Gene regulation through messenger RNA stability.
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Animals↗
Biomedical subjects
D W Cleveland
Publications and source records attributed to D W Cleveland.
Autoregulatory control of beta-tubulin mRNA stability is linked to translation elongation.
Tubulin synthesis in animal cells is controlled in part by an autoregulatory mechanism that modulates the stability of ribosome-bound tubulin mRNAs. For beta tubulin, the initial recognition event for this selective RNA instability has previously been shown to be a cotranslational binding (presumably by tubulin itself) to the nascent amino-terminal beta-tubulin tetrapeptide just after it emerges from the ribosome. Although this "autoregulation" of tubulin expression is thus obligatorily linked to the translation process, the mechanism of how a cotranslational protein-protein binding event ultimately triggers RNA degradation is unknown. Using protein synthesis inhibitors to slow and ultimately to block translation elongation, we now show that the mRNA destabilization pathway requires ongoing ribosome translocation.
Animals↗
Expression of NF-L and NF-M in fibroblasts reveals coassembly of neurofilament and vimentin subunits.
We have used transient and stable DNA transfection to force synthesis of the mouse NF-L and NF-M genes in nonneuronal cultured animal cells. When the authentic NF-L gene (containing 1.7 kb of sequences 5' to the transcription initiation site) was transfected into L cells, correctly initiated NF-L mRNA was produced from the transfected gene but not the endogenous NF-L genes. Therefore, the normal restriction of NF-L expression to neurons cannot derive exclusively from absence in nonneuronal cells of neuron-specific transcription factors. When the NF-L coding region was linked to the strong promoter from Moloney Murine Sarcoma virus, we obtained high levels of synthesis of NF-L subunits (accumulating to as much as 9% of cell protein in stable cell lines). Although NF-L and NF-M polypeptides are normally expressed exclusively in postmitotic neurons, NF-L or NF-M polypeptides expressed in fibroblasts were efficiently assembled into intermediate filament arrays, thus demonstrating the competence of both NF-L and NF-M to assemble in vivo in the absence of additional neuron-specific factors. As judged by immunofluorescence localization and by the alteration in the solubility of the endogenous vimentin filaments, filaments containing NF-L appeared to be copolymers with vimentin. Neither the alteration in the properties of the vimentin array nor the accumulation of NF-L to a level that made it the second most abundant cellular protein (after actin) had any observable effect on cell viability or growth rate.
Animals↗
Differential utilization of beta-tubulin isotypes in differentiating neurites.
beta-Tubulin is encoded in vertebrate genomes by a family of six to seven functional genes that produce six different polypeptide isotypes. We now document that although rat PC-12 cells express five of these isotypes, only two (classes II and III) accumulate significantly as a consequence of nerve growth factor-stimulated neurite outgrowth. In contrast to previous efforts that have failed to detect in vivo distinctions among different beta-tubulin isotypes, we demonstrate using immunoblotting with isotype-specific antibodies that three beta-tubulin polypeptides (classes I, II, and IV) are used preferentially for assembly of neurite microtubules (with approximately 70% of types I and II assembled but only approximately 50% of type III in polymer). Immunofluorescence localization shows that an additional isotype (V) is partially excluded from neurites. Distinctions in in vivo localization of the neuron-specific, class III isotype have also been directly observed using immunofluorescence and immunogold electron microscopy. The sum of these efforts documents that some in vivo functional differences between tubulin isotypes do exist.
Animals↗
Multiple determinants of eukaryotic mRNA stability.
Regulated changes in mRNA stability play an important role in modulating the level of expression of many eukaryotic genes. In several systems, specific sequence determinants that dictate mRNA instability have been identified. Thus, the presence of instability determinants, and not the absence of sequences that dictate stability, appears to be required for regulated mRNA degradation. These instability determinants presumably interact with specific nucleases or other trans-acting factors that regulate the accessibility of the domain to nucleases. Although each RNA destabilization pathway has unique features, in many cases RNA degradation requires ongoing protein synthesis. In some of the systems discussed, the mRNAs are degraded co-translationally, perhaps by a ribosome-associated ribonuclease. For other messages, the mechanistic reasons for the dependence of mRNA degradation on ongoing protein synthesis are still unknown.
