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Results for “Translational repression”

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PubMed2006

PUF protein-mediated deadenylation is catalyzed by Ccr4p.

PUF proteins control gene expression by binding to the 3'-untranslated regions of specific mRNAs and triggering mRNA decay or translational repression. Here we focus on the mechanism of PUF-mediated regulation. The yeast PUF protein, Mpt5p, regulates HO mRNA and stimulates removal of its poly(A) tail (i.e. deadenylation). Mpt5p repression in vivo is dependent on POP2, a component of the cytoplasmic Ccr4p-Pop2p-Not complex that deadenylates mRNAs. In this study, we elucidate the individual roles of the Ccr4p and Pop2p deadenylases in Mpt5p-regulated deadenylation. Both in vivo and in vitro, Pop2p and Ccr4p proteins are required for Mpt5p-regulated deadenylation of HO. However, the requirements for the two proteins differ dramatically: the enzymatic activity of Ccr4p is essential, whereas that of Pop2p is dispensable. We conclude that Pop2p is a bridge through which the PUF protein recruits the Ccr4p enzyme to the target mRNA, thereby stimulating deadenylation. Our data suggest that PUF proteins may enhance mRNA degradation and repress expression by both deadenylation-dependent and -independent mechanisms, using the same Pop2p bridge to recruit a multifunctional Pop2p complex to the mRNA.

Binding Sites↗

PubMed1986

Translational control of mRNA expression during the early mitogenic response in Swiss mouse 3T3 cells: identification of specific proteins.

Addition of serum or epidermal growth factor to quiescent Swiss mouse 3T3 cells in culture leads to a number of specific changes in the pattern of protein synthesis. Earlier experiments with actinomycin D suggested that the altered expression of these proteins was controlled at either the pretranslational or translational level. Here we have identified and further characterized the regulation of mRNA expression for ten of these proteins, including protein synthesis elongation factor eEF-1 alpha, poly A binding protein, vimentin, the multiple forms of the actin protein family, and alpha- and beta-tubulin. Using an in vitro translation system, we determined the change in the level of mRNA encoding for each of these proteins after serum stimulation. The results showed that the amount of mRNA coding for eEF-1 alpha, poly A binding protein, vimentin, and alpha- and beta-tubulin remains unchanged during this time, whereas that of the actin family increases. Thus, with the exception of the actin family, the results argue that the expression of all the proteins identified is regulated at the translational level. The importance of this latter group of proteins in cell growth and the abundance of their cognate mRNAs should prove them useful tools in elucidating the mechanisms involved in the activation of translationally repressed mRNA during the mitogenic response.

Actins↗

PubMed1989

The sea urchin multicatalytic protease: purification, biochemical analysis, subcellular distribution, and relationship to snRNPs.

We have purified and extensively characterized a 19-S particle from sea urchin eggs. This particle is the sea urchin homologue of the "prosome", a particle originally identified in duck erythroblasts. We now show that these sea urchin prosomes contain multiple proteolytic activities. As shown for analogous particles from other cells, these particles hydrolyze synthetic substrates containing neutral hydrophobic or basic amino acids at the carboxy terminus of the synthetic peptides. They contain 16-20 small proteins ranging in molecular weight from 20,000 to 32,000. Peptide mapping shows that most of the polypeptides are unique, however, three exist in two isoelectric forms. We have investigated the possible function of the sea urchin multicatalytic proteases (MCPs) by determining their subcellular distribution, their relationship to egg snRNPs, and their possible role in translational repression. There are almost as many MCPs (2 x 10(8] as ribosomes (6.6 x 10(8] or mRNPs (1.8 x 10(7] per egg. This suggests that like ribosomes, the MCPs are stored in the egg for use during later development. We find that a substantial proportion of egg MCPs move into nuclei by the late blastula stage. Using a specific antibody against one of the sea urchin MCP proteins and antibodies against U1-U6, La, and Ro RNPs, we show that the sea urchin particle is distinct from these RNPs, although the anti-U1-U6 RNP antibody cross-reacts with a single MCP protein. In addition, the sea urchin MCP appears to be associated with a large structure in the cytoplasm of unfertilized eggs and is released under the same conditions that activate egg mRNPs in vitro.

Animals↗

PubMed2005

A role for the eIF4E-binding protein 4E-T in P-body formation and mRNA decay.

