Biomedical subjects
Publications and source records attributed to J R Greenberg.
The ability of the restorative dentist to communicate the location and orientation of the patient's pertinent facial landmarks to the dental laboratory technician has great bearing on the esthetic success of final anterior dental restorations. This article describes a new device designed to facilitate this process.
Nip1p is an essential Saccharomyces cerevisiae protein that was identified in a screen for temperature conditional (ts) mutants exhibiting defects in nuclear transport. New results indicate that Nip1p has a primary role in translation initiation. Polysome profiles indicate that cells depleted of Nip1p and nip1-1 cells are defective in translation initiation, a conclusion that is supported by a reduced rate of protein synthesis in Nip1p-depleted cells. Nip1p cosediments with free 40 S ribosomal subunits and polysomal preinitiation complexes, but not with free or elongating 80 S ribosomes or 60 S subunits. Nip1p can be isolated in an about 670-kDa complex containing polyhistidine-tagged Prt1p, a subunit of translation initiation factor 3, by binding to Ni2+-NTA-agarose beads in a manner completely dependent on the tagged form of Prt1p. The nip1-1 ts growth defect was suppressed by the deletion of the ribosomal protein, RPL46. Also, nip1-1 mutant cells are hypersensitive to paromomycin. These results suggest that Nip1p is a subunit of eukaryotic initiation factor 3 required for efficient translation initiation.
Only five of the nine subunits of human eukaryotic translation initiation factor 3 (eIF3) have recognizable homologs encoded in the Saccharomyces cerevisiae genome, and only two of these (Prt1p and Tif34p) were identified previously as subunits of yeast eIF3. We purified a polyhistidine-tagged form of Prt1p (His-Prt1p) by Ni2+ affinity and gel filtration chromatography and obtained a complex of approximately 600 kDa composed of six polypeptides whose copurification was completely dependent on the polyhistidine tag on His-Prt1p. All five polypeptides associated with His-Prt1p were identified by mass spectrometry, and four were found to be the other putative homologs of human eIF3 subunits encoded in S. cerevisiae: YBR079c/Tif32p, Nip1p, Tif34p, and YDR429c/Tif35p. The fifth Prt1p-associated protein was eIF5, an initiation factor not previously known to interact with eIF3. The purified complex could rescue Met-tRNAiMet binding to 40S ribosomes in defective extracts from a prt1 mutant or extracts from which Nip1p had been depleted, indicating that it possesses a known biochemical activity of eIF3. These findings suggest that Tif32p, Nip1p, Prt1p, Tif34p, and Tif35p comprise an eIF3 core complex, conserved between yeast and mammals, that stably interacts with eIF5. Nip1p bound to eIF5 in yeast two-hybrid and in vitro protein binding assays. Interestingly, Sui1p also interacts with Nip1p, and both eIF5 and Sui1p have been implicated in accurate recognition of the AUG start codon. Thus, eIF5 and Sui1p may be recruited to the 40S ribosomes through physical interactions with the Nip1p subunit of eIF3.
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Previous work in our laboratories (Slobin, L. I., and Greenberg, J.R. (1988) Eur. J. Biochem. 173, 305-310) showed that messenger ribonucleoprotein (mRNP) particles possess a polypeptide component of approximately 62 kDa that appears to share a common epitope with eucaryotic elongation factor 1 alpha (EF-1 alpha). We report here that the previously unidentified mRNP constituent corresponds to seryl-tRNA synthetase (SerRS). Furthermore, we show that SerRS contains a sequence motif that is shared by both EF-1 alpha and glutaminyl-tRNA synthetase. We also find that the association of SerRS with mRNA depends on the functional state of the latter. Our data suggest that SerRS may participate directly in the initiation phase of protein synthesis.
The Assessment of Living Skills and Resources (ALSAR), an innovative instrumental activities of daily living tool, systematically evaluates the accomplishment of 11 tasks by separately rating patient skill and resource levels for each task and combining these levels to determine risk. The ALSAR was administered to 75 elderly veterans in a home care program. It predicted change to more supportive living arrangements and a more structured living environment, nursing home placement, hospitalization, and death during the 6-month study period. The ALSAR has proven useful for interdisciplinary problem solving and treatment planning.
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This laboratory previously detected by UV crosslinking a number of proteins associated with cytoplasmic mRNA in mammalian cells, and the data suggested that they are involved in translation. To find out which proteins are associated with caps we made use of reticulocyte mRNA specifically labeled in the cap with 32P together with a cell-free translation system and UV crosslinking. Approximately 8 bands corresponding to proteins crosslinked to the cap itself have been detected by polyacrylamide gel electrophoresis after UV crosslinking and digestion with RNases or tobacco pyrophosphatase. All but one were specific for methylated caps. One was similar in size and partial peptide map to a cap-binding protein, CBP I, previously identified in other laboratories, and most of the others corresponded to proteins previously known to be associated with mRNA but not known to be associated with caps. The results suggest that most mRNA-associated proteins are associated with caps or poly(A). Also, the number of cap-associated proteins may be greater than previously suspected.
