40 and 60 S ribosomal subunits have been reconstituted in vitro from purified ribosomal RNA and ribosomal proteins of Dictyostelium discoideum. The functionality of the reconstituted ribosomes was ...demonstrated inin vitro mRNA-directed protein synthesis. The reassembly proceeded well with immature precursors of ribosomal RNA but poorly if at all with mature cytoplasmic RNA species. Reassembly also required a preparation of small nuclear RNA(s), acting as morphopoietic factor(s).
Phosphorylation of ribosomal protein S6 leads to the stabilization of pre-spore specific mRNAs during development of Dictyostelium discoideum. The purification of S6 kinase has allowed the ...identification of protein S11 as the mRNase specific for pre-spore mRNAs. Methylation of ribosomal protein S31 leads to the destabilization of ribosomal protein mRNAs. The purification of S31 methyltransferase has allowed the identification of protein S29 as the mRNAse specific for ribosomal protein mRNAs.
The nuclei of Dictyostelium discoideum cells have been found to contain polyribosomes active in protein synthesis. mRNA molecules enter nuclear polyribosomes while they are still being synthesized. ...“Non sense mediated mRNA decay” occurs in the nucleus, through the interaction of the mRNAs containing a nonsense codon with newly formed nuclear ribosomes, rather than with cytoplasmic ribosomes, as previously generally supposed.
The assembly of ribosomal subunits starting from free ribosomal RNA and protein of Dictyostelium discoideum was induced in vitro in the presence of several oligoribonucleotides complementary to ...defined sequences of ribosomal RNA. The reconstituted particles had a full complement of ribosomal proteins, but did not function in an in vitro protein synthesis system and were disassembled following interaction with mRNA. The same result was obtained in vivo by fusing the oligodeossiribonucleotides coding for the selected oligoribonucleotides to the promoter of the gene coding for contact site A protein. This gene is expressed only in the first part of development. Transfected growing cells, transferred in developing buffer in the presence of pulses of cAMP, accumulated significant amounts of the oligoribonucleotides. When retransferred to the growth medium, they grew progressively more slowly, until their doubling time doubled, apparently due to the availability of a limiting amount of functional ribosomes. To avoid disassembly of misassembled subunits (G. Mangiarotti et al., 1997, J. Biol. Chem. 272, 27818–27822), two oligoribonucleotides complementary to sequences present at the 5′ ends of pre-17S and pre-26S RNAs were also induced to accumulate during early development with the same technique. When transfected cells were retransferred to the growth medium, their rate of growth declined rapidly to zero and cells died, apparently because they were unable to disassemble misassembled ribosomal subunits and avoid their entry into polyribosomes. This technique to perturb protein synthesis, arrest cell growth, and cause cell suicide will be tested in abnormally growing animal cells.
Ribosomal protein mRNAs left over from growth are selectively excluded from polyribosomes in the first half of Dictyostelium discoideum development. This is due to the fact that they are sequestered ...by a class of free 40S ribosomal subunits, characterized by possessing a methylated S24 protein. At the time of formation of tight cell aggregates, the methylated S24 is substituted by an unmethylated S24, while protein S31 of the same or other 40S subunits becomes methylated. This leads to a rapid degradation of the ribosomal protein mRNAs.Key words: synthesis of ribosomal proteins, Dictyostelium, methylation of S24, methylation of S31.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
During the development of Dictyostelium discoideum, several thousand new mRNA species appear in the cytoplasm after the cells have formed stable aggregates. Here we show that six of these late mRNAs, ...corresponding to six clones randomly chosen from a genomic library, are synthesized from the very beginning of development at a rate comparable to that observed late in development but that transcripts do not accumulate until after aggregation. The early- and late-synthesized mRNAs are identical in size and compete with each other for hybridization to the genomic clones. The early-synthesized mRNAs do not accumulate in the cytoplasm in the preaggregation stage because they are very unstable. Their stability, estimated from the kinetics of incorporation during continuous labeling with32P, increases by perhaps an order of magnitude in the postaggregation stage. We conclude that mRNA stabilization is the major controlling factor of the expression of these genes.
40 and 60 S ribosomal subunits have been reconstituted in vitro from purified ribosomal RNA and ribosomal proteins of Dictyostelium discoideum. The functionality of the reconstituted ribosomes was ...demonstrated in in vitro mRNA-directed protein synthesis. The reassembly proceeded well with immature precursors of ribosomal RNA but poorly if at all with mature cytoplasmic RNA species. Reassembly also required a preparation of small nuclear RNA(s), acting as morphopoietic factor(s).
AC914 mRNA, a pre-spore-specific mRNA that accumulates only in the post-aggregation stage of development, is transcribed constitutively
as shown by nuclear run-off experiments and by fusing its ...promoter to the luciferase reporter gene. The same mRNA disappears
quickly from disaggregated cells. If the 5â²-untranslated region (5â²UTR) of the constitutively expressed Actin 15 mRNA is substituted
for the 5â²UTR of AC914 mRNA, this can no longer be destabilized and accumulates both in growing and disaggregated cells. If
the 5â²UTR of AC914 mRNA is substituted for the 5â²UTR of Actin 15 mRNA, the latter accumulates only in aggregated cells. Pactamycin,
but not other inhibitors of protein synthesis, prevents AC914 mRNA from being destabilized in disaggregated cells, suggesting
a role of 40âS subunits in the destabilization. This has been confirmed by using an in vitro system in which the in vivo stability of different mRNAs is reproduced. A protein kinase A-dependent phosphorylation of ribosomal protein S6 determines
whether 40âS subunits are capable or not of destabilizing AC914 mRNA in the in vitro system.
AC914 mRNA, a pre-spore-specific mRNA that accumulates only in the post-aggregation stage of development, is transcribed constitutively as shown by nuclear run-off experiments and by fusing its ...promoter to the luciferase reporter gene. The same mRNA disappears quickly from disaggregated cells. If the 5′-untranslated region (5′UTR) of the constitutively expressed Actin 15 mRNA is substituted for the 5′UTR of AC914 mRNA, this can no longer be destabilized and accumulates both in growing and disaggregated cells. If the 5′UTR of AC914 mRNA is substituted for the 5′UTR of Actin 15 mRNA, the latter accumulates only in aggregated cells. Pactamycin, but not other inhibitors of protein synthesis, prevents AC914 mRNA from being destabilized in disaggregated cells, suggesting a role of 40 S subunits in the destabilization. This has been confirmed by using an in vitro system in which the in vivo stability of different mRNAs is reproduced. A protein kinase A-dependent phosphorylation of ribosomal protein S6 determines whether 40 S subunits are capable or not of destabilizing AC914 mRNA in the in vitro system.
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