scholarly journals Pre-mRNA splicing and the nuclear matrix.

1987 ◽  
Vol 7 (1) ◽  
pp. 111-120 ◽  
Author(s):  
S Zeitlin ◽  
A Parent ◽  
S Silverstein ◽  
A Efstratiadis
Keyword(s):  
Hela Cell ◽  
Nuclear Matrix ◽  
Globin Gene ◽  
Mrna Splicing ◽  
Micrococcal Nuclease ◽  
Northern Analysis ◽  
Ribonucleoprotein Complexes ◽  
In Vitro Splicing

We examined the relationship between pre-mRNA splicing and the nuclear matrix by using an in vivo system that we have developed. Plasmids containing the inducible herpesvirus tk gene promoter linked to an intron-containing segment of the rabbit beta-globin gene were transfected into HeLa cells, and then the promoter was transactivated by infection with a TK- virus. Northern analysis revealed that the globin pre-mRNA and all its splicing intermediates and products are associated with the nuclear matrix prepared from such transfected cells. When the nuclear matrix was incubated with a HeLa cell in vitro splicing extract in the presence of ATP, the amount of matrix-associated precursor progressively decreased without a temporal lag in the reaction, with a corresponding increase in free intron lariat. Thus, most of the events of the splicing process (endonucleolytic cuts and branching) occur in this in vitro complementation reaction. However, ligation of exons cannot be monitored in this system because of the abundance of preexisting mature mRNA. Since the matrix is not a self-splicing entity, whereas the in vitro splicing system cannot process efficiently deproteinized matrix RNA, we conclude from our in vitro complementation results (which can be reproduced by using micrococcal nuclease-treated splicing extract) that the nuclear matrix preparation retains parts of preassembled ribonucleoprotein complexes that have the potential to function when supplemented with soluble factors (presumably other than most of the small nuclear ribonucleoproteins known to participate in splicing) present in the HeLa cell extract.

Pre-mRNA splicing and the nuclear matrix

1987 ◽  
Vol 7 (1) ◽  
pp. 111-120
Author(s):  
S Zeitlin ◽  
A Parent ◽  
S Silverstein ◽  
A Efstratiadis
Keyword(s):  
Hela Cell ◽  
Nuclear Matrix ◽  
Globin Gene ◽  
Mrna Splicing ◽  
Micrococcal Nuclease ◽  
Northern Analysis ◽  
Ribonucleoprotein Complexes ◽  
In Vitro Splicing

We examined the relationship between pre-mRNA splicing and the nuclear matrix by using an in vivo system that we have developed. Plasmids containing the inducible herpesvirus tk gene promoter linked to an intron-containing segment of the rabbit beta-globin gene were transfected into HeLa cells, and then the promoter was transactivated by infection with a TK- virus. Northern analysis revealed that the globin pre-mRNA and all its splicing intermediates and products are associated with the nuclear matrix prepared from such transfected cells. When the nuclear matrix was incubated with a HeLa cell in vitro splicing extract in the presence of ATP, the amount of matrix-associated precursor progressively decreased without a temporal lag in the reaction, with a corresponding increase in free intron lariat. Thus, most of the events of the splicing process (endonucleolytic cuts and branching) occur in this in vitro complementation reaction. However, ligation of exons cannot be monitored in this system because of the abundance of preexisting mature mRNA. Since the matrix is not a self-splicing entity, whereas the in vitro splicing system cannot process efficiently deproteinized matrix RNA, we conclude from our in vitro complementation results (which can be reproduced by using micrococcal nuclease-treated splicing extract) that the nuclear matrix preparation retains parts of preassembled ribonucleoprotein complexes that have the potential to function when supplemented with soluble factors (presumably other than most of the small nuclear ribonucleoproteins known to participate in splicing) present in the HeLa cell extract.

scholarly journals Effects of exon sequences on splicing of model pre-mRNA substrates in vitro.

1996 ◽  
Vol 43 (1) ◽  
pp. 161-173 ◽  
Author(s):  
Z Dominski ◽  
R Kole
Keyword(s):  
Globin Gene ◽  
Beta Globin ◽  
Splice Site Selection ◽  
Sequence Composition ◽  
Recognition Element ◽  
Internal Exon ◽  
Alternatively Spliced ◽  
In Vitro Splicing