4E-transporter (4E-T) is one of several proteins that bind the mRNA 5'cap-binding protein, eukaryotic initiation factor 4E (eIF4E), through a conserved binding motif. We previously showed that 4E-T is a nucleocytoplasmic shuttling protein, which mediates the import of eIF4E into the nucleus. At steady state, 4E-T is predominantly cytoplasmic and is concentrated in bodies that conspicuously resemble the recently described processing bodies (P-bodies), which are believed to be sites of mRNA decay. In this paper, we demonstrate that 4E-T colocalizes with mRNA decapping factors in bona fide P-bodies. Moreover, 4E-T controls mRNA half-life, because its depletion from cells using short interfering RNA increases mRNA stability. The 4E-T binding partner, eIF4E, also is localized in P-bodies. 4E-T interaction with eIF4E represses translation, which is believed to be a prerequisite for targeting of mRNAs to P-bodies. Collectively, these data suggest that 4E-T interaction with eIF4E is a priming event in inducing messenger ribonucleoprotein rearrangement and transition from translation to decay.

Blotting, Western↗

PubMed2005

Prediction of regulatory modules comprising microRNAs and target genes.

MOTIVATION: MicroRNAs (miRNAs) are small endogenous RNAs that can play important regulatory roles via the RNA-interference pathway by targeting mRNAs for cleavage or translational repression. We propose a computational method to predict miRNA regulatory modules (MRMs) or groups of miRNAs and target genes that are believed to participate cooperatively in post-transcriptional gene regulation. RESULTS: We tested our method with the human genes and miRNAs, predicting 431 MRMs. We analyze a module with genes: BTG2, WT1, PPM1D, PAK7 and RAB9B, and miRNAs: miR-15a and miR-16. Review of the literature and annotation with Gene Ontology terms reveal that the roles of these genes can indeed be closely related in specific biological processes, such as gene regulation involved in breast, renal and prostate cancers. Furthermore, it has been reported that miR-15a and miR-16 are deleted together in certain types of cancer, suggesting a possible connection between these miRNAs and cancers. Given that most known functionalities of miRNAs are related to negative gene regulation, extending our approach and exploiting the insight thus obtained may provide clues to achieving practical accuracy in the reverse-engineering of gene regulatory networks. AVAILABILITY: A list of predicted modules is available from the authors upon request.

Algorithms↗

PubMed1998

EDEN and EDEN-BP, a cis element and an associated factor that mediate sequence-specific mRNA deadenylation in Xenopus embryos.

During Xenopus early development, gene expression is regulated mainly at the translational level by the length of the poly(A) tail of mRNAs. The Eg family and c-mos maternal mRNAs are deadenylated rapidly and translationally repressed after fertilization. Here, we characterize a short sequence element (EDEN) responsible for the rapid deadenylation of Eg5 mRNA. Determining the core EDEN sequence permitted us to localize the c-mos EDEN sequence. The c-mos EDEN confered a rapid deadenylation to a reporter gene. The EDEN-specific RNA-binding protein (EDEN-BP) was purified and a cDNA obtained. EDEN-BP is highly homologous to a human protein possibly involved in myotonic dystrophy. Immunodepleting EDEN-BP from an egg extract totally abolished the EDEN-mediated deadenylation activity, but did not affect the default deadenylation activity. Therefore, EDEN-BP constitutes the first trans-acting factor for which an essential role in the specificity of mRNA deadenylation has been directly demonstrated.

Adenine↗

PubMed1990

The rIIA gene of bacteriophage T4. I. Its DNA sequence and discovery of a new open reading frame between genes 60 and rIIA.

We have determined the DNA sequence of the rIIA gene and have discovered a small open reading frame, rIIA.1, between genes 60 and rIIA. The predicted molecular weights of these proteins are 82,840 for rIIA and 8,124 for rIIA.1. The rIIA protein has a repeated motif which suggests that the gene has evolved by duplication. It also has a motif which suggests that it belongs to a group of ompR-like proteins that control regulation of gene expression in response to changes in the external environment. We have sequenced three different missense mutants whose mutations lie in the Ala segment of the rIIA genetic map. All three changes are found within the first 35 bp of the rIIA coding sequence. The region of control of protein synthesis is identical in the rIIA gene and in gene 44 of T4. We relate this finding to the high sensitivity of both RNAs to translational repression by the T4 regA gene product.

Amino Acid Sequence↗

PubMed1993

Encapsidation of heterologous RNAs by bacteriophage MS2 coat protein.

