共查询到20条相似文献,搜索用时 31 毫秒
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Johann W. Bauer Clemens Brandl Olaf Haubenreisser Bjoern Wimmer Manuela Weber Thomas Karl Alfred Klausegger Michael Breitenbach Helmut Hintner Tobias von der Haar Mick F. Tuite Lore Breitenbach-Koller 《PloS one》2013,8(7)
Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered ‘specialized’ ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin β3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered. 相似文献
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Analyses of ribosomes extracted from spores of Bacillus cereus T by a dryspore disruption technique indicated that previously reported defects in ribosomes from spores may arise during the ribosome extraction process. The population of ribosomes from spores is shown to cotain a variable quantity of free 50S subunits which are unstable, giving rise to slowly sedimenting particles in low-Mg2+ sucrose gradients and showing extremely low activity in in vitro protein synthesis. The majority of the ribosomal subunits in spores, obtained by dissociation of 70S ribosomes and polysomes, are shown to be as stable as subunits from vegetative cells, though the activity of spore polysomes was lower than that of vegetative ribosomes. In spite of the instability and inactivity of a fraction of the spore's ribosomal subunits, the activity of the total population obtained from spores by the dry disruption technique was 32% of vegetative ribosome activity, fivefold higher than previously obtained with this species. The improvement in activity and the observed variability of subunit destabilization are taken as evidence for partial degradation of spore ribosomes during extraction. 相似文献
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We have identified a new class of ribosomal protein (RP) genes that appear to have been retrotransposed from X-linked RP genes. Mammalian ribosomes are composed of four RNA species and 79 different proteins. Unlike RNA constituents, each protein is typically encoded by a single intron- containing gene. Here we describe functional autosomal copies of the X-linked human RP genes, which we designated RPL10L (ribosomal protein L10-like gene), RPL36AL and RPL39L after their progenitors. Because these genes lack introns in their coding regions, they were likely retrotransposed from X-linked genes. The identities between the retrotransposed genes and the original X-linked genes are 89-95% in their nucleotide sequences and 92-99% in their amino acid sequences, respectively. Northern blot and PCR analyses revealed that RPL10L and RPL39L are expressed only in testis, whereas RPL36AL is ubiquitously expressed. Although the role of the autosomal RP genes remains unclear, they may have evolved to compensate for the reduced dosage of X-linked RP genes. 相似文献
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Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle. 总被引:5,自引:0,他引:5
Smooth muscle cells are able to adapt rapidly to chemical and mechanical signals impinging on the cell surface. It has been suggested that dynamic changes in the actin cytoskeleton contribute to the processes of contractile activation and mechanical adaptation in smooth muscle. In this review, evidence for functionally important changes in actin polymerization during smooth muscle contraction is summarized. The functions and regulation of proteins associated with "focal adhesion complexes" (membrane-associated dense plaques) in differentiated smooth muscle, including integrins, focal adhesion kinase (FAK), c-Src, paxillin, and the 27-kDa small heat shock protein (HSP27) are described. Integrins in smooth muscles are key elements of mechanotransduction pathways that communicate with and are regulated by focal adhesion proteins that include FAK, c-Src, and paxillin as well as proteins known to mediate cytoskeletal remodeling. Evidence that functions of FAK and c-Src protein kinases are closely intertwined is discussed as well as evidence that focal adhesion proteins mediate key signal transduction events that regulate actin remodeling and contraction. HSP27 is reviewed as a potentially significant effector protein that may regulate actin dynamics and cross-bridge function in response to activation of p21-activated kinase and the p38 mitogen-activated protein kinase signaling pathway by signaling pathways linked to integrin proteins. These signaling pathways are only part of a large number of yet to be defined pathways that mediate acute adaptive responses of the cytoskeleton in smooth muscle to environmental stimuli. 相似文献
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Effect of various methods of preparation on the apparent protein composition of eukaryotic ribosomes. An essential preliminary to stoicheiometric measurements.