We used several related pre-mRNA substrates consisting of two introns and three exons to study effects of exon sequences on in vitro splicing. By varying the sequence of the internal exon and measuring the frequency of its skipping we confirmed that 26-nucleotide exon element naturally existing in beta-globin gene and previously analysed in vivo, has a strong stimulatory effect on splicing. Sequence analysis of this element suggests that it belongs to a family of purine-rich splicing elements found in exons of several alternatively spliced pre-mRNAs. The 26-nucleotide element can efficiently function in enhancing inclusion of internal exons regardless of their size and sequence composition, suggesting that it plays a role of a general exon recognition element. The purine-rich element is dispensable in exons flanked by strong splice sites, which promote efficient inclusion of otherwise poorly recognized exons. A row of six cytidines inserted into the internal exon (GC2 mutation) initially considered to stimulate exon inclusion to a similar extent as the purine-rich element (Dominski & Kole, 1994, J. Biol. Chem. 269, 23590-23596), appears not to affect splice site selection in vitro, and in vivo it is likely to act by stabilizing mRNA that includes the internal exon against rapid cytoplasmic degradation.

scholarly journals PRP18, a protein required for the second reaction in pre-mRNA splicing.

1990 ◽  
Vol 10 (1) ◽  
pp. 324-332 ◽  
Author(s):  
U Vijayraghavan ◽  
J Abelson
Keyword(s):  
Micrococcal Nuclease ◽  
Heat Inactivation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Exon 1 ◽  
In Vitro Splicing

We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.

scholarly journals PRP18, a protein required for the second reaction in pre-mRNA splicing

1990 ◽  
Vol 10 (1) ◽  
pp. 324-332 ◽  
Author(s):  
U Vijayraghavan ◽  
J Abelson
Keyword(s):  
Micrococcal Nuclease ◽  
Heat Inactivation ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Wild Type ◽  
Exon 1 ◽  
In Vitro Splicing

We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.

scholarly journals Autonomous splicing and complementation of in vivo-assembled spliceosomes.

1989 ◽  
Vol 108 (3) ◽  
pp. 765-777 ◽  
Author(s):  
S Zeitlin ◽  
R C Wilson ◽  
A Efstratiadis
Keyword(s):  
Nuclear Extract ◽  
Physiological Concentration ◽  
Ribonucleoprotein Complexes ◽  
Matrix Bound ◽  
The Relationship ◽  
Matrix Preparation ◽  
Cell Nuclear Extract ◽  
Biochemical Features

We have used an in vivo system generating assayable amounts of a specific pre-mRNA to study the relationship between splicing and an operationally defined nuclear matrix preparation (NM). When NM is prepared by extraction of DNase I-treated nuclei with an approximately physiological concentration of KCl (0.1 M), a portion of NM-associated precursor can be spliced in vitro in the presence of ATP and Mg2+ and in the absence of splicing extract ("autonomous splicing"). We propose that the autonomous reaction, which does not exhibit a temporal lag and is half-complete in 5 min, occurs in fully assembled, matrix-bound ribonucleoprotein complexes (in vivo spliceosomes). Extraction of the NM with concentrations of KCl greater than 0.4 M eliminates autonomous splicing but leaves behind preassembled complexes that can be complemented for splicing with HeLa cell nuclear extract. The splicing complementing factor, representing one or more activities present in the nuclear extract and also in the cytoplasmic S100 fraction, is relatively heat resistant, devoid of an RNA component, and does not bind to DEAE-Sepharose in 0.1 M KCl. It exists in the nucleus in two forms; bound to autonomous spliceosomes and free in the nucleoplasm. Biochemical features of the complementation reaction, and conditions for reversible uncoupling of the two splicing steps are described and discussed.

The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro

1986 ◽  
Vol 6 (7) ◽  
pp. 2317-2323
Author(s):  
D Zarkower ◽  
P Stephenson ◽  
M Sheets ◽  
M Wickens
Keyword(s):  
Hela Cell ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
Polyadenylation Site ◽  
Single Base ◽  
Cleavage And Polyadenylation ◽  
Cell Nuclear Extract

The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This sequence is required for accurate and efficient cleavage and polyadenylation of pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late pre-mRNAs are cleaved and polyadenylated in vitro in a HeLa cell nuclear extract, and that cleavage in vitro is abolished by each of four different single-base changes in AAUAAA. In this same extract, precleaved RNAs (RNAs with 3' termini at the polyadenylation site) are efficiently polyadenylated. This in vitro polyadenylation reaction also requires the AAUAAA sequence.