Amino Acid Sequence↗
Autoregulated instability of beta-tubulin mRNAs by recognition of the nascent amino terminus of beta-tubulin.
Tubulin synthesis in animal cells is controlled by an autoregulatory mechanism that modulates the stability of polysome-bound tubulin messenger RNAs. The beta-tubulin RNAs are selectively targeted as substrates for destabilization not through the recognition of specific RNA sequences, but rather through co-translational recognition of the amino-terminal beta-tubulin tetrapeptide after its emergence from the ribosome. This motif is likely to be used in other systems where RNA degradation is coupled to ribosome attachment and translation.
Amino Acid Sequence↗
A monoclonal antibody against the type II isotype of beta-tubulin. Preparation of isotypically altered tubulin.
Mammalian brain tubulin consists of several isotypes of alpha and beta subunits that separate on polyacrylamide gels into three electrophoretic classes, designated alpha, beta 1, and beta 2. It has not been possible hitherto to resolve the different isotypes in a functional form. To this end, we have now isolated a monoclonal antibody, using as an immunogen a chemically synthesized peptide corresponding to the carboxyl-terminal sequence of the major tubulin isotype (type II) found in the beta 1-tubulin electrophoretic fraction. The antibody binds to beta 1 but not to alpha or beta 2. When pure tubulin from bovine brain is passed through an immunoaffinity column made from the anti-type II antibody, the tubulin that elutes in the unbound fraction is enriched greatly for the beta 2 electrophoretic variant. The tubulin that binds to the column appears to contain only alpha and beta 1, not beta 2. When these tubulin fractions are characterized by immunoblotting using the anti-type II antibody, the antibody binds only to the beta 1 band in the bound fraction, not to the beta 1 band in the unbound fraction. Using polyclonal antibodies generated against the carboxyl-termini of types I, III, and IV, we demonstrate that the beta 1 electrophoretic species is comprised of isotypes I, II, and IV, whereas the beta 2 variant is comprised exclusively of type III beta-tubulin. Further, we calculate that beta-tubulin in purified bovine brain tubulin is comprised of 3% type I, 58% type II, 25% type III, and 13% type IV tubulins.
Animals↗
Sequence of chicken c beta 7 tubulin. Analysis of a complete set of vertebrate beta-tubulin isotypes.
In chicken, beta-tubulin is encoded by a family of seven genes. We have now isolated and sequenced overlapping cDNA clones corresponding to gene c beta 7 (previously designated c beta 4'), the only chicken beta-tubulin not previously characterized. The inferred amino acid sequence of c beta 7 tubulin is identical with the class I beta-tubulin isotype found in human, mouse and rat. Moreover, c beta 7 is highly expressed in almost all tissue and cell types in chicken, a pattern similar to those of the genes for class I beta-tubulin isotypes in other vertebrates. Comparison of the complete family of chicken beta-tubulin gene sequences reveals that the heterogeneity of beta-tubulin polypeptides encoded in a higher eukaryote is confined to six distinct beta-tubulin isotypes. Five of these are members of evolutionarily conserved isotypic classes (I to V), whereas the sixth represents a divergent erythroid-specific tubulin whose sequence has not been conserved.
Amino Acid Sequence↗
A survey of the alpha-tubulin gene family in chicken: unexpected sequence heterogeneity in the polypeptides encoded by five expressed genes.
