The ins and outs of translation

A report on the 2nd EMBO Conference on Protein Synthesis and Translational Control, Heidelberg, Germany, 12-16 September 2007

Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA

The modified base 5-methylcytosine (m5C) is well studied in DNA, but investigations of its prevalence in cellular RNA have been largely confined to tRNA and rRNA. In animals, the two m5C methyltransferases NSUN2 and TRDMT1 are known to modify specific tRNAs and have roles in the control of cell growth and differentiation. To map modified cytosine sites across a human transcriptome, we coupled bisulfite conversion of cellular RNA with next-generation sequencing. We confirmed 21 of the 28 previously known m5C sites in human tRNAs and identified 234 novel tRNA candidate sites, mostly in anticipated structural positions. Surprisingly, we discovered 10 275 sites in mRNAs and other non-coding RNAs. We observed that distribution of modified cytosines between RNA types was not random; within mRNAs they were enriched in the untranslated regions and near Argonaute binding regions. We also identified five new sites modified by NSUN2, broadening its known substrate range to another tRNA, the RPPH1 subunit of RNase P and two mRNAs. Our data demonstrates the widespread presence of modified cytosines throughout coding and non-coding sequences in a transcriptome, suggesting a broader role of this modification in the post-transcriptional control of cellular RNA function

The estrogen and c-Myc target gene HSPC111 is over-expressed in breast cancer and associated with poor patient outcome

Introduction: Estrogens play a pivotal role in the initiation and progression of breast cancer. The genes that mediate these processes are not fully defined, but potentially include the known mammary oncogene MYC. Characterization of estrogen-target genes may help to elucidate further the mechanisms of estrogen-induced mitogenesis and endocrine resistance.Methods: We used a transcript profiling approach to identify targets of estrogen and c-Myc in breast cancer cells. One previously uncharacterized gene, namely HBV pre-S2 trans-regulated protein 3 (HSPC111), was acutely upregulated after estrogen treatment or inducible expression of c-Myc, and was selected for further functional analysis using over-expression and knock-down strategies. HSPC111 expression was also analyzed in relation to MYC expression and outcome in primary breast carcinomas and published gene expression datasets.Results: Pretreatment of cells with c-Myc small interfering RNA abrogated estrogen induction of HSPC111, identifying HSPC111 as a potential c-Myc target gene. This was confirmed by the demonstration of two functional E-box motifs upstream of the transcription start site. HSPC111 mRNA and protein were over-expressed in breast cancer cell lines and primary breast carcinomas, and this was positively correlated with MYC mRNA levels. HSPC111 is present in a large, RNA-dependent nucleolar complex, suggesting a possible role in ribosomal biosynthesis. Neither over-expression or small interfering RNA knock-down of HSPC111 affected cell proliferation rates or sensitivity to estrogen/antiestrogen treatment. However, high expression of HSPC111 mRNA was associated with adverse patient outcome in published gene expression datasets.Conclusion: These data identify HSPC111 as an estrogen and c-Myc target gene that is over-expressed in breast cancer and is associated with an adverse patient outcome

The role of poly(A) modifying enzymes as mRNA regulators in germ cells

RNA-modifizierende Enzyme sind ein wichtiger Teil der Genexpressionskaskade in eukaryotischen Zellen. Besonders interrassant sind hierbei die Enzymeklassen der Deadenylasen (DeAd) und zytoplasmatische poly(A) Polymerasen (cytoPAP) die direkt den poly(A) Schwanz am 3‘ Ende von mRNAs modifizieren. DeAds verkürzen und zytoPAPs verlängern diese Struktur und deren biologische Rollen wurden bisher nur unzureichend untersucht. In meiner Arbeit benutzte ich den Modellorganismus Caenorhabditis elegans, um die Bedeutung von DeAds und zytoPAPs für die Genexpressionsregulierung in sich entwickelnden Keimzellen zu studieren. Durch die Kombinierung von klassisch genetischen mit biochemischen und genomischen Herangehensweisen konnte ich die Schlüsselenzyme welche für die Regulierung von mRNAs in Keimzellen wichtig sind identifizieren und beschreiben. Meine gesammelten Daten zeigen wie RNA-modifizierende Enzyme gezielt in der Entwicklungsbiologie eingesetzt werden können.RNA modifying enzymes are an important part of the gene expression cascade in eukaryotic cells. Especially interesting are two opposing classes of enzymes that directly modify the mRNA 3’ poly(A) tail, known as deadenylases (DeAds) and cytoplasmic poly(A) polymerases (cytoPAP). DeAds shorten and cytoPAPs elongate the tail structure and their biological roles in multi-cellular organisms were only scarcely characterized. In my work, Caenorhabditis elegans is used as a model to investigate the roles of deadenylases and cytoplasmic poly(A) polymerases in regulating gene expression in developing germ cells. By combining classical genetic with biochemical and genomics approaches, I identified the specific enzymes that are the keys for mRNA regulation in germ cells. My combined data suggests that the opposing activities of tail modifiers provide an enzymatic framework that is employed by various gene-specific RNA-binding proteins to guide gene expression programs in germ cells

