchc6-deficient macrophages exhibited similar increases in cytokine expression due to bacterial stimulation. Although loss of Zcchc6 also led to increased neutrophil emigration to the airways during pneumonia, these responses were not sufficient to impact host defense against infection.

Essential role for non-canonical poly(A) polymerase GLD4 in cytoplasmic polyadenylation and carbohydrate metabolism.

Regulation of gene expression at the level of cytoplasmic polyadenylation is important for many biological phenomena including cell cycle progression, mitochondrial respiration, and learning and memory. GLD4 is one of the non-canonical poly(A) polymerases that regulates cytoplasmic polyadenylation-induced translation, but its target mRNAs and role in cellular physiology is not well known. To assess the full panoply of mRNAs whose polyadenylation is controlled by GLD4, we performed an unbiased whole genome-wide screen using poy(U) chromatography and thermal elution. We identified hundreds of mRNAs regulated by GLD4, several of which are involved in carbohydrate metabolism including GLUT1, a major glucose transporter. Depletion of GLD4 not only reduced GLUT1 poly(A) tail length, but also GLUT1 protein. GLD4-mediated translational control of GLUT1 mRNA is dependent of an RNA binding protein, CPEB1, and its binding elements in the 3΄ UTR. Through regulating GLUT1 level, GLD4 affects glucose uptake into cells and lactate levels. Moreover, GLD4 depletion impairs glucose deprivation-induced GLUT1 up-regulation. In addition, we found that GLD4 affects glucose-dependent cellular phenotypes such as migration and invasion in glioblastoma cells. Our observations delineate a novel post-transcriptional regulatory network involving carbohydrate metabolism and glucose homeostasis mediated by GLD4.

TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms.

Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single-molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre-miRNAs) in the absence of Lin28. We find that the overhang of a pre-miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre-miRNAs, which have a 1-nt 3' overhang, TUT7 restores the canonical end structure (2-nt 3' overhang) through mono-uridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 3' end is further recessed into the stem (as in 3' trimmed pre-miRNAs), TUT7 generates an oligo-U tail that leads to degradation. In contrast to Lin28-stimulated oligo-uridylation, which is processive, a distributive mode is employed by TUT7 for both mono- and oligo-uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7-RNA interaction, thus explaining how TUT7 differentiates pre-miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre-miRNAs.

TDP-43 toxicity and the usefulness of junk.

A new study shows that loss of the lariat debranching enzyme Dbr1 suppresses TDP-43 toxicity. The accumulated intronic lariat RNAs, which are normally degraded after splicing, likely act as decoys to sequester TDP-43 away from binding to and disrupting functions of other RNAs.

Lin28-mediated control of let-7 microRNA expression by alternative TUTases Zcchc11 (TUT4) and Zcchc6 (TUT7).

The pluripotency factor Lin28 recruits a 3' terminal uridylyl transferase (TUTase) to selectively block let-7 microRNA biogenesis in undifferentiated cells. Zcchc11 (TUTase4/TUT4) was previously identified as an enzyme responsible for Lin28-mediated pre-let-7 uridylation and control of let-7 expression. Here we investigate the protein and RNA determinants for this interaction. Biochemical dissection and reconstitution assays reveal the TUTase domains necessary and sufficient for Lin28-enhanced pre-let-7 uridylation. A single C2H2-type zinc finger domain of Zcchc11 was found to be responsible for the functional interaction with Lin28. We identify Zcchc6 (TUTase7) as an alternative TUTase that functions with Lin28 in vitro, and accordingly, we find Zcchc11 and Zcchc6 redundantly control let-7 biogenesis in embryonic stem cells. Our study indicates that Lin28 uses two different TUTases to control let-7 expression and has important implications for stem cell biology as well as cancer.

Reengineering CCA-adding enzymes to function as (U,G)- or dCdCdA-adding enzymes or poly(C,A) and poly(U,G) polymerases.