To characterize the alpha-tubulin gene family in chicken, we have isolated five chicken alpha-tubulin genes and determined the majority of the sequences of the encoded polypeptides. Three of these (c alpha 3, c alpha 5/6 and c alpha 8) encode novel, expressed alpha-tubulins that have not previously been analyzed, whereas one gene segment is a pseudogene and another appears capable of encoding a functional subunit (although we were unable to document its expression in a survey of chicken tissues). Together with two additional expressed, functional alpha-tubulins reported earlier, we conclude that the chicken alpha-tubulin family is comprised of at least five functional genes whose polypeptide products are substantially more heterogeneous than found in preceding analyses of vertebrate alpha-tubulins. Comparison of the amino acid sequences reveals that the five polypeptides are between 96 and 83% identical, with the extreme carboxy-terminal residues representing a highly heterogeneous variable domain. Since some alpha-tubulins undergo cyclic post-translational removal and readdition of a carboxy-terminal tyrosine, the notable sequence divergence in this domain suggests that individual tubulins probably participate to different extents in this modification cycle.
Alleles↗
Autoregulated instability of tubulin mRNAs: a novel eukaryotic regulatory mechanism.
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Gene Expression Regulation↗
Neurofilament and tubulin expression recapitulates the developmental program during axonal regeneration: induction of a specific beta-tubulin isotype.
We examined the differential expression of genes encoding three beta-tubulin isotypes (classes I, II, and IV) and the 68-kDa neurofilament protein (NF68) in rat sensory neurons during development, maturation, and axonal regeneration. Expression of the specific beta-tubulin gene encoding the class II isotype was induced to high levels during development and axonal regeneration, whereas the expression of genes encoding the two other isotypes (classes I and IV) remained comparable to mature levels. Conversely, expression of the NF68 gene was relatively low during development and regeneration. Thus, the developmental program for cytoskeletal gene expression is recapitulated during axonal regeneration. The high level of class II beta-tubulin expression found in developing and regenerating neurons occurs during the longitudinal growth of axons. In contrast, induction of NF68 gene expression is associated with the radial growth of axons in maturing neurons.
Aging↗
cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7-kDa carboxyl-terminal fragment.
cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.
Amino Acid Sequence↗
Autoregulated changes in stability of polyribosome-bound beta-tubulin mRNAs are specified by the first 13 translated nucleotides.
The expression of tubulin polypeptides in animal cells is controlled by an autoregulatory mechanism whereby increases in the tubulin subunit concentration result in rapid and specific degradation of tubulin mRNAs. We have now determined that the sequences that are necessary and sufficient to specify mouse beta-tubulin mRNAs as substrates for this autoregulated instability reside within the first 13 translated nucleotides (which encode the first four beta-tubulin amino acids Met-Arg-Glu-Ile). This domain has been functionally conserved throughout evolution, inasmuch as sequences isolated from the analogous region of human, chicken, and yeast beta-tubulin mRNAs also confer autoregulation. Further, for an RNA to be a substrate for regulation, not only must it carry the 13-nucleotide coding sequence, but it must also be ribosome bound and its translation must proceed 3' to codon 41.
Amino Acid Sequence↗
Evidence of discontinuous transcription in the trypanosomatid Crithidia fasciculata.
In an effort to exploit the advantages of Crithidia fasciculata for detailed analysis of the mechanisms of discontinuous transcription in the trypanosomatid family, we have cloned, sequenced, and characterized the mini-exon gene repeat in Crithidia and mapped the termini of its primary transcript. We find that Crithidia contains approximately 500 mini-exon genes, present almost exclusively as tandemly repeated arrays on a single chromosome. Transcripts derived from these genes are approximately 90 bases in length with heterogeneity at both the 5' and 3' ends. Primer extension experiments reveal a putative splicing intermediate. Specific inhibition of in vitro translation of Crithidia mRNAs by an oligonucleotide complementary to the mini-exon suggests that all Crithidia mRNAs contain the mini-exon at their 5' termini. Comparison of mini-exon gene sequences from various trypanosomatids reveals several regions of conservation that imply functional constraints on the transcription of mini-exon genes and the processing of their transcripts.
Animals↗
Autoregulation of tubulin expression is achieved through specific degradation of polysomal tubulin mRNAs.
