More than just scanning: the importance of cap-independent mRNA translation initiation for cellular stress response and cancer

The scanning model for eukaryotic mRNA translation initiation states that the small ribosomal subunit, along with initiation factors, binds at the cap structure at the 5′ end of the mRNA and scans the 5′ untranslated region (5′UTR) until an initiation codon is found. However, under conditions that impair canonical cap-dependent translation, the synthesis of some proteins is kept by alternative mechanisms that are required for cell survival and stress recovery. Alternative modes of translation initiation include cap- and/or scanning-independent mechanisms of ribosomal recruitment. In most cap-independent translation initiation events there is a direct recruitment of the 40S ribosome into a position upstream, or directly at, the initiation codon via a specific internal ribosome entry site (IRES) element in the 5′UTR. Yet, in some cellular mRNAs, a different translation initiation mechanism that is neither cap- nor IRES-dependent seems to occur through a special RNA structure called cap-independent translational enhancer (CITE). Recent evidence uncovered a distinct mechanism through which mRNAs containing N ⁶-methyladenosine (m⁶A) residues in their 5′UTR directly bind eukaryotic initiation factor 3 (eIF3) and the 40S ribosomal subunit in order to initiate translation in the absence of the cap-binding proteins. This review focuses on the important role of cap-independent translation mechanisms in human cells and how these alternative mechanisms can either act individually or cooperate with other cis-acting RNA regulons to orchestrate specific translational responses triggered upon several cellular stress states, and diseases such as cancer. Elucidation of these non-canonical mechanisms reveals the complexity of translational control and points out their potential as prospective novel therapeutic targets.     

Nucleotide sequence of the gene for the mitochondrial 15S ribosomal RNA of yeast

We have determined the nucleotide sequence of a DNA segment carrying the entire 15S ribosomal RNA gene of yeast mitochondrial genome. Many stretches of sequence are present which are homologous to the E. coli 16S ribosomal RNA gene. The gene sequence can be folded into a secondary structure according to the [1] model on bacterial ribosomal RNAs. The structure reveals a striking similarity between the two RNAs despite the large difference in their base compositions. In the middle of the gene, we found a guanine-cytosine rich sequence that is also present in several other regions of the mitochondrial genome.     

Structure-function relationships of the polypyrimidine tract binding protein

The polypyrimidine tract binding protein (PTB) is a 58-kDa RNA binding protein involved in multiple aspects of mRNA metabolism including splicing regulation, polyadenylation, 3′end formation, internal ribosomal entry site-mediated translation, RNA localization and stability. PTB contains four RNA recognition motifs (RRMs) separated by three linkers. In this review we summarize structural information on PTB in solution that has been gathered during the past 7 years using NMR spectroscopy and small-angle X-ray scattering. The structures of all RRMs of PTB in their free state and in complex with short pyrimidine tracts, as well as a structural model of PTB RRM2 in complex with a peptide, revealed unusual structural features that provided new insights into the mechanisms of action of PTB in the different processes of RNA metabolism and in particular splicing regulation. Received 16 August 2007; received after revision 18 September 2007; accepted 2 October 2007     

Ribosomal gene numbers in Microseridinae (Compositae: Cichorieae)

Summary 8 species of the subtribe Microseridinae contain between 1100 and 3400 genes for 25 and 18 S ribosomal RNA. The gene numbers seem to evolve by discrete steps. Their trend follows a general reduction in genome size during the evolution of the annual species ofMicroseris, but numbers remain high in one of them and inAgoseris grandiflora. 2 species ofPyrrhopappus differ by a duplication of the ribosomal gene numbers; 5 S ribosomal RNA genes in 4 species are repeated roughly 10,000 times.We thank Miss S. Werner, Miss A. Roth and Miss U. Krehan for help with some of the experiments. This paper is part of a project supported by grant Ba 536/1-5 from the Deutsche Forschungsgemeinschaft.     

The reduction of genic activity in the tetraploid amphibianOdontophrynus americanus is not due to loss of ribosomal DNA

Summary Genic activity in tetraploid members of the amphibian speciesOdontophrymus americanus is reduced to that of diploid ones. Loss of ribosomal genes, a mechanism suggested by others as a means of decreasing genetic activity, could be ruled out. The diploids and the tetraploids have almost identical proportions of their genomes complementary to (28s+18s) ribosomal RNA.     

