Autoregulated changes in stability of polyribosome-bound beta-tubulin mRNAs are specified by the first 13 translated nucleotides

1988 ◽  
Vol 8 (3) ◽  
pp. 1224-1235
Author(s):  
T J Yen ◽  
D A Gay ◽  
J S Pachter ◽  
D W Cleveland
Keyword(s):  
Amino Acids ◽  
Animal Cells ◽  
Beta Tubulin ◽  
Coding Sequence ◽  
Tubulin Subunit ◽  
Concentration Result ◽  
Necessary And Sufficient ◽  
Specific Degradation ◽  
Autoregulatory Mechanism

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.

scholarly journals Autoregulated changes in stability of polyribosome-bound beta-tubulin mRNAs are specified by the first 13 translated nucleotides.

1988 ◽  
Vol 8 (3) ◽  
pp. 1224-1235 ◽  
Author(s):  
T J Yen ◽  
D A Gay ◽  
J S Pachter ◽  
D W Cleveland
Keyword(s):  
Amino Acids ◽  
Animal Cells ◽  
Beta Tubulin ◽  
Coding Sequence ◽  
Tubulin Subunit ◽  
Concentration Result ◽  
Necessary And Sufficient ◽  
Specific Degradation ◽  
Autoregulatory Mechanism

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.

Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation

1992 ◽  
Vol 12 (2) ◽  
pp. 791-799
Author(s):  
N G Theodorakis ◽  
D W Cleveland
Keyword(s):  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Selective Degradation ◽  
Cellular Factors ◽  
Nascent Peptide ◽  
Tubulin Mrna ◽  
The Stability ◽  
Necessary And Sufficient ◽  
Specific Degradation

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.

scholarly journals Physical evidence for cotranslational regulation of beta-tubulin mRNA degradation.

1992 ◽  
Vol 12 (2) ◽  
pp. 791-799 ◽  
Author(s):  
N G Theodorakis ◽  
D W Cleveland
Keyword(s):  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Selective Degradation ◽  
Cellular Factors ◽  
Nascent Peptide ◽  
Tubulin Mrna ◽  
The Stability ◽  
Necessary And Sufficient ◽  
Specific Degradation

Tubulin synthesis is controlled by an autoregulatory mechanism through which an increase in the intracellular concentration of tubulin subunits leads to specific degradation of tubulin mRNAs. The sequence necessary and sufficient for the selective degradation of a beta-tubulin mRNA in response to changes in the level of free tubulin subunits resides within the first 13 translated nucleotides that encode the amino-terminal sequence of beta-tubulin, Met-Arg-Glu-Ile (MREI). Previous results have suggested that the sequence responsible for autoregulation resides in the nascent peptide rather than in the mRNA per se, raising the possibility that the regulation of the stability of tubulin mRNA is mediated through binding of tubulin or some other cellular factor to the nascent amino-terminal tubulin peptide. We now show that this putative cotranslational interaction is not mediated by tubulin alone, as no meaningful binding is detectable between tubulin subunits and the amino-terminal beta-tubulin polypeptide. However, microinjection of a monoclonal antibody that binds to the beta-tubulin nascent peptide selectively disrupts the regulation of beta-tubulin, but not alpha-tubulin, synthesis. This finding provides direct evidence for cotranslational degradation of beta-tubulin mRNA mediated through binding of one or more cellular factors to the beta-tubulin nascent peptide.

scholarly journals Sir3p Domains Involved in the Initiation of Telomeric Silencing in Saccharomyces cerevisiae

Genetics ◽  
1998 ◽  
Vol 150 (3) ◽  
pp. 977-986 ◽  
Author(s):  
Yangsuk Park ◽  
John Hanish ◽  
Arthur J Lustig
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Fusion Proteins ◽  
Initiation Site ◽  
Negative Control ◽  
Model System ◽  
Mutant Protein ◽  
Regulatory Domain ◽  
C Terminus ◽  
Necessary And Sufficient

Abstract Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3N205 mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD “active” site is under both positive and negative control mediated by multiple Sir3p domains.

scholarly journals EBNA3C Coactivation with EBNA2 Requires a SUMO Homology Domain

2004 ◽  
Vol 78 (1) ◽  
pp. 367-377 ◽  
Author(s):  
Adam Rosendorff ◽  
Diego Illanes ◽  
Gregory David ◽  
Jeffrey Lin ◽  
Elliott Kieff ◽  
...  
Keyword(s):  
Amino Acids ◽  
Gene Activation ◽  
Point Mutations ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Epstein Barr ◽  
Necessary And Sufficient ◽  
Regulated Promoters

