Nuclear proteins that bind the pre-mRNA 3' splice site sequence r(UUAG/G) and the human telomeric DNA sequence d(TTAGGG)n

1993 ◽  
Vol 13 (7) ◽  
pp. 4301-4310 ◽  
Author(s):  
F Ishikawa ◽  
M J Matunis ◽  
G Dreyfuss ◽  
T R Cech
Keyword(s):  
Splice Site ◽  
Peptide Sequencing ◽  
Nuclear Proteins ◽  
Beta Globin ◽  
Telomeric Dna ◽  
Two Dimensional Gel Electrophoresis ◽  
Mrna Metabolism ◽  
Dna Repeat ◽  
Splice Site Sequence ◽  
Heterogeneous Nuclear Ribonucleoproteins

HeLa cell nuclear proteins that bind to single-stranded d(TTAGGG)n, the human telomeric DNA repeat, were identified and purified by a gel retardation assay. Immunological data and peptide sequencing experiments indicated that the purified proteins were identical or closely related to the heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2-B1, D, and E and to nucleolin. These proteins bound to RNA oligonucleotides having r(UUAGGG) repeats more tightly than to DNA of the same sequence. The binding was sequence specific, as point mutation of any of the first 4 bases [r(UUAG)] abolished it. The fraction containing D and E hnRNPs was shown to bind specifically to a synthetic oligoribonucleotide having the 3' splice site sequence of the human beta-globin intervening sequence 1, which includes the sequence UUAGG. Proteins in this fraction were further identified by two-dimensional gel electrophoresis as D01, D02, D1*, and E0; intriguingly, these members of the hnRNP D and E groups are nuclear proteins that are not stably associated with hnRNP complexes. These studies establish the binding specificities of these D and E hnRNPs. Furthermore, they suggest the possibility that these hnRNPs could perhaps bind to chromosome telomeres, in addition to having a role in pre-mRNA metabolism.

scholarly journals Nuclear proteins that bind the pre-mRNA 3' splice site sequence r(UUAG/G) and the human telomeric DNA sequence d(TTAGGG)n.

1993 ◽  
Vol 13 (7) ◽  
pp. 4301-4310 ◽  
Author(s):  
F Ishikawa ◽  
M J Matunis ◽  
G Dreyfuss ◽  
T R Cech
Keyword(s):  
Splice Site ◽  
Peptide Sequencing ◽  
Nuclear Proteins ◽  
Beta Globin ◽  
Telomeric Dna ◽  
Two Dimensional Gel Electrophoresis ◽  
Mrna Metabolism ◽  
Dna Repeat ◽  
Splice Site Sequence ◽  
Heterogeneous Nuclear Ribonucleoproteins

HeLa cell nuclear proteins that bind to single-stranded d(TTAGGG)n, the human telomeric DNA repeat, were identified and purified by a gel retardation assay. Immunological data and peptide sequencing experiments indicated that the purified proteins were identical or closely related to the heterogeneous nuclear ribonucleoproteins (hnRNPs) A1, A2-B1, D, and E and to nucleolin. These proteins bound to RNA oligonucleotides having r(UUAGGG) repeats more tightly than to DNA of the same sequence. The binding was sequence specific, as point mutation of any of the first 4 bases [r(UUAG)] abolished it. The fraction containing D and E hnRNPs was shown to bind specifically to a synthetic oligoribonucleotide having the 3' splice site sequence of the human beta-globin intervening sequence 1, which includes the sequence UUAGG. Proteins in this fraction were further identified by two-dimensional gel electrophoresis as D01, D02, D1*, and E0; intriguingly, these members of the hnRNP D and E groups are nuclear proteins that are not stably associated with hnRNP complexes. These studies establish the binding specificities of these D and E hnRNPs. Furthermore, they suggest the possibility that these hnRNPs could perhaps bind to chromosome telomeres, in addition to having a role in pre-mRNA metabolism.

Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes

1987 ◽  
Vol 7 (1) ◽  
pp. 281-293
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Branch Site ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Branch Formation ◽  
Splice Site Sequence ◽  
Multiple Interactions ◽  
Small Nuclear Ribonucleoproteins

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

scholarly journals Multiple interactions between the splicing substrate and small nuclear ribonucleoproteins in spliceosomes.