Interphase studies with a simplified method of silver staining of nucleoli.

A simple silver staining method is presented providing a rapid and reliable technique for the selective staining of nuclear structures synthesizing ribosomal RNA (18S and 28S RNA).     

Morphology of ribosomal RNA in chicken fibroblasts]

It is shown with the electron microscope that the 28 S RNA component of the ribosomal RNA extracted from Chicken fibroblasts contains secondary structures which are not present in the 18S component.     

Interphase studies with a simplified method of silver staining of nucleoli

Summary A simple silver staining method is presented providing a rapid and reliable technique for the selective staining of nuclear structures synthesizing ribosomal RNA (18S and 28S RNA).The critical advice of Prof. Dr W. Krone is gratefully acknowledged.     

Denaturation map of the ribosomal DNA of Lytechinus variegatus sperm.

Electron microscopy of the partially heat denatured ribosomal DNA (rDNA) from sea urchin (Lytechinus variegatus) sperm has demonstrated that it consists of repeating units of 3.6 +/- 0.2 micron, corresponding to a mol.wt of 7.2 +/- 0.4 x 10(6). Based on differential denaturability, each repeat unit is divided into 2 regions. The larger region of 2.47 +/- 0.11 micron (mol.wt 4.9 +/- 0.22 x 10(6)) corresponds in length to the ribosomal precursor RNA of sea urchins and the smaller, GC-rich, subunit of 1.16 +/- 0.09 micrometer (mol.wt 2.3 +/- 0.18 x 10(6)) is presumed to contain non-transcribed spacer sequences.     

A creature with a hundred waggly tails: intrinsically disordered proteins in the ribosome

Intrinsic disorder (i.e., lack of a unique 3-D structure) is a common phenomenon, and many biologically active proteins are disordered as a whole, or contain long disordered regions. These intrinsically disordered proteins/regions constitute a significant part of all proteomes, and their functional repertoire is complementary to functions of ordered proteins. In fact, intrinsic disorder represents an important driving force for many specific functions. An illustrative example of such disorder-centric functional class is RNA-binding proteins. In this study, we present the results of comprehensive bioinformatics analyses of the abundance and roles of intrinsic disorder in 3,411 ribosomal proteins from 32 species. We show that many ribosomal proteins are intrinsically disordered or hybrid proteins that contain ordered and disordered domains. Predicted globular domains of many ribosomal proteins contain noticeable regions of intrinsic disorder. We also show that disorder in ribosomal proteins has different characteristics compared to other proteins that interact with RNA and DNA including overall abundance, evolutionary conservation, and involvement in protein–protein interactions. Furthermore, intrinsic disorder is not only abundant in the ribosomal proteins, but we demonstrate that it is absolutely necessary for their various functions.     

Degradation of ribosomal RNA from Escherichia coli by ribonuclease U2 and in vitro transcription of "RNA-fragments" into complementary DNA]

Under well defined conditions ribosomal RNAs purified from Escherichia coli can be degraded by ribonuclease U2 giving rise to RNA fragments of 60--70 nucleotides. In vitro, these fragments are efficiently transcribed into a complementary DNA by DNA polymerase RNA dependent, partially purified from extracts of E. coli. In vivo, "RNA-fragments-U2" inhibit the development of plant tumors.     

RNA fragments, in vivo inhibitors of Shope fibroma and vaccinia virus multiplication]

RNA-fragments rich in purine nucleotides and resulting from degradation of ribosomal RNA from E. coli M 500 Sho-R with pancreatic RNase exhibit only in vivo an inhibitory effect on Shope fibroma and vaccinia virus multiplication.     

Unchanged r-RNA-gene dose in mice liver cells of different developmental stages.

The content of ribosomal DNA in mice liver at the beginning as well as near the end of the hematopoietic period was measured by RNA/DAN-hybridization in solution. At both stages the amount of ribosomal DNA was the same and comparable to that of postnatal liver.     