A 62-kDa polypeptide, which reacts with antibodies directed against a peptide corresponding to a portion of the amino-terminal structure of eucaryotic elongation factor Tu (eEF-Tu), was purified from the 0.5 M NaCl wash of rabbit reticulocyte polysomes. Previous work has shown that this polypeptide is a constituent of messenger ribonucleoprotein particles (mRNPs) from a variety of mammalian cell types [Greenberg, J.R. and Carroll, E. C. (1985) Mol. Cell Biol. 5, 342-351]. The purified polypeptide bound mRNA as well as rRNA using a nitrocellulose-filter assay. The same nitrocellulose-filter assay failed to detect binding to GTP. Using a competition-binding assay, it was established that the purified polypeptide interacts with poly(U) and poly(G) but not with poly(A). This preference for synthetic polynucleotides was the same as found for eEF-Tu [Slobin, L.I. (1983) J. Biol. Chem. 258, 4895-4900]. Furthermore, treatment of the purified RNA-binding protein with trypsin resulted in a rapid cleavage of two peptide bonds resulting in fragments of 60 kDa and 53 kDa. Trypsin also cleaves eEF-Tu rapidly at two bonds resulting in two large polypeptide fragments [Slobin, L.I., Clark, R.V. & Olson, M.O.J. (1981) Biochemistry 20, 5761-5767]. The amino acid sequence of the first 39 residues of the purified RNA-binding protein was determined and found to possess no homology to eEF-Tu.
We tested the effects of a series of synthetic oligonucleotides (hybridons) complementary to the 5' noncoding and coding regions of rabbit beta-globin mRNA on endogenous protein synthesis in a rabbit reticulocyte cell-free translation system. With highly purified hybridons inhibition was completely specific for beta-globin. The sites most sensitive to inhibition are the beginning of the 5' noncoding region and a sequence including the initiation codon and several upstream bases. The region between these was relatively insensitive to inhibition. The sites of maximum sensitivity coincide with known protein binding sites, suggesting that hybridons exert their effects in part by blocking the binding of proteins required for translation. Their effectiveness seems related to the ease with which they are displaced by ribosomes.
By two-dimensional gel electrophoresis, partial peptide mapping, and antibody binding we have shown that eukaryotic elongation factor Tu is in close contact with mRNA in rabbit reticulocytes. It can be crosslinked to mRNA by irradiating both polysomes and 40-80 S mRNA-protein complexes with short-wave UV light. To our knowledge this is the first case in which a known translation factor has been shown to be associated with mRNA in native ribonucleoproteins.
A variety of evidence suggests that the cytoplasmic mRNA-associated proteins of eucaryotic cells are derived from the cytoplasm and function there, most likely in protein synthesis or some related process. Furthermore, the evidence suggests that protein-free mRNA added to a cell-free translation system should become associated with a set of proteins similar to those associated with mRNA in native polyribosomes. To test this hypothesis, we added deproteinized rabbit reticulocyte mRNA to a homologous cell-free translation system made dependent on exogenous mRNA by treatment with micrococcal nuclease. The resulting reconstituted complexes were irradiated with UV light to cross-link the proteins to mRNA, and the proteins were analyzed by gel electrophoresis. The proteins associated with polyribosomal mRNA in the reconstituted complexes were indistinguishable from those associated with polyribosomal mRNA in intact reticulocytes. Furthermore, reticulocyte mRNA-associated proteins were very similar to those of cultured mammalian cells. The composition of the complexes varied with the translational state of the mRNA; that is, certain proteins present in polyribosomal mRNA-protein complexes were absent or reduced in amount in 40S to 80S complexes and in complexes formed in the absence of translation. However, other proteins, including a 78-kilodalton protein associated with polyadenylate, were present irrespective of translational state, or else they were preferentially associated with untranslated mRNA. These findings are in agreement with previous data suggesting that proteins associated with cytoplasmic mRNA are derived from the cytoplasm and that they function in translation or some other cytoplasmic process, rather than transcription, RNA processing, or transport from the nucleus to the cytoplasm.
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The proteins associated with poly(A) and other regions of mRNA and hnRNA molecules in mouse L cells were investigated with the aid of ultraviolet light-induced crosslinking of proteins to RNA. The poly(A)s of polyribosomal and free cytoplasmic mRNAs are associated with a protein, p78A. In contrast, the poly(A) of hnRNA is associated with a smaller protein, p60A, that differs from p78A in its partial peptide map. p78A occurs free in the cytoplasm, but p60A does not. There is a second 78 kd protein, p78X, associated with mRNA sequences other than poly(A). p78X differs from p78A in its partial peptide map. The total proteins crosslinked to polyribosomal and free cytoplasmic mRNAs are similar. However, the total proteins crosslinked to hnRNA are quite different from those crosslinked to mRNA. We suggest that newly synthesized mRNA molecules emerging from the nucleus into the cytoplasm shed the proteins with which they were associated in the nucleus and become associated with a new set of proteins derived from the cytosol. Furthermore, the cytoplasmic mRNA-associated proteins continue to exchange with free proteins.