1987 ◽  
Vol 7 (1) ◽  
pp. 281-293 ◽  
Author(s):  
B Chabot ◽  
J A Steitz
Keyword(s):  
Splice Site ◽  
Beta Globin ◽  
Branch Site ◽  
U2 Snrnp ◽  
U1 Snrnp ◽  
Branch Formation ◽  
Splice Site Sequence ◽  
Multiple Interactions ◽  
Small Nuclear Ribonucleoproteins

Protection experiments with antibodies against small nuclear ribonucleoproteins (snRNPs) have elucidated the location of and requirements for interactions between snRNPs and human beta-globin transcripts during splicing in vitro. U2 snRNP association with the intron branch site continues after branch formation, requires intact U2 RNA, and is affected by some alterations of the 3' splice site sequence. U2 snRNP binding to the branched intermediate and U1 snRNP protection of an extended 5' splice region are detected exclusively in spliceosome fractions, indicating that both snRNPs are spliceosome components. While each snRNP associates specifically with the pre-mRNA, they also appear to interact with each other. The recovery of fragments mapping upstream of the 5' splice site suggests how the excised exon is held in the spliceosome.

Nuclear pre-mRNA processing in plants: distinct modes of 3'-splice-site selection in plants and animals

1988 ◽  
Vol 8 (5) ◽  
pp. 2042-2051
Author(s):  
K Wiebauer ◽  
J J Herrero ◽  
W Filipowicz
Keyword(s):  
Splice Site ◽  
Hela Cells ◽  
Site Selection ◽  
Human Growth ◽  
Mrna Processing ◽  
Beta Globin ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Consensus Sequences ◽  
Intron 2

The report that human growth hormone pre-mRNA is not processed in transgenic plant tissues (A. Barta, K. Sommergruber, D. Thompson, K. Hartmuth, M.A. Matzke, and A.J.M. Matzke, Plant Mol. Biol. 6:347-357, 1986) has suggested that differences in mRNA splicing processes exist between plants and animals. To gain more information about the specificity of plant pre-mRNA processing, we have compared the splicing of the soybean leghemoglobin pre-mRNA with that of the human beta-globin pre-mRNA in transfected plant (Orychophragmus violaceus and Nicotiana tabacum) protoplasts and mammalian (HeLa) cells. Of the three introns of leghemoglobin pre-mRNA, only intron 2 was correctly and efficiently processed in HeLa cells. The 5' splice sites of the remaining two introns were faithfully recognized, but correct processing of the 3' sites took place only rarely (intron 1) or not at all (intron 3); cryptic 3' splice sites were used instead. While the first intron in human beta-globin pre-mRNA was not spliced in transfected plant protoplasts, intron 2 processing occurred at a low level, indicating that some mammalian introns can be recognized by the plant intron-splicing machinery. However, excision of intron 2 proved to be incorrect, involving the authentic 5' splice site and a cryptic 3' splice site. Our results indicate that the mechanism of 3'-splice-site selection during intron excision differs between plants and animals. This conclusion is supported by analysis of the 3'-splice-site consensus sequences in animal and plant introns which revealed that polypyrimidine tracts, characteristic of animal introns, are not present in plant pre-mRNAs. It is proposed that an elevated AU content of plant introns is important for their processing.

scholarly journals Probing G-quadruplex topologies and recognition concurrently in real time and 3D using a dual-app nucleoside probe

2019 ◽  
Vol 47 (12) ◽  
pp. 6059-6072 ◽  
Author(s):  
Ashok Nuthanakanti ◽  
Ishtiyaq Ahmed ◽  
Saddam Y Khatik ◽  
Kayarat Saikrishnan ◽  
Seergazhi G Srivatsan
Keyword(s):  
Nucleic Acid ◽  
Ligand Binding ◽  
Real Time ◽  
Telomeric Dna ◽  
X Ray ◽  
X Ray Crystallography ◽  
New Class ◽  
Loop Region ◽  
Dna Repeat ◽  
G Quadruplex