RNA fragments rich in G and A nucleotides obligatory for in vitro DNA replication of phages phi-X 174 and lambda]

Evidence was presented that in vitro conversion of single-stranded DNA of phage phi X 174 to the double-stranded replicative form by partially purified DNA-dependent DNA polymerase I requires a specific RNA fragment acting as primer (25-50 nucleotides). RNA fragments highly rich in nucleotides A and G were obtained by partial degradation of E. coli M 500 Sho-R ribosomal RNA with pancreatic ribonuclease. They become covalently bound to the newly synthesized DNA chain of the replicative form of phage phi X 174. These RNA fragments are also required for in vitro replication of lambda phage DNA.     

PCR jumping in clones of 30-million-year-old DNA fragments from amber preserved termites (Mastotermes electrodominicus)

DNA from 30-million-year-old amber preserved termites (Mastotermes electrodominicus) was PCR amplified with nuclear ribosomal RNA small subunit primers and cloned into the TA vector (INVITROGEN). We obtained several classes of recombinant clones as a result. AuthenticMastotermes electrodominicus clones were identified. The source of other classes of clones was identified as contaminants of the ancient DNA template. Several of the clones appeared to be chimeric in structure with half of the clone identical to the termite sequence and the other half identical to contaminant sequences. The phenomenon of PCR jumping was identified as a possible source for the chimeric clones.     

Incorporation of tritiated uridine during pachytene and diplotene stages in the oocytes of the Japanese quail (Coturnix coturnix japonica)

Summary Incorporation of³H-uridine was studied during pachytene and diplotene stages of quail oocytes. No labelling could be detected during early pachytene. During advanced and late pachytene, labelling simultaneously appeared on the macrochromosomes and on certain michromosomes in the zone where they emerge from the chromocentric surface periphery. The latter localization corresponds to the region of ribosomal RNA synthesis. At diplotene the same localizations were labelled with a considerably increased intensity.     

Experimentelle Untersuchungen zum Wirkungsmechanismus der beiden entwicklungsphysiologisch aktiven Fraktionen des «Gehirnhormons» der Insekten (Aktivationsfaktor I und II) auf die Prothoracaldrüse

Summary 2 prothoracotropical factors (activation factor I and II) have been obtained by gel filtration techniques from brains and corpora cardiaca of the cockroachPeriplaneta americana. In contrast to activation factor II, activation factor I caused significant influence of RNA synthesis. The RNA pattern of prothoracic glands stimulated by activation factor I as demonstrated by polyacrylamide gel electrophoresis consists of different kinds of RNA. Short time incubation revealed effects on sRNA synthesis, while long time incubation demonstrated predominantly increase of ribosomal RNA synthesis. Measurements of the membrane potential of the prothoracic gland cells of the wax mothGalleria mellonella indicated an increase by activation factor II; activation factor I was without any visible effect. Our results demonstrate for the first time that the two activation factors induce different effects at cellular level.

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Für technische Unterstützung danken wir FräuleinA. Zinsser und FrauR. Meissner.

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Durchgeführt mit Mitteln des Ministeriums für Wissenschaft und Technik und mit Unterstützung durch die Sächsische Akademie der Wissenschaften zu Leipzig.     

Structure and function of Zika virus NS5 protein: perspectives for drug design

Zika virus (ZIKV) belongs to the positive-sense single-stranded RNA-containing Flaviviridae family. Its recent outbreak and association with human diseases (e.g. neurological disorders) have raised global health concerns, and an urgency to develop a therapeutic strategy against ZIKV infection. However, there is no currently approved antiviral against ZIKV. Here we present a comprehensive overview on recent progress in structure–function investigation of ZIKV NS5 protein, the largest non-structural protein of ZIKV, which is responsible for replication of the viral genome, RNA capping and suppression of host interferon responses. Structural comparison of the N-terminal methyltransferase domain and C-terminal RNA-dependent RNA polymerase domain of ZIKV NS5 with their counterparts from related viruses provides mechanistic insights into ZIKV NS5-mediated RNA replication, and identifies residues critical for its enzymatic activities. Finally, a collection of recently identified small molecule inhibitors against ZIKV NS5 or its closely related flavivirus homologues are also discussed.