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is critical for EBV immortalization of infected B lymphocytes and can coactivate the EBV LMP1 promoter with EBNA2. EBNA3C amino acids 365 to 545 are necessary and sufficient for coactivation and are required for SUMO-1 and SUMO-3 interaction. We found that EBNA3C but not EBNA3CΔ343-545 colocalized with SUMO-1 in nuclear bodies and was modified by SUMO-2, SUMO-3, and SUMO-1. EBNA3C amino acids 545 to 628 and amino acids 30 to 365 were also required for EBNA3C sumolation and nuclear body localization but were dispensable for coactivation, indicating that EBNA3C sumolation is not required for coactivation. Furthermore, EBNA3C amino acids 476 to 992 potently coactivated with EBNA2 but EBNA3C amino acids 516 to 922 lacked activity, indicating that amino acids 476 to 515 are critical for coactivation. EBNA3C amino acids 476 to 515 include DDDVIEV507-513, which are similar to SUMO-1 EEDVIEV84-90. EBNA3C m1 and m2 point mutations, DDD507-509 mutated to AAA and DVIEVID509-513 mutated to AVIAVIA, respectively, diminished SUMO-1 and SUMO-3 interaction in directed yeast two-hybrid and glutathione S-transferase pulldown assays. Furthermore, EBNA3C m1 and m2 did not coactivate the LMP1 promoter with EBNA2. Overexpression of wild-type SUMO-1, SUMO-3, and the SUMO-conjugating enzyme UBC9 coactivated the LMP1 promoter with EBNA2. Since EBNA2 activation is dependent on p300/CBP, the possible effect of EBNA3C on p300-mediated transcription was assayed. EBNA3C potentiated transcription of p300 fused to a heterologous DNA binding domain, whereas EBNA3C m1 and m2 did not. All of these data are consistent with a model in which EBNA3C upregulates EBNA2-mediated gene activation by binding to a sumolated repressor and inhibiting repressive effects on p300/CBP and other transcription factor(s) at EBNA2-regulated promoters.

Dissecting interactions between EB1, microtubules and APC in cortical clusters at the plasma membrane

2002 ◽  
Vol 115 (8) ◽  
pp. 1583-1590 ◽  
Author(s):  
Angela I. M. Barth ◽  
Kathleen A. Siemers ◽  
W. James Nelson
Keyword(s):  
Amino Acids ◽  
Cluster Formation ◽  
Adenomatous Polyposis ◽  
Polyposis Coli ◽  
Binding Domains ◽  
C Terminus ◽  
Endogenous Proteins ◽  
Apc Protein ◽  
Necessary And Sufficient ◽  
Armadillo Repeats

End-binding protein (EB) 1 binds to the C-terminus of adenomatous polyposis coli (APC) protein and to the plus ends of microtubules (MT) and has been implicated in the regulation of APC accumulation in cortical clusters at the tip of extending membranes. We investigated which APC domains are involved in cluster localization and whether binding to EB1 or MTs is essential for APC cluster localization. Armadillo repeats of APC that lack EB1- and MT-binding domains are necessary and sufficient for APC localization in cortical clusters; an APC fragment lacking the armadillo repeats, but containing MT-and EB1-binding domains, does not localize to the cortical clusters but instead co-aligns with MTs throughout the cell. Significantly, analysis of endogenous proteins reveals that EB1 does not accumulate in the APC clusters. However, overexpressed EB1 does accumulate in APC clusters; the APC-binding domain in EB1 is located in the C-terminal region of EB1 between amino acids 134 and 268. Overexpressed APC- or MT-binding domains of EB1 localize to APC cortical clusters and MT, respectively, without affecting APC cluster formation itself. These results show that localization of APC in cortical clusters is different from that of EB1 at MT plus ends and appears to be independent of EB1.

scholarly journals Wachstumsgeschwindigkeit der Peptidkette im Tabakmosaikvirus

1967 ◽  
Vol 22 (12) ◽  
pp. 1280-1291 ◽  
Author(s):  
H. Diringer ◽  
F. A. Anderer ◽  
G. Schramm
Keyword(s):  
Amino Acids ◽  
Growth Rate ◽  
Protein Synthesis ◽  
Peptide Chain ◽  
Cell Protein ◽  
Virus Protein ◽  
Animal Cells ◽  
Tobacco Leaves ◽  
Soluble Cell ◽  
C Terminus

The rate of incorporation of labelled amino acids into the complete tobacco mosaic virus (TMV), into soluble virus protein and into soluble cell proteins has been determined in discs of infected and healthy tobacco leaves. The rate of overall protein synthesis is increased by 50% in the infected leaves. At least 60% of the increase derives from the synthesis of virus-specific proteins and the synthesis of cellular proteins is not inhibited. The virus protein synthesis is strongly temperature dependent and shows a maximum at 28 °C.The exchange of free labelled amino acids between the external medium and the inner cellular pool reaches equilibrium within ten minutes. The influence of the exchange rate on the measurement of the kinetics of peptide chain synthesis is discussed in detail.Discs from infected leaves were incubated for short periods at low temperatures in media containing 3H-tyrosine or 3H-proline. Peptides isolated after 5 minutes incubation at 15 °C were found to be uniformly labelled with no apparent gradient of radioactivity from the N- to the C-terminus. The results indicate that the growth rate of the peptide chain at 15 °C is probably higher than 2 - 3 amino acids/sec and at 28 °C higher than 20 amino acids/sec. These values are higher than those for animal cells and similar to those for protein synthesis in Escherichia coli.Comparison of the growth rate of TMV protein with rate of total protein synthesis and the number of ribosomes in the tobacco leaves indicate that only a small portion of the ribosomes takes part in cell protein synthesis.

scholarly journals An amino-terminal tetrapeptide specifies cotranslational degradation of beta-tubulin but not alpha-tubulin mRNAs.