Abstract Comprehensive understanding of structure and recognition properties of regulatory nucleic acid elements in real time and atomic level is highly important to devise efficient therapeutic strategies. Here, we report the establishment of an innovative biophysical platform using a dual-app nucleoside analog, which serves as a common probe to detect and correlate different GQ structures and ligand binding under equilibrium conditions and in 3D by fluorescence and X-ray crystallography techniques. The probe (SedU) is composed of a microenvironment-sensitive fluorophore and an excellent anomalous X-ray scatterer (Se), which is assembled by attaching a selenophene ring at 5-position of 2′-deoxyuridine. SedU incorporated into the loop region of human telomeric DNA repeat fluorescently distinguished subtle differences in GQ topologies and enabled quantify ligand binding to different topologies. Importantly, anomalous X-ray dispersion signal from Se could be used to determine the structure of GQs. As the probe is minimally perturbing, a direct comparison of fluorescence data and crystal structures provided structural insights on how the probe senses different GQ conformations without affecting the native fold. Taken together, our dual-app probe represents a new class of tool that opens up new experimental strategies to concurrently investigate nucleic acid structure and recognition in real time and 3D.

scholarly journals Cell-specific expression of heat shock proteins in chicken reticulocytes and lymphocytes.

1984 ◽  
Vol 99 (4) ◽  
pp. 1316-1323 ◽  
Author(s):  
R Morimoto ◽  
E Fodor
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Gel Electrophoresis ◽  
Elevated Temperatures ◽  
Major Protein ◽  
Beta Globin ◽  
Specific Expression ◽  
Two Dimensional Gel Electrophoresis ◽  
Globin Synthesis ◽  
Shock Proteins

We have found that chicken reticulocytes respond to elevated temperatures by the induction of only one heat shock protein, HSP70, whereas lymphocytes induce the synthesis of all four heat shock proteins (89,000 mol wt, HSP89; 70,000 mol wt, HSP70; 23,000 mol wt, HSP23; and 22,000 mol wt, HSP22). The synthesis of HSP70 in lymphocytes was rapidly induced by small increases in temperature (2 degrees-3 degrees C) and blocked by preincubation with actinomycin D. Proteins normally translated at control temperatures in reticulocytes or lymphocytes were not efficiently translated after incubation at elevated temperatures. The preferential translation of mRNAs that encode the heat shock proteins paralleled a block in the translation of other cellular proteins. This effect was most prominently observed in reticulocytes where heat shock almost completely repressed alpha- and beta-globin synthesis. HSP70 is one of the major nonglobin proteins in chicken reticulocytes, present in the non-heat-shocked cell at approximately 3 X 10(6) molecules per cell. We compared HSP70 from normal and heat-shocked reticulocytes by two-dimensional gel electrophoresis and by digestion with Staphylococcus aureus V8 protease and found no detectable differences to suggest that the P70 in the normal cell is different from the heat shock-induced protein, HSP70. P70 separated by isoelectric focusing gel electrophoresis into two major protein spots, an acidic P70A (apparent pl = 5.95) and a basic P70B (apparent pl = 6.2). We observed a tissue-specific expression of P70A and P70B in lymphocytes and reticulocytes. In lymphocytes, P70A is the major 70,000-mol-wt protein synthesized at normal temperatures whereas only P70B is synthesized at normal temperatures in reticulocytes. Following incubation at elevated temperatures, the synthesis of both HSP70A and HSP70B was rapidly induced in lymphocytes, but synthesis of only HSP70B was induced in reticulocytes.

scholarly journals The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream.

1997 ◽  
Vol 17 (8) ◽  
pp. 4667-4676 ◽  
Author(s):  
R C Chan ◽  
D L Black
Keyword(s):  
Splice Site ◽  
Binding Protein ◽  
Cell Extract ◽  
Neural Cell ◽  
Splicing Regulation ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Splice Site Sequence ◽  
Hela Extract

The neural cell-specific N1 exon of the c-src pre-mRNA is both negatively regulated in nonneural cells and positively regulated in neurons. We previously identified conserved intronic elements flanking N1 that direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. We now show that these upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds.

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