scholarly journals In vitro splicing of influenza viral NS1 mRNA and NS1-beta-globin chimeras: possible mechanisms for the control of viral mRNA splicing.

1986 ◽  
Vol 83 (15) ◽  
pp. 5444-5448 ◽  
Author(s):  
S. J. Plotch ◽  
R. M. Krug
Keyword(s):  
Mrna Splicing ◽  
Viral Mrna ◽  
Beta Globin ◽  
In Vitro Splicing

scholarly journals The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro.

1988 ◽  
Vol 8 (2) ◽  
pp. 860-866 ◽  
Author(s):  
P J Furdon ◽  
R Kole
Keyword(s):  
Splice Site ◽  
Mrna Splicing ◽  
Beta Globin ◽  
Specific Sequence ◽  
In Vitro Splicing ◽  
Intron 2 ◽  
Specific Sequences

We have shown previously that truncation of the human beta-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit beta-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.

scholarly journals The length of the downstream exon and the substitution of specific sequences affect pre-mRNA splicing in vitro

1988 ◽  
Vol 8 (2) ◽  
pp. 860-866
Author(s):  
P J Furdon ◽  
R Kole
Keyword(s):  
Splice Site ◽  
Mrna Splicing ◽  
Beta Globin ◽  
Specific Sequence ◽  
In Vitro Splicing ◽  
Intron 2 ◽  
Specific Sequences

We have shown previously that truncation of the human beta-globin pre-mRNA in the second exon, 14 nucleotides downstream from the 3' splice site, leads to inhibition of splicing but not cleavage at the 5' splice site. We now show that several nonglobin sequences substituted at this site can restore splicing and that the efficiency of splicing depends on the length of the second (downstream) exon and not a specific sequence. Deletions in the first exon have no effect on the efficiency of in vitro splicing. Surprisingly, an intron fragment from the 5' region of the human or rabbit beta-globin intron 2, when placed 14 nucleotides downstream from the 3' splice site, inhibited all the steps in splicing beginning with cleavage at the 5' splice site. This result suggests that the intron 2 fragment carries a "poison" sequence that can inhibit the splicing of an upstream intron.

In vitro splicing pathways of pre-mRNAs containing multiple intervening sequences?

1987 ◽  
Vol 7 (10) ◽  
pp. 3428-3437
Author(s):  
K M Lang ◽  
R A Spritz
Keyword(s):  
Temporal Order ◽  
Exon Skipping ◽  
Beta Globin ◽  
Splice Site Selection ◽  
Interleukin 3 ◽  
Intervening Sequences ◽  
In Vitro Splicing ◽  
Lag Times ◽  
Alpha Lactalbumin

We analyzed the in vitro splicing pathways of three multi-intervening-sequence (IVS) pre-mRNAs: human beta-globin, which contains two IVSs (K. M. Lang, V. L. van Santen, and R. A. Spritz, EMBO J. 4:1991-1996, 1985); rat alpha-lactalbumin, which contains three IVSs; and murine interleukin-3, which contains four IVSs. We found that there are highly preferred pathways of IVS removal from these multi-IVS pre-mRNAs in vitro. The three IVSs of rat alpha-lactalbumin pre-mRNA were excised sequentially from 5' to 3'; in most molecules, IVS1 was removed first, followed by IVS2 and finally by IVS3. The splicing pathway of interleukin-3 pre-mRNA in vitro was more complex. The four IVSs were excised in a highly preferred temporal order, but the order was not strictly sequential or directional. In most molecules, IVS1 and IVS4 were removed first, either simultaneously or in rapid succession. Subsequently, IVS2 was excised, followed by IVS3. The observed splicing pathways apparently resulted from differences in lag times and maximum excision rates of the different IVSs. We detected no exon skipping during splicing of these transcripts in vitro. These observations have implication for proposed models of splice site selection.

scholarly journals Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing.

1989 ◽  
Vol 9 (12) ◽  
pp. 5273-5280 ◽  
Author(s):  
G C Conway ◽  
A R Krainer ◽  
D L Spector ◽  
R J Roberts
Keyword(s):  
Rna Splicing ◽  
De Novo ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Macromolecular Complex ◽  
Large Structures ◽  
Nuclear Extracts ◽  
In Vitro Splicing ◽  
Nuclear Ribonucleoprotein

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.

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.

scholarly journals Nonconsensus branch-site sequences in the in vitro splicing of transcripts of mutant rabbit beta-globin genes.

1985 ◽  
Vol 82 (24) ◽  
pp. 8349-8353 ◽  
Author(s):  
R. A. Padgett ◽  
M. M. Konarska ◽  
M. Aebi ◽  
H. Hornig ◽  
C. Weissmann ◽  
...  
Keyword(s):  
Globin Genes ◽  
Beta Globin ◽  
Branch Site ◽  
In Vitro Splicing

Psoromic Acid Derivatives: A New Family of Small-Molecule Pre-mRNA Splicing Inhibitors Discovered by a Stage-Specific High-Throughput in Vitro Splicing Assay

ChemBioChem ◽  
2012 ◽  
Vol 13 (5) ◽  
pp. 640-644 ◽  
Author(s):  
Timur R. Samatov ◽  
Alexander Wolf ◽  
Peter Odenwälder ◽  
Sergey Bessonov ◽  
Céline Deraeve ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Mrna Splicing ◽  
New Family ◽  
In Vitro Splicing

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 Analyzing mechanisms of alternative pre-mRNA splicing using in vitro splicing assays

Methods ◽  
2005 ◽  
Vol 37 (4) ◽  
pp. 306-313 ◽  
Author(s):  
Martin J. Hicks ◽  
Bianca J. Lam ◽  
Klemens J. Hertel
Keyword(s):  
Mrna Splicing ◽  
In Vitro Splicing

scholarly journals Multiple splicing factors are released from endogenous complexes during in vitro pre-mRNA splicing

1989 ◽  
Vol 9 (12) ◽  
pp. 5273-5280
Author(s):  
G C Conway ◽  
A R Krainer ◽  
D L Spector ◽  
R J Roberts
Keyword(s):  
Rna Splicing ◽  
De Novo ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Macromolecular Complex ◽  
Large Structures ◽  
Nuclear Extracts ◽  
In Vitro Splicing ◽  
Nuclear Ribonucleoprotein

Pre-mRNA splicing occurs in a macromolecular complex called the spliceosome. Efforts to isolate spliceosomes from in vitro splicing reactions have been hampered by the presence of endogenous complexes that copurify with de novo spliceosomes formed on added pre-mRNA. We have found that removal of these large complexes from nuclear extracts prevents the splicing of exogenously added pre-mRNA. We therefore examined these complexes for the presence of splicing factors and proteins known or thought to be involved in RNA splicing. These fast-sedimenting structures were found to contain multiple small nuclear ribonucleoproteins (snRNPs) and a fragmented heterogeneous nuclear ribonucleoprotein complex. At least two splicing factors other than the snRNPs were also associated with these large structures. Upon incubation with ATP, these splicing factors as well as U1 and U2 snRNPs were released from these complexes. The presence of multiple splicing factors suggests that these complexes may be endogenous spliceosomes released from nuclei during preparation of splicing extracts. The removal of these structures from extracts that had been preincubated with ATP yielded a splicing extract devoid of large structures. This extract should prove useful in the fractionation of splicing factors and the isolation of native spliceosomes formed on exogenously added pre-mRNA.

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