1994 ◽  
Vol 14 (6) ◽  
pp. 4076-4086 ◽  
Author(s):  
C J Bachurski ◽  
N G Theodorakis ◽  
R M Coulson ◽  
D W Cleveland
Keyword(s):  
Degradation Mechanism ◽  
Full Range ◽  
Mrna Degradation ◽  
Mrna Levels ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Amino Terminal ◽  
Tubulin Subunit ◽  
Tubulin Mrna

The steady-state level of alpha- and beta-tubulin synthesis is autoregulated by a posttranscriptional mechanism that selectively alters alpha- and beta-tubulin mRNA levels in response to changes in the unassembled tubulin subunit concentration. For beta-tubulin mRNAs, previous efforts have shown that this is the result of a selective mRNA degradation mechanism which involves cotranslational recognition of the nascent amino-terminal beta-tubulin tetrapeptide as it emerges from the ribosome. Site-directed mutagenesis is now used to determine that the minimal sequence requirement for conferring the full range of beta-tubulin autoregulation is the amino-terminal tetrapeptide MR(E/D)I. Although tubulin-dependent changes in alpha-tubulin mRNA levels are shown to result from changes in cytoplasmic mRNA stability, transfection of wild-type and mutated alpha-tubulin genes reveals that alpha- and beta-tubulin mRNA degradation is not mediated through a common pathway. Not only does the amino-terminal alpha-tubulin tetrapeptide MREC fail to confer regulated mRNA degradation, neither wild-type alpha-tubulin transgenes nor an alpha-tubulin gene mutated to encode an amino-terminal MREI yields mRNAs that are autoregulated. Further, although slowing ribosome transit accelerates the autoregulated degradation of endogenous alpha- and beta-tubulin mRNAs, degradation of alpha-tubulin transgene mRNAs is not enhanced, and in one case, the mRNA is actually stabilized. We conclude that, despite similarities, alpha- and beta-tubulin mRNA destabilization pathways utilize divergent determinants to link RNA instability to tubulin subunit concentrations.

scholarly journals Chroniques génomiques

2020 ◽  
Vol 36 (6-7) ◽  
pp. 675-677
Author(s):  
Bertrand Jordan
Keyword(s):  
Amino Acids ◽  
Functional Significance ◽  
Open Reading Frames ◽  
Systematic Search ◽  
Biological Processes ◽  
Coding Sequence ◽  
Small Proteins ◽  
Human Dna ◽  
Small Orfs ◽  
Reading Frames

A systematic search for non-conventional open reading frames in human DNA reveals a large number of small ORFs encoding peptides generally smaller than 100 amino-acids. These ORFs are transcribed and translated into small proteins, which are demonstrated to have functional significance by bulk CRISPR inactivation. Evidence is also found for bicistronic mRNAs including such a small ORF upstream of a canonical coding sequence. These findings add a new facet to our understanding of biological processes.

scholarly journals Identification of a novel functional specificity signal within the GPI anchor signal sequence of carcinoembryonic antigen

2007 ◽  
Vol 177 (2) ◽  
pp. 211-218 ◽  
Author(s):  
Thomas B. Nicholson ◽  
Clifford P. Stanners
Keyword(s):  
Amino Acids ◽  
Carcinoembryonic Antigen ◽  
Cell Adhesion Molecule ◽  
Signal Sequence ◽  
Mature Protein ◽  
Functional Specificity ◽  
Gpi Anchor ◽  
Neural Cell Adhesion ◽  
Tumorigenic Activity ◽  
Necessary And Sufficient

Exchanging the glycophosphatidylinositol (GPI) anchor signal sequence of neural cell adhesion molecule (NCAM) for the signal sequence of carcinoembryonic antigen (CEA) generates a mature protein with NCAM external domains but CEA-like tumorigenic activity. We hypothesized that this resulted from the presence of a functional specificity signal within this sequence and generated CEA/NCAM chimeras to identify this signal. Replacing the residues (GLSAG) 6–10 amino acids downstream of the CEA anchor addition site with the corresponding NCAM residues resulted in GPI-anchored proteins lacking the CEA-like biological functions of integrin modulation and differentiation blockage. Transferring this region from CEA into NCAM in conjunction with the upstream proline (PGLSAG) was sufficient to specify the addition of the CEA anchor. Therefore, this study identifies a novel specificity signal consisting of six amino acids located within the GPI anchor attachment signal, which is necessary and sufficient to specify the addition of a particular functional GPI anchor and, thereby, the ultimate function of the mature protein.

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