The phylogenetically invariant ACAGAGA and AGC sequences of U6 small nuclear RNA are more tolerant of mutation in human cells than in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (9) ◽  
pp. 5377-5382
Author(s):  
B Datta ◽  
A M Weiner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transient Expression ◽  
Human Cells ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Transient Expression Assay ◽  
U6 Snrna ◽  
Nuclear Rna

U6 small nuclear RNA (snRNA) is the most highly conserved of the five spliceosomal snRNAs that participate in nuclear mRNA splicing. The proposal that U6 snRNA plays a key catalytic role in splicing [D. Brow and C. Guthrie, Nature (London) 337:14-15, 1989] is supported by the phylogenetic conservation of U6, the sensitivity of U6 to mutation, cross-linking of U6 to the vicinity of the 5' splice site, and genetic evidence for extensive base pairing between U2 and U6 snRNAs. We chose to mutate the phylogenetically invariant 41-ACAGAGA-47 and 53-AGC-55 sequences of human U6 because certain point mutations within the homologous regions of Saccharomyces cerevisiae U6 selectively block the first or second step of mRNA splicing. We found that both sequences are more tolerant to mutation in human cells (assayed by transient expression in vivo) than in S. cerevisiae (assayed by effects on growth or in vitro splicing). These differences may reflect different rate-limiting steps in the particular assays used or differential reliance on redundant RNA-RNA or RNA-protein interactions. The ability of mutations in U6 nucleotides A-45 and A-53 to selectively block step 2 of splicing in S. cerevisiae had previously been construed as evidence that these residues might participate directly in the second chemical step of splicing; an indirect, structural role seems more likely because the equivalent mutations have no obvious phenotype in the human transient expression assay.

scholarly journals The DNA-binding protein HTa from Thermoplasma acidophilum is an archaeal histone analog

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Antoine Hocher ◽  
Maria Rojec ◽  
Jacob B Swadling ◽  
Alexander Esin ◽  
Tobias Warnecke
Keyword(s):  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Coli Strain ◽  
Micrococcal Nuclease ◽  
Thermoplasma Acidophilum ◽  
Chromatin Architecture ◽  
Archaeal Histone

Histones are a principal constituent of chromatin in eukaryotes and fundamental to our understanding of eukaryotic gene regulation. In archaea, histones are widespread but not universal: several lineages have lost histone genes. What prompted or facilitated these losses and how archaea without histones organize their chromatin remains largely unknown. Here, we elucidate primary chromatin architecture in an archaeon without histones, Thermoplasma acidophilum, which harbors a HU family protein (HTa) that protects part of the genome from micrococcal nuclease digestion. Charting HTa-based chromatin architecture in vitro, in vivo and in an HTa-expressing E. coli strain, we present evidence that HTa is an archaeal histone analog. HTa preferentially binds to GC-rich sequences, exhibits invariant positioning throughout the growth cycle, and shows archaeal histone-like oligomerization behavior. Our results suggest that HTa, a DNA-binding protein of bacterial origin, has converged onto an architectural role filled by histones in other archaea.

scholarly journals Sickle Cell Anemia and Babesia Infection

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1435
Author(s):  
Divya Beri ◽  
Manpreet Singh ◽  
Marilis Rodriguez ◽  
Karina Yazdanbakhsh ◽  
Cheryl Ann Lobo
Keyword(s):  
Sickle Cell ◽  
Sickle Cell Anemia ◽  
Current Knowledge ◽  
Globin Gene ◽  
Environmental Niche ◽  
Parasite Resistance ◽  
The Usa ◽  
Human Infections

Babesia is an intraerythrocytic, obligate Apicomplexan parasite that has, in the last century, been implicated in human infections via zoonosis and is now widespread, especially in parts of the USA and Europe. It is naturally transmitted by the bite of a tick, but transfused blood from infected donors has also proven to be a major source of transmission. When infected, most humans are clinically asymptomatic, but the parasite can prove to be lethal when it infects immunocompromised individuals. Hemolysis and anemia are two common symptoms that accompany many infectious diseases, and this is particularly true of parasitic diseases that target red cells. Clinically, this becomes an acute problem for subjects who are prone to hemolysis and depend on frequent transfusions, like patients with sickle cell anemia or thalassemia. Little is known about Babesia’s pathogenesis in these hemoglobinopathies, and most parallels are drawn from its evolutionarily related Plasmodium parasite which shares the same environmental niche, the RBCs, in the human host. In vitro as well as in vivo Babesia-infected mouse sickle cell disease (SCD) models support the inhibition of intra-erythrocytic parasite proliferation, but mechanisms driving the protection of such hemoglobinopathies against infection are not fully studied. This review provides an overview of our current knowledge of Babesia infection and hemoglobinopathies, focusing on possible mechanisms behind this parasite resistance and the clinical repercussions faced by Babesia-infected human hosts harboring mutations in their globin gene.

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