Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3

Calzone, Frank J.; Angerer, Robert C.; Gorovsky, Martin A.

doi:10.1093/nar/10.6.2145

Cited by 43 publications

(49 citation statements)

References 11 publications

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“…5A. We should naturally bear in mind that these results refer to relative amounts of mRNA used as samples in the translation system and that, according to our own results and of others [30], the specilk yield for thc starved cells is 50 ; < less than for normal cells. To determine whether mRNAs for some of these proteins arc also present in non-polyadenylated RNA, the same type of analysis as described above was carried out.…”

Section: Transkition Qfpolj~somal and Subpolysoniril In Rnas In Vitrosupporting

confidence: 50%

Response of Tetrahymena pyriformis to stress induced by starvation

Galego

Barahona

Rodrigues-Pousada

1984

European Journal of Biochemistry

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mRNA synthesis was studied in exponentially growing and starved Tetrakymenap~viformis. Poly(A)-containing RNAs separated from total RNA by affinity chromatography on oligo(dT)-cellulose were characterized by polyacrylamide gel electrophoresis ; their template activity was assayed in a rabbit reticulocyte lysate system and their translation products were analysed using two-dimensional electrophoresis according to O'Farrell. 1. Polysome profiles show that the bulk of ribosomes are in 80s monosomes in starved cells, whereas less than 8 are present in the form of monosomes in exponentially growing cells, the rest being engaged in polysomes.Polysomes are almost complctcly reformed 30 min after addition of enriched medium to suspensions of starved cells. This polysome reformation is dependent on mRNA synthcsis since we have shown that it is inhibited by actinomycin D.2. Electrophoretic profiles of poly(A)-rich RNA isolated from cytoplasmic fractions of exponential and starved cells are indistinguishable except that in the latter state significant amounts of low-molecular-mass species arc observed.3. Poly(A)-rich RNAs isolated from polysomal and non-polysomal (subpolysomal) fractions of exponential cells are equally able to promote protein synthesis. The corresponding poly(A)-rich RNAs isolated from starved cells also possess equal template activitics which are, however, 15 7; lowcr than those of the poly(A)-rich RNAs of exponentially growing cells. We also present evidence that in the system uscd in aitro, polyadenylated RNA isolated from heavy polysomes of starved cells directs the synthesis of four sets of proteins with molecular masses around 100 kDa, 70 kDa, 50 kDa and 30 kDa. The formcr two groups of proteins are more abundant in the translation products of poly(A)-rich RNA of starved than of normal cells, whereas the latter two groups are present only in the translation products of poly(A)-rich RNA of starved cells. The tluorograms of the translation products obtained in i,>itro from subpolysomal poly(A)-rich RNA are identical to those obtained from polysomal poly(A)-rich RNA.4. Studies on starved cells in viao show that polypeptides of 100 kDa, 70 kDa and 38 kDa are more strongly labelled and also revealed the specific presence of 85 kDa, 55 kDa, 50 kDa and 25 kDa proteins. These results lead us to the conclusion that this microorganism responds to depleted environmental conditions by regulating gene expression at thc transcriptional level, but also at the translational level.

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Section: Transkition Qfpolj~somal and Subpolysoniril In Rnas In Vitrosupporting

confidence: 50%

Response of Tetrahymena pyriformis to stress induced by starvation

Galego

Barahona

Rodrigues-Pousada

1984

European Journal of Biochemistry

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“…was used at a concentration of 15 mM, which has been shown to cause the dissociation of ribosomes from mRNAs without disrupting the majority of nonribosomal RNA-protein complexes (Calzone et al 1982;Johannes and Sarnow 1998). These results suggest that Tap stays associated with the CTE-containing RNA in polysomes or binds to ribosomal components, whereas NXT1 is dissociated from the translation complex prior to, or at the start of translation.…”

Section: Tap But Not Nxt1 Associates With Polyribosomes In Transfecmentioning

confidence: 91%

“…(B) The same as in A, except that 15 mM EDTA was added to the lysate before gradient analysis. This has been shown to cause the dissociation of ribosomes from mRNAs without disrupting the majority of nonribosomal RNA-protein complexes (Calzone et al 1982;Johannes and Sarnow 1998).…”

Section: Tap But Not Nxt1 Associates With Polyribosomes In Transfecmentioning

confidence: 99%

“…After the addition of an equal volume of RSB containing 1% Triton X-100, 1% deoxycholate, and 2% Tween 20, the suspension was incubated for 10 min on ice and centrifuged for 10 min at 10,000g. In some experiments, EDTA was added to a final concentration of 15 mM in order to disrupt the polysomes (Calzone et al 1982;Johannes and Sarnow 1998). The supernatant lysates were loaded onto gradients of 10% to 50% (w/v) sucrose in 10 mM Tris-HCl (pH 7.6), 75 mM KCl, and 3 mM MgCl 2 .…”

Section: Polyribosome Analysismentioning

confidence: 99%

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Tap and NXT promote translation of unspliced mRNA

Jin¹,

Guzik²,

Bor³

et al. 2003

Genes Dev.