CCA-adding enzymes build and repair the 3'-terminal CCA sequence of tRNA. These unusual RNA polymerases use either a ribonucleoprotein template (class I) or pure protein template (class II) to form mock base pairs with the Watson-Crick edges of incoming CTP and ATP. Guided by the class II Bacillus stearothermophilus CCA-adding enzyme structure, we introduced mutations designed to reverse the polarity of hydrogen bonds between the nucleobases and protein template. We were able to transform the CCA-adding enzyme into a (U,G)-adding enzyme that incorporates UTP and GTP instead of CTP and ATP; we transformed the related Aquifex aeolicus CC- and A-adding enzymes into UU- and G-adding enzymes and Escherichia coli poly(A) polymerase into a poly(G) polymerase; and we transformed the B. stearothermophilus CCA-adding enzyme into a poly(C,A) polymerase by mutations in helix J that appear, based on the apoenzyme structure, to sterically limit addition to CCA. We also transformed the B. stearothermophilus CCA-adding enzyme into a dCdCdA-adding enzyme by mutating an arginine that interacts with the incoming ribose 2' hydroxyl. Most importantly, we found that mutations in helix J can affect the specificity of the nucleotide binding site some 20 A away, suggesting that the specificity of both class I and II enzymes may be dictated by an intricate network of hydrogen bonds involving the protein, incoming nucleotide, and 3' end of the tRNA. Collaboration between RNA and protein in the form of a ribonucleoprotein template may help to explain the evolutionary diversity of the nucleotidyltransferase family.

Dead-box proteins: a family affair--active and passive players in RNP-remodeling.

DEAD-box proteins are characterized by nine conserved motifs. According to these criteria, several hundreds of these proteins can be identified in databases. Many different DEAD-box proteins can be found in eukaryotes, whereas prokaryotes have small numbers of different DEAD-box proteins. DEAD-box proteins play important roles in RNA metabolism, and they are very specific and cannot mutually be replaced. In vitro, many DEAD-box proteins have been shown to have RNA-dependent ATPase and ATP-dependent RNA helicase activities. From the genetic and biochemical data obtained mainly in yeast, it has become clear that these proteins play important roles in remodeling RNP complexes in a temporally controlled fashion. Here, I shall give a general overview of the DEAD-box protein family.

A story with a good ending: tRNA 3'-end maturation by CCA-adding enzymes.

CCA-adding enzymes (tRNA nucleotidyltransferases) are responsible for the maturation or repair of the functional 3' end of tRNAs. These enzymes are remarkable because they polymerize the essential nucleotides CCA onto the 3' terminus of tRNA precursors without using a nucleic acid template. Recent crystal structures, plus three decades of enzymology, have revealed the elegant mechanisms by which CCA-adding enzymes achieve their substrate specificity in a nucleic acid template independent fashion. The class I CCA-adding enzyme employs both an arginine sidechain and backbone phosphates of the bound tRNA to recognize incoming nucleotides. It switches from C to A addition through changes in the size and shape of the nucleotide-binding pocket, which is progressively altered by the elongating 3' terminus of the tRNA. By contrast, the class II CCA-adding enzyme uses only amino acid sidechains, which form a protein template for incoming nucleotide selection.

General translational repression by activators of mRNA decapping.

Translation and mRNA degradation are affected by a key transition where eukaryotic mRNAs exit translation and assemble an mRNP state that accumulates into processing bodies (P bodies), cytoplasmic sites of mRNA degradation containing non-translating mRNAs, and mRNA degradation machinery. We identify the decapping activators Dhh1p and Pat1p as functioning as translational repressors and facilitators of P body formation. Strains lacking both Dhh1p and Pat1p show strong defects in mRNA decapping and P body formation and are blocked in translational repression. Contrastingly, overexpression of Dhh1p or Pat1p causes translational repression, P body formation, and arrests cell growth. Dhh1p, and its human homolog, RCK/p54, repress translation in vitro, and Dhh1p function is bypassed in vivo by inhibition of translational initiation. These results identify a broadly acting mechanism of translational repression that targets mRNAs for decapping and functions in translational control. We propose this mechanism is competitively balanced with translation, and shifting this balance is an important basis of translational control.

Directed evolution as a tool for understanding and optimizing nucleic acid polymerase function.