The RNA bacteriophages of E. coli specifically encapsidate a single copy of the viral genome in a protein shell composed mainly of 180 molecules of coat protein. Coat protein is also a translational repressor and shuts off viral replicase synthesis by interaction with a RNA stem-loop containing the replicase initiation codon. We wondered whether the translational operator also serves as the viral pac site, the signal which mediates the exclusive encapsidation of viral RNA by its interaction with coat protein. To test this idea we measured the ability of lacZ RNA fused to the translational operator to be incorporated into virus-like particles formed from coat protein expressed from a plasmid. The results indicate that the operator-lacZ RNA is indeed encapsidated and that nucleotide substitutions in the translational operator which reduce the tightness of the coat protein-operator interaction also reduce or abolish encapsidation of the hybrid RNA. When coat protein is expressed in excess compared to the operator-lacZ RNA, host RNAs are packaged as well. However, elevation of the level of operator-lacZ RNA relative to coat protein results in its selective encapsidation at the expense of cellular RNAs. Our results are consistent with the proposition that this single protein-RNA interaction accounts both for translational repression and viral genome encapsidation.

Base Sequence↗

PubMed1997

RNA-protein interactions within the 3 ' untranslated region of vimentin mRNA.

Several functions have been attributed to protein binding within the 3'untranslated region (3'UTR) of mRNA, including mRNA localization, stability, and translational repression. Vimentin is an intermediate filament protein whose 3'untranslated sequence is highly conserved between species. In order to identify sequences that might play a role in vimentin mRNA function, we synthesized32P-labeled RNA from different regions of vimentin's 3'UTR and assayed for protein binding with HeLa extracts using band shift assays. Sequences required for binding are contained within a region 61-114 nucleotides downstream of the stop codon, a region which is highly conserved from Xenopus to man. As judged by competition assays, binding is specific. Solution probing studies of 32P-labeled RNA with various nucleases and lead support a complex stem and loop structure for this region. Finally, UV cross-linking of the RNA-protein complex identifies an RNA binding protein of 46 kDa. Fractionation of a HeLa extract on a sizing column suggests that in addition to the 46 kDa protein, larger complexes containing additional protein(s) can be identified. Vimentin mRNA has been shown to be localized to the perinuclear region of the cytoplasm, possibly at sites of intermediate filament assembly. To date, all sequences required for localization of various mRNAs have been confined to the 3'UTR. Therefore, we hypothesize that this region and associated protein(s) might be important for vimentin mRNA function such as in localization.

Animals↗

PubMed1997

An efficient protocol for linker scanning mutagenesis: analysis of the translational regulation of an Escherichia coli RNA polymerase subunit gene.

A protocol has been developed that is capable of saturating regions hundreds of basepairs in length with linker scanning mutations. The efficacy of this method stems from the design of the linker scanning mutagenesis (LSM) cassette which is composed of a selectable marker flanked by two oligonucleotides, each of which contains a recognition site for a different restriction endonuclease. The cleavage site for one endonuclease is within its recognition site, while the second endonuclease cleaves in the target DNA beyond the end of the cassette. Digestion with these endonucleases and subsequent ligation results in the replacement of 12 bp of the original target sequence with 12 bp of the linker scanning oligonucleotide. We have used this protocol to mutagenize a span of approximately 400 bp surrounding the start site of the gene for the beta subunit (rpoB) of Escherichia coli RNA polymerase. The translation of the beta mRNA has been shown previously to be regulated by the intracellular concentration of either beta or beta'. Analysis of the linker scanning mutations indicates that sequences extending a considerable distance both upstream and downstream of the start site are required for normal translation. Also a site that appears to be involved in translational repression by excess beta' has been identified.

Bacteriophage lambda↗

PubMed1999

The mouse Y-box protein, MSY2, is associated with a kinase on non-polysomal mouse testicular mRNAs.

In male germ cells many mRNAs are sequestered by proteins into translationally silent messenger ribo-nucleoprotein (mRNP) particles. These masked paternal mRNAs are stored and translated at specific times of germ cell development. Little is known about the mammalian testicular mRNA masking proteins bound to non-polysomal mRNAs. In this report, the major proteins binding to non-polysomal testicular mRNAs were isolated and analyzed. The two predominant proteins identified were: a Y-box protein (MSY2), the mammalian homolog to the Xenopus oocyte masking protein FRGY2/mRNP3+4, and a poly(A) binding protein. A kinase activity was also found associated with these non-polysomal RNAs. The kinase co-immunoprecipitates with MSY2 and phosphorylates MSY2 in vitro. The MSY2 associated kinase is not casein kinase 2, the kinase believed to phosphorylate mRNP3+4 in oocytes, but a yet unidentified kinase. MSY2 was found to be phosphorylated in vivo and MSY2 dephosphorylation led to a decrease in its affinity to bind RNA as judged by northwestern blotting. Therefore, testicular masked mRNAs may be regulated by the phosphorylation state of MSY2. Reconstitution experiments in which non-polysomal mRNA-binding proteins are dissociated from their RNAs and allowed to bind to exogenous mRNAs suggest that MSY2 binds RNA in a sequence-independent fashion. Furthermore, association of the non-polysomal derived proteins to exogenous non-specific mRNAs led to their translational repression in vitro.