The role of the translational regulator p97 in mammalian cells

Members of the eukaryotic initiation factor 4G (eIF4G) family play a central role in the translation initiation process. One member of this family is p97 (also called DAP5 and NAT1), a protein that is highly homologous to the C-terminal two thirds of eIF4G. Overexpression studies suggested that p97 is a pure translational repressor that has to be cleaved into a shorter form called p86, in order to show translational activity. In this study a series of experiments indicated that full length p97 has a number elF property such as association with active translating ribosomes, stimulatory effects in the Direct Initiation Factor assay and accumulation in stress granules. Additionally the endogenous p97 complex was isolated from HeLa cells and mRNA as well as the protein components were characterized. P97 associated mRNAs were described by a custom made 5'UTR focus array, showing that the protein binds to a broad range of mRNA. The relative lack of mRNA specificity argues for a general role of p97 in translation, which does not seems to be essential in unchallenged cells, because a down regulation of p97 protein levels has no effect on the translational status of the bulk of mRNAs. Mass spectrometry analysis revealed a novel protein-protein interaction between p97 and DNA methyltransferase 1 (Dnmt1), which does not rely on a nucleic acid. For this interaction the C- and N-terminus of p97 play a critical role. Further, Dnmt1 has the ability to interact with elF4G and the small ribosomal subunit, which might provide evidence for a novel function of Dnmt1 in RNA metabolism

GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult <i>C. elegans</i> Germ Line

<div><p>To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult <i>Caenorhabditis elegans</i> reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that <i>gld-4</i> activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with <i>glp-1</i> mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of <i>glp-1</i> mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.</p></div

The eIF4G–homolog p97 can activate translation independent of caspase cleavage

The eukaryotic initiation factor (eIF) 4G family plays a central role during translation initiation, bridging between the 5′ and 3′ ends of the mRNA via its N-terminal third while recruiting other factors and ribosomes through its central and C-terminal third. The protein p97/NAT1/DAP5 is homologous to the central and C-terminal thirds of eIF4G. p97 has long been considered to be a translational repressor under normal cellular conditions. Further, caspase cleavage liberates a p86 fragment that is thought to mediate cap-independent translation in apoptotic cells. We report here that, surprisingly, human p97 is polysome associated in proliferating cells and moves to stress granules in stressed, nonapoptotic cells. Tethered-function studies in living cells show that human p97 and p86 both can activate translation; however, we were unable to detect polysome association of p86 in apoptotic cells. We further characterized the zebrafish orthologs of p97, and found both to be expressed throughout embryonic development. Their simultaneous knockdown by morpholino injection led to impaired mesoderm formation and early embryonic lethality, indicating conservation of embryonic p97 function from fish to mammals. These data indicate that full-length p97 is a translational activator with essential role(s) in unstressed cells, suggesting a reassessment of current models of p97 function

Differential GLD-4 and GLD-2 expression in the proliferative zone is FBF dependent.

<p>(A) GLD-4 expression is equal across the distal germ line. GLD-2 intensities increase from low-to-high in a distal-to-proximal manner. Extruded gonads of indicated genotype stained with DAPI, α-GLD-2, α-GLD-4, and α-GLH-2 as a positive tissue penetration control (not shown). Asterisk, distal tip; arrowhead, mitosis-to-meiosis boundary. (B,C) Distal GLD-2 expression is repressed by <i>fbf</i> activity. (B) Example of an <i>fbf</i>(RNAi) immunostained extruded gonad. For the complete RNAi experiment see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647.s001" target="_blank">Figure S1</a>. (C) Quantification of the complete <i>fbf</i>(RNAi) experiment. Four different regions of nine germ lines per genotype were analyzed in their median, primarily cytoplasmic area. Error bars are SEM. ***, p<0.001; **, p<0.01; *, p<0.05; bars without indicated p value are statistically not significant (Student's t-test). (D, E) FBF binds specifically to at least one of the five predicted sequence elements in the <i>gld-2</i> 3′UTR. (D) Schematic drawing of the 1094 nt long <i>gld-2</i> 3′UTR. Sequence alignment of FBF-binding element consensus (FBE cons.) sequence <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#pgen.1004647-Lamont1" target="_blank">[14]</a> and the conserved FBE4 element in three <i>Caenorhabditis</i> species: <i>ce</i>, <i>C. elegans</i>; <i>cb</i>, <i>C. briggsae</i>; <i>cr</i>, <i>C. remanei</i>. pA indicates beginning of the poly(A) tail. (E) Yeast three-hybrid assay. RNA hybrid and Gal4-protein fusions are indicated. FBF-1, FBF-2 and PUF-5 belong to same RNA-binding protein family. Note, the wild-type (wt) and mutant (mut) sequence of FBE4 tested is larger than the given sequences (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004647#s4" target="_blank">Materials and Methods</a>). A positive and negative control RNA was included (not shown) and protein expression was confirmed by western blotting (not shown). (F) LAP-tagged FBF-2 associates with endogenous <i>gld-2</i> mRNA in RNA-coimmunoprecipitation experiments (RIPs) directed against the GFP portion of the fusion protein.</p