Polynucleotide polymerases play a crucial role in transmitting genetic information from generation to generation, and they are the most important reagents in biotechnology. Although classical crystal structure analyses as well as biochemical studies have significantly contributed to our understanding of how DNA polymerases function, surprising new insights regarding the importance of certain residues and protein motifs, or of their mutability have been achieved in recent years by evolutionary approaches. Directed evolution has also facilitated the generation of polymerases with tailored substrate repertoires or with stabilities and activities beyond those of their naturally evolved counterparts. Recent new insights in polymerase structure-function relationships and new achievements in the development of tailored polymerases for current methods of nucleic acid synthesis will be summarized in this article.

An Arabidopsis RNA lariat debranching enzyme is essential for embryogenesis.

An embryo-defective mutant of Arabidopsis thaliana was isolated that arrests development at a variety of stages, from as early as the globular stage of embryogenesis to as late as formation of an abnormal bent cotyledon stage embryo. Defects in the suspensor, a normally transient structure derived from the fertilized egg, were often associated with the arrested embryo. The lesion was within a gene encoding a protein with domains characteristic of lariat debranching enzymes, which has been named AtDBR1 (for Arabidopsis thaliana Debranching enzyme 1). Cleavage of the 2'-5'-phosphodiester bond found in excised intron lariats ("debranching") is essential for turnover of intronic sequences as well as generation of some small nucleolar RNAs. The mutation within AtDBR1 was confirmed by complementation as being responsible for the embryo-lethal phenotype, and the activity of the encoded protein in cleavage of 2'-5'-phosphodiester bonds was verified using an in vitro debranching assay.

A eubacterial origin for the human tRNA nucleotidyltransferase?

tRNA CCA-termini are generated and maintained by tRNA nucleotidyltransferases. Together with poly(A) polymerases and other enzymes they belong to the nucleotidyltransferase superfamily. However, sequence alignments within this family do not allow to distinguish between CCA-adding enzymes and poly(A) polymerases. Furthermore, due to the lack of sequence information about animal CCA-adding enzymes, identification of corresponding animal genes was not possible so far. Therefore, we looked for the human homolog using the baker's yeast tRNA nucleotidyltransferase as a query sequence in a BLAST search. This revealed that the human gene transcript CGI-47 (#AF151805) deposited in GenBank is likely to encode such an enzyme. To identify the nature of this protein, the cDNA of the transcript was cloned and the recombinant protein biochemically characterized, indicating that CGI-47 encodes a bona fide CCA-adding enzyme and not a poly(A) polymerase. This confirmed animal CCA-adding enzyme allowed us to identify putative homologs from other animals. Calculation of a neighbor-joining tree, using an alignment of several CCA-adding enzymes, revealed that the animal enzymes resemble more eubacterial ones than eukaryotic plant and fungal tRNA nucleotidyltransferases, suggesting that the animal nuclear cca genes might have been derived from the endosymbiotic progenitor of mitochondria and are therefore of eubacterial origin.

cgh-1, a conserved predicted RNA helicase required for gametogenesis and protection from physiological germline apoptosis in C. elegans.

A high frequency of apoptosis is a conserved hallmark of oocyte development. In C. elegans, about half of all developing oocytes are normally killed by a physiological germline-specific apoptosis pathway, apparently so that they donate cytoplasm to the survivors. We have investigated the functions of CGH-1, the C. elegans ortholog of the predicted RNA helicase ste13/ME31B/RCK/p54, which is germline-associated in metazoans and required for sexual reproduction in yeast. We show that CGH-1 is expressed specifically in the germline and early embryo, and is localized to P granules and other possible mRNA-protein particles. cgh-1 is required for oocyte and sperm function. It is also needed to prevent the physiological germline apoptosis mechanism killing essentially all developing oocytes, making lack of cgh-1 function the first stimulus identified that can trigger this mechanism. We conclude that cgh-1 and its orthologs may perform conserved functions during gametogenesis, that in C. elegans certain aspects of oocyte development are monitored by the physiological germline apoptosis pathway, and that similar surveillance mechanisms may contribute to germline apoptosis in other species.