We have utilized protein synthesis inhibitors to investigate the autoregulatory mechanism that uses the concentration of unpolymerized tubulin subunits to specify tubulin mRNA content in animal cells. Puromycin and pactamycin, both of which remove RNAs from polysomes, completely unlink tubulin RNA content from the level of free subunits, whereas pretreatment of cells with cycloheximide, which traps mRNAs onto stalled polyribosomes, enhances the specific degradation of tubulin RNAs in response to increases in the subunit content. Moreover, in the absence of protein synthesis inhibitors, the tubulin RNAs that are lost from cells with elevated free tubulin subunit levels are those that are associated with polyribosomes. Further, beta-tubulin mRNAs encoding a truncated translation product of only 26 amino acids (and that cannot be polyribosomal) are not substrates for autoregulation. We conclude that autoregulation of tubulin synthesis is achieved by specifically altering the stability of tubulin RNAs that are bound to polyribosomes.
Animals↗
The sequence and expression of the divergent beta-tubulin in chicken erythrocytes.
We report here the complete sequence of a highly divergent chicken erythrocyte beta-tubulin, c beta 6, which appears to represent a major exception to the observation that the primary sequences and sites of expression of beta-tubulin isotypes are conserved within vertebrates. The amino acid sequence was deduced from overlapping cloned cDNAs identified in a chicken erythroblast cDNA library contained in the expression vector, lambda gt11. Compared with other chicken beta-tubulins, among which the maximum sequence divergence is only 8%, c beta 6-tubulin is more hydrophobic, contains seven fewer net negative charges, and exhibits a surprising 17% overall divergence in its amino acid sequence. DNA and RNA blot analyses show that c beta 6-tubulin is present as a single gene copy in the chicken genome and is specifically expressed in the bone marrow. Comparisons of RNA blots and immunoblots of various cells and tissues confirm that this beta-tubulin isotype is contained specifically in erythrocytes and thrombocytes and accounts for 75% of the beta-tubulin mRNA species contained in developing erythroblasts. Interestingly, c beta 6-tubulin exhibits 18% amino acid sequence divergence relative to MB1, the analogous hematopoietic beta-tubulin contained in mouse.
Amino Acid Sequence↗
Sequences that confer beta-tubulin autoregulation through modulated mRNA stability reside within exon 1 of a beta-tubulin mRNA.
Synthesis of alpha- and beta-tubulin is controlled in animal cells by a novel autoregulatory mechanism: the concentration of unpolymerized subunits specifies the level of tubulin mRNAs. Using transient DNA transfection, we have localized the sequences that identify a beta-tubulin RNA as a substrate for autoregulation. Insertion of as few as 106 nucleotides (57 bases of 5' untranslated region and 49 coding nucleotides) from a beta-tubulin gene into a thymidine kinase gene is sufficient to make expression of the resultant chimeric RNA regulated as if it were an authentic beta-tubulin mRNA. Furthermore, all 5' untranslated region sequences can be deleted without disrupting regulation. We conclude that this novel autoregulatory pathway is specified by cytoplasmic events that modulate mRNA stability through sequences lying within the first 16 translated codons of a beta-tubulin mRNA.
Animals↗
A combination of RNase H and S1 nuclease circumvents an artefact inherent to conventional S1 analysis of RNA splicing.
S1 nuclease mapping is commonly used to analyze transcription and processing of unlabelled RNAs. However, the S1 protocol that appears best suited to demonstrate splicing of a particular RNA (using an intronless probe that is 5' end-labelled in the downstream exon) is not diagnostic as expected. Rather, both intron-containing RNA and intronless RNA confer protection of probe across the splice juncture. To unambiguously demonstrate correctly spliced RNAs that begin at a specific initiation site, we present a procedure in which unspliced RNA molecules are first cleaved by RNase H following annealing to an intronic DNA fragment and the remaining RNA is then subjected to S1 analysis using an intronless probe present in vast excess. Only spliced, correctly initiated transcripts can protect the probe across the splice junction and up to residue +1. This RNase H/S1 method provides a broadly applicable technique with which to demonstrate splicing and initiation of a variety of transcripts, especially ones from transfected genes that can arise both from the normal and from activated cryptic initiation sites.
Endonucleases↗