Animals↗

PubMed2000

La protein has a positive effect on the translation of TOP mRNAs in vivo.

In vertebrates, the mRNAs encoding ribosomal proteins, as well as other proteins implicated in translation, are characterized by a 5'-untranslated region (5'-UTR), including a stretch of pyrimidines at the 5'-end. The 5'-terminal oligopyrimidine (5'-TOP) sequence, which is involved in the growth-dependent translational regulation characteristic of this class of genes (so-called TOP genes), has been shown to specifically bind the La protein in vitro, suggesting that La might be implicated in translational regulation in vivo. In order to substantiate this hypothesis, we have examined the effect of La on TOP mRNA translational control in both stable and transient transfection experiments. In particular we have constructed and analyzed three stably transfected Xenopus cell lines inducible for overexpression of wild-type La or of putative dominant negative mutated forms. Moreover, La-expressing plasmids have been transiently co-transfected together with a plasmid expressing a reporter TOP mRNA in a human cell line. Our results suggest that in vivo La protein plays a positive role in the translation of TOP mRNA. They also suggest that the function of La is to counteract translational repression exerted by a negative factor, possibly cellular nucleic acid binding protein (CNBP), which has been previously shown to bind the 5'-UTR downstream from the 5'-TOP sequence.

5' Untranslated Regions↗

PubMed2000

Puralpha: a multifunctional single-stranded DNA- and RNA-binding protein.

Puralpha is a ubiquitous, sequence-specific DNA- and RNA-binding protein which is highly conserved in eukaryotic cells. Puralpha has been implicated in diverse cellular functions, including transcriptional activation and repression, translation and cell growth. Moreover, this protein has been shown to be involved in regulating several human viruses which replicate in the central nervous system (CNS), including human immunodeficiency virus type I (HIV-1) and JC virus (JCV). Puralpha exerts part of its activity by interacting with cellular proteins, including pRb, E2F, cyclin A, Sp1 and members of the Y-box family of proteins, including YB-1 and MSY1, as well as viral proteins such as polyomavirus large T-antigen and HIV-1 Tat. The ability of Puralpha to interact with its target DNA sequence and to associate with several viral and cellular proteins is modulated by RNA. Puralpha has also been shown to be involved in cell growth and proliferation. Its association with pRb, E2F and cyclin A coupled with its fluctuating levels throughout the cell cycle, position Puralpha as a crucial factor in the cell cycle. Moreover, microinjection studies demonstrate that Puralpha causes either a G(1) or G(2) arrest depending on the cell cycle time of injection. The gene encoding Puralpha has been localized to a human locus which is frequently deleted in myelogenous leukemias and other cancers and Puralpha gene deletions have been detected in many cases of lymphoid cancers. The following review details the structural characteristics of Puralpha, its family members and the involvement of this protein in regulating various cellular and viral genes, viral replication and cell growth.

Amino Acid Sequence↗

PubMed2000

Nucleotide-sequence-specific and non-specific interactions of T4 DNA polymerase with its own mRNA.

The DNA-binding DNA polymerase (gp43) of phage T4 is also an RNA-binding protein that represses translation of its own mRNA. Previous studies implicated two segments of the untranslated 5'-leader of the mRNA in repressor binding, an RNA hairpin structure and the adjacent RNA to the 3' side, which contains the Shine-Dalgarno sequence. Here, we show by in vitro gp43-RNA binding assays that both translated and untranslated segments of the mRNA contribute to the high affinity of gp43 to its mRNA target (translational operator), but that a Shine-Dalgarno sequence is not required for specificity. Nucleotide sequence specificity appears to reside solely in the operator's hairpin structure, which lies outside the putative ribosome-binding site of the mRNA. In the operator region external to the hairpin, RNA length rather than sequence is the important determinant of the high binding affinity to the protein. Two aspects of the RNA hairpin determine specificity, restricted arrangement of purine relative to pyrimidine residues and an invariant 5'-AC-3' in the unpaired (loop) segment of the RNA structure. We propose a generalized structure for the hairpin that encompasses these features and discuss possible relationships between RNA binding determinants of gp43 and DNA binding by this replication enzyme.