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1. We investigated whether there is any change in the relative amounts of ribosomal proteins during the isolation or extraction of the ribosomes by different methods, or during electrophoresis of the proteins. 2. To see whether proteins are lost (or gained) during the preparation of the ribosome we compared the two-dimensional protein pattern of three preparations: (a) ribosomes conventionally prepared by ultracentrifugation; (b) crude ribosomes obtained by pH5 precipitation; (c) crude ribosomes prepared by gel filtration. 3. To see whether proteins were lost during protein extraction we compared the two-dimensional pattern of ribosomes by using three different extraction methods (LiCl/urea, acetic acid and guanidine hydrochloride). 4. In all experiments listed above the relative amounts of the great majority of the proteins remained unchanged. We interpret this as showing that the relative amounts of ribosomal proteins (as we observed them on a two-dimensional gel) correspond to the proportions existing in the particle in vivo. 相似文献
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Partitioning and translation of mRNAs encoding soluble proteins on membrane-bound ribosomes 总被引:4,自引:0,他引:4
Lerner RS Seiser RM Zheng T Lager PJ Reedy MC Keene JD Nicchitta CV 《RNA (New York, N.Y.)》2003,9(9):1123-1137
In eukaryotic cells, it is generally accepted that protein synthesis is compartmentalized; soluble proteins are synthesized on free ribosomes, whereas secretory and membrane proteins are synthesized on endoplasmic reticulum (ER)-bound ribosomes. The partitioning of mRNAs that accompanies such compartmentalization arises early in protein synthesis, when ribosomes engaged in the translation of mRNAs encoding signal-sequence-bearing proteins are targeted to the ER. In this report, we use multiple cell fractionation protocols, in combination with cDNA microarray, nuclease protection, and Northern blot analyses, to assess the distribution of mRNAs between free and ER-bound ribosomes. We find a broad representation of mRNAs encoding soluble proteins in the ER fraction, with a subset of such mRNAs displaying substantial ER partitioning. In addition, we present evidence that membrane-bound ribosomes engage in the translation of mRNAs encoding soluble proteins. Single-cell in situ hybridization analysis of the subcellular distribution of mRNAs encoding ER-localized and soluble proteins identify two overall patterns of mRNA distribution in the cell-endoplasmic reticular and cytosolic. However, both partitioning patterns include a distinct perinuclear component. These results identify previously unappreciated roles for membrane-bound ribosomes in the subcellular compartmentalization of protein synthesis and indicate possible functions for the perinuclear membrane domain in mRNA sorting in the cell. 相似文献
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Membrane-bound ribosomes in kidney: methods of estimation and effect of compensatory renal growth 总被引:2,自引:1,他引:1
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Membrane-bound ribosomes are thought to secrete protein for export and free ribosomes to secrete protein for intracellular use. The proportion of the total ribosomes that is bound to membranes in normal mouse kidneys has been estimated by three different methods, and the results have been compared with those obtained by a fourth method used by us previously. The most valid estimates appear to be those obtained (a) by comparison of radioactivity in peaks representing free and membrane-bound ribosomes on linear sucrose gradients after labeling for 24 hr with 14C-orotic acid, and (b) by measurements of optical density in free and bound ribosomes that had been separated by centrifugation on discontinuous gradients of 0.5 M/2.0 M sucrose. Analyses by these methods show that about 20–25% of the ribosomes in a postnuclear supernatant prepared from mouse kidneys, but only 10–15% of the ribosomes in a post-mitochondrial supernatant, are membrane-bound. About 75% of the bound ribosomes sediment as polysomes of many different sizes. The proportion of membrane-bound ribosomes and their aggregation into polysomes were unchanged in kidneys undergoing compensatory hypertrophy after removal of the opposite kidney. These experiments show that, unlike liver, kidney has a predominance of free ribosomes compared to bound ribosomes; those ribosomes that are membrane-bound do not become free during compensatory renal growth. 相似文献
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Noll H 《BioEssays : news and reviews in molecular, cellular and developmental biology》2008,30(11-12):1220-1234
By the early 1960s, evidence had accumulated that proteins were synthesized from special RNA copies of genes, named "messenger RNAs" (mRNAs), not directly from the stable RNAs found in the ribosomes of the cytoplasm. Yet, precisely how the protein chains were assembled along the RNA and, in particular, the relationship between the mRNAs and the ribosomes during protein synthesis, was obscure. In this account, I discuss how my laboratory found that multiple ribosomes traverse each mRNA, yielding the structures known as polysomes. This work led on to the first physical determination of the coding ratio, new insights into how protein chains are initiated, and an early suggestion that chloroplasts and mitochondria in eukaryotic cells might ultimately have been derived from symbiotic bacteria. 相似文献
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Jonathan D. Dinman 《The Journal of biological chemistry》2009,284(18):11761-11765
Despite having been identified first, their greater degree of complexity
has resulted in our understanding of eukaryotic ribosomes lagging behind that
of their bacterial and archaeal counterparts. A much more complicated
biogenesis program results in ribosomes that are structurally, biochemically,
and functionally more complex. However, recent advances in molecular genetics
and structural biology are helping to reveal the intricacies of the eukaryotic
ribosome and to address many longstanding questions regarding its many roles
in the regulation of gene expression.Since its initial discovery using differential ultracentrifugation of rat
liver homogenates (reviewed in Ref.
1), the ribosome has remained a
foundational platform upon which our understanding of the relationship between
structure and function at the molecular level has been built. There is a rich
history of biochemistry and genetics of eukaryotic ribosomes, including the
discovery in the 1950s that they 32 are the site of protein synthesis, the
elucidation of the function of the nucleolus, and even the discovery of the
first eukaryotic RNA polymerase (reviewed in Ref.
2). Whereas early studies using
mammalian ribosomes defined the “integral requirements” for
protein synthesis, a switch to bacterial ribosomes in the 1960s facilitated
the identification of the “minimal requirements” for the
translational machinery, giving rise to a “golden age” of
translation. In particular, the greater degree of structural and functional
complexity makes eukaryotic ribosomes more challenging to work with than their
bacterial and archaeal counterparts. For example, whereas bacterial
translation initiation requires only a small set of trans-acting
factors and is facilitated by the Shine-Dalgarno sequence, this process in
eukaryotes requires a multifactorial complex of trans-acting factors
that is almost as massive as the ribosome itself (reviewed in Ref.
3). Here, some of the current
topics and challenges in the study of the eukaryotic ribosome are
reviewed. 相似文献
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Jacco C. Karper Mark M. Ewing Margreet R. de Vries Saskia C. A. de Jager Erna A. B. Peters Hetty C. de Boer Anton-Jan van Zonneveld Johan Kuiper Eric G. Huizinga T. Harma C. Brondijk J. Wouter Jukema Paul H. A. Quax 《PloS one》2013,8(7)