141

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Tap has been proposed to play a role in general mRNA export and also functions in expression of RNA with retained introns that contain the MPMV CTE (constitutive transport element). Tap forms a functional heterodimer with NXT/p15. We have previously demonstrated that unspliced intron-containing CTE RNA is efficiently exported to the cytoplasm in mammalian cells. Here we show that Tap and NXT proteins function together to enhance translation of proteins from the exported CTE RNA. Pulse chase experiments show that Tap/NXT significantly increases the rate of protein synthesis. Sucrose gradient analysis demonstrates that Tap and NXT efficiently shift the unspliced RNA into polyribosomal fractions. Furthermore, Tap, but not NXT is detected in polyribosomes. Taken together, our results indicate that Tap and NXT serve a role in translational regulation of RNA after export to the cytoplasm. They further suggest that Tap/NXT may play a role in remodeling of cytoplasmic RNP complexes, providing a link between export pathways and cytoplasmic fate. In higher eukaryotes, the majority of genes produce nascent mRNA transcripts that contain several introns. Export of incompletely spliced RNAs with retained introns from these genes would potentially result in translation of aberrant proteins that could have deleterious effects on the cells. However, cells appear to have developed checkpoints to ensure that only fully spliced mRNAs are exported and expressed (Chang and Sharp 1989;Legrain and Rosbash 1989).Retroviruses use special mechanisms that allow unspliced and incompletely spliced (that is, intron-containing) viral transcripts to exit the nucleus and escape cellular proofreading mechanisms (Hammarskjöld 1997(Hammarskjöld , 2001). Replication of all of these viruses requires the cytoplasmic expression of intron-containing forms of the initial, genome-length viral RNA transcript, because the unspliced RNA serves both as the mRNA for the viral Gag and GagPol proteins and as the RNA that is packaged into viral particles (Berkowitz et al. 1996). In complex retroviruses such as HIV-1, export of unspliced and incompletely spliced RNA relies on the interaction of the viral Rev protein with a structured RNA sequence present in these RNAs, the Rev responsive element (RRE; Hadzopoulou-Cladaras et al. 1989; Hammarskjold et al. 1989;Malim et al. 1989; Pollard and Malim 1998).The resulting mRNP complex is exported by virtue of the nuclear export signal (NES) in Rev that interacts with the cellular export receptor Crm1 (Fornerod et al. 1997;Neville et al. 1997).Simple retroviruses do not encode an Rev-like transacting protein, and export of their unspliced RNA relies on the interaction of cis-acting RNA elements directly with cellular factors. The first element of this kind was identified in the simian type D retrovirus Mason Pfizer Monkey Virus (MPMV) and was given the name CTE (constitutive transport element; Bray et al. 1994; Ernst et al. 1997a,b; Hammarskjold 2001), because its interaction with cellular factors results in constitut...

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“….u ribosomal subunits (27,94). These experiments were performed on cells transfected with the plasmid expressing the full-length 9G8 protein (Fig.…”

Section: Mn>104 Welternmentioning

confidence: 99%

The SR Protein 9G8 and the Wilms' Tumor Suppressor Protein WT1 Promote Translation of mRNAs with Retained Introns

Swartz

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Recent data indicate that the various steps in post-transcriptional gene expression are linked. In the studies presented in this thesis, we show further evidence of the interconnection between the multiple steps of mRNA metabolism. In one study, we show a link between splicing with nucleocytoplasmic export and translation. In another study, we show that the products of an alternatively spliced pre-mRNA have different roles in mRNA metabolism. Specifically, SR proteins, Tap, and WT1(+KTS) function similarly to link nuclear and cytoplasmic events in the expression of mRNAs with retained introns.The first study examines the cytoplasmic role of the splicing regulatory SR protein, 9G8, which has recently been proposed to function in mRNA export in conjunction with the export protein, Tap/NXF1.We investigated the effect of 9G8 on cytoplasmic RNA fate. 9G8 was shown to enhance expression of unspliced RNA containing either the MPMV-CTE or the recently discovered Tap-CTE. 9G8 also enhanced polyribosome association of unspliced RNA containing a CTE. Hyperphosphorylated 9G8 was present in monosomes and small polyribosomes, whereas soluble fractions contained only hypophosphorylated

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Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3

Cited by 43 publications

References 11 publications

Response of Tetrahymena pyriformis to stress induced by starvation

Response of Tetrahymena pyriformis to stress induced by starvation

Tap and NXT promote translation of unspliced mRNA

The SR Protein 9G8 and the Wilms' Tumor Suppressor Protein WT1 Promote Translation of mRNAs with Retained Introns

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Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3

Cited by 43 publications

References 11 publications

Response of Tetrahymena pyriformis to stress induced by starvation

Response of Tetrahymena pyriformis to stress induced by starvation

Tap and NXT promote translation of unspliced mRNA

The SR Protein 9G8 and the Wilms&#39; Tumor Suppressor Protein WT1 Promote Translation of mRNAs with Retained Introns

Contact Info

Product

Resources

About

The SR Protein 9G8 and the Wilms' Tumor Suppressor Protein WT1 Promote Translation of mRNAs with Retained Introns