Base Sequence↗

PubMed2002

RNA determinants of translational operator recognition by the DNA polymerases of bacteriophages T4 and RB69.

The DNA polymerases (gp43s) of the two related phages T4 and RB69 are DNA-binding proteins that also function as mRNA-binding autogenous translational repressors. As repressors, T4 gp43 is narrowly specific to its own mRNA whereas RB69 gp43 is equally effective against mRNA for either protein. We used in vitro RNase-sensitivity and RNA footprinting assays to identify features of the non-identical T4 and RB69 mRNA targets (translational operators) that allow for their identical binding affinities and biological responses to RB69 gp43. We observed that T4 gp43 and RB69 gp43 produce identical footprints on RNA substrates bearing the T4-derived operator, suggesting that the two gp43s make identical contacts with this operator. In contrast, the footprint produced by RB69 gp43 on its autogenous RNA target was shorter than its footprint on operator RNA from T4. As expected, we also observed only weak protection of RB69-derived operator RNA from RNase by T4 gp43; however, photocross-linking studies suggested that T4 gp43 recognizes structural features of the RB69-derived operator that are not detected by RNase- sensitivity assays. The results suggest that RB69 gp43 and T4 gp43 differ in their abilities to use RNA-sequence-independent interactions to configure potential RNA targets for translational repression.

Bacteriophages↗

PubMed2002

An analysis of the sequence requirements of EDEN-BP for specific RNA binding.

EDEN-BP (embryo deadenylation element-binding protein) binds specifically to the EDEN motif in the 3'-untranslated regions of maternal mRNAs and targets these mRNAs for deadenylation and translational repression in Xenopus laevis embryos. EDEN-BP contains three RNA recognition motifs (RRMs) and is related to the elav family of RNA-binding proteins. In the present study we show that the two N-terminal RRMs of EDEN-BP are necessary for the interaction with EDEN as well as a part of the linker region (between RRM2 and RRM3). Using a band shift assay we show that two different complexes are formed according to the size and, therefore, the functional nature of the EDEN motif. Finally, we show that EDEN-BP can form a dimer in a two-hybrid assay. Accordingly, we suggest that the functional configuration of EDEN-BP is a dimer.

3' Untranslated Regions↗

PubMed2004

Coenzyme B12 riboswitches are widespread genetic control elements in prokaryotes.

Recent studies have begun to reveal that numerous fundamental metabolic pathways in bacteria are regulated by riboswitches residing within certain messenger RNAs. These riboswitches selectively bind metabolites and modulate gene expression in response to changing ligand concentrations. Previously, we provided evidence that the btuB mRNAs of Escherichia coli and Salmonella typhimurium each carry a coenzyme B12-dependent riboswitch that causes repressed translation of the encoded cobalamin-transport protein at elevated coenzyme concentrations. Herein, we use a phylogenetic analysis to define a consensus sequence and secondary structure model for the ligand- binding domain of this riboswitch class. RNA structures that conform to this model are widespread in both Gram-positive and Gram-negative organisms. In addition, we find that the 5'-untranslated region (5'-UTR) of the cobalamin biosynthesis (cob) operon of S.typhimurium carries an RNA motif that matches this consensus sequence. Biochemical and genetic characterization of this motif confirms that the RNA directly binds coenzyme B12, and that it likely serves as a genetic control element for regulating expression of the 25-gene operon for cobalamin production in this pathogen.

5' Untranslated Regions↗

PubMed2006

Global analysis of microRNA target gene expression reveals that miRNA targets are lower expressed in mature mouse and Drosophila tissues than in the embryos.

MicroRNAs (miRNAs) are non-coding small RNAs of approximately 22 nt that regulate the gene expression by base pairing with target mRNAs, leading to mRNA cleavage or translational repression. It is currently estimated that miRNAs account for approximately 1% of predicted genes in higher eukaryotic genomes and that up to 30% of genes might be regulated by miRNAs. However, only very few miRNAs have been functionally characterized and the general functions of miRNAs are not globally studied. In this study, we systematically analyzed the expression patterns of miRNA targets using several public microarray profiles. We found that the expression levels of miRNA targets are lower in all mouse and Drosophila tissues than in the embryos. We also found miRNAs more preferentially target ubiquitously expressed genes than tissue-specifically expressed genes. These results support the current suggestion that miRNAs are likely to be largely involved in embryo development and maintaining of tissue identity.

Animals↗