scholarly journals Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing

1994 ◽  
Vol 14 (11) ◽  
pp. 7445-7454
Author(s):  
Z Dominski ◽  
R Kole
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Consensus Sequence ◽  
Aberrant Splice ◽  
Splice Sites ◽  
Branch Point Sequence ◽  
Splice Site Sequence ◽  
Internal Exon ◽  
Cryptic Splice Sites

Certain thalassemic human beta-globin pre-mRNAs carry mutations that generate aberrant splice sites and/or activate cryptic splice sites, providing a convenient and clinically relevant system to study splice site selection. Antisense 2'-O-methyl oligoribonucleotides were used to block a number of sequences in these pre-mRNAs and were tested for their ability to inhibit splicing in vitro or to affect the ratio between aberrantly and correctly spliced products. By this approach, it was found that (i) up to 19 nucleotides upstream from the branch point adenosine are involved in proper recognition and functioning of the branch point sequence; (ii) whereas at least 25 nucleotides of exon sequences at both 3' and 5' ends are required for splicing, this requirement does not extend past the 5' splice site sequence of the intron; and (iii) improving the 5' splice site of the internal exon to match the consensus sequence strongly decreases the accessibility of the upstream 3' splice site to antisense 2'-O-methyl oligoribonucleotides. This result most likely reflects changes in the strength of interactions near the 3' splice site in response to improvement of the 5' splice site and further supports the existence of communication between these sites across the exon.

scholarly journals Identification and characterization by antisense oligonucleotides of exon and intron sequences required for splicing.

1994 ◽  
Vol 14 (11) ◽  
pp. 7445-7454 ◽  
Author(s):  
Z Dominski ◽  
R Kole
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Consensus Sequence ◽  
Aberrant Splice ◽  
Splice Sites ◽  
Branch Point Sequence ◽  
Splice Site Sequence ◽  
Internal Exon ◽  
Cryptic Splice Sites

Certain thalassemic human beta-globin pre-mRNAs carry mutations that generate aberrant splice sites and/or activate cryptic splice sites, providing a convenient and clinically relevant system to study splice site selection. Antisense 2'-O-methyl oligoribonucleotides were used to block a number of sequences in these pre-mRNAs and were tested for their ability to inhibit splicing in vitro or to affect the ratio between aberrantly and correctly spliced products. By this approach, it was found that (i) up to 19 nucleotides upstream from the branch point adenosine are involved in proper recognition and functioning of the branch point sequence; (ii) whereas at least 25 nucleotides of exon sequences at both 3' and 5' ends are required for splicing, this requirement does not extend past the 5' splice site sequence of the intron; and (iii) improving the 5' splice site of the internal exon to match the consensus sequence strongly decreases the accessibility of the upstream 3' splice site to antisense 2'-O-methyl oligoribonucleotides. This result most likely reflects changes in the strength of interactions near the 3' splice site in response to improvement of the 5' splice site and further supports the existence of communication between these sites across the exon.

scholarly journals A Splice Site Mutant of Maize Activates Cryptic Splice Sites, Elicits Intron Inclusion and Exon Exclusion, and Permits Branch Point Elucidation

1999 ◽  
Vol 121 (2) ◽  
pp. 411-418 ◽  
Author(s):  
Shailesh Lal ◽  
Jae-Hyuk Choi ◽  
Janine R. Shaw ◽  
L. Curtis Hannah
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Splice Sites ◽  
Cryptic Splice Sites

scholarly journals A 5′ Splice Site-Proximal Enhancer Binds SF1 and Activates Exon Bridging of a Microexon

2000 ◽  
Vol 20 (11) ◽  
pp. 3988-3995 ◽  
Author(s):  
Troy Carlo ◽  
Rebecca Sierra ◽  
Susan M. Berget
Keyword(s):  
Splice Site ◽  
Troponin T ◽  
Repeated Sequence ◽  
Single Copy ◽  
Splice Sites ◽  
Exon Definition ◽  
Internal Exon ◽  
Splicing Factor 1

ABSTRACT Internal exon size in vertebrates occurs over a narrow size range. Experimentally, exons shorter than 50 nucleotides are poorly included in mRNA unless accompanied by strengthened splice sites or accessory sequences that act as splicing enhancers, suggesting steric interference between snRNPs and other splicing factors binding simultaneously to the 3′ and 5′ splice sites of microexons. Despite these problems, very small naturally occurring exons exist. Here we studied the factors and mechanism involved in recognizing a constitutively included six-nucleotide exon from the cardiac troponin T gene. Inclusion of this exon is dependent on an enhancer located downstream of the 5′ splice site. This enhancer contains six copies of the simple sequence GGGGCUG. The enhancer activates heterologous microexons and will work when located either upstream or downstream of the target exon, suggesting an ability to bind factors that bridge splicing units. A single copy of this sequence is sufficient for in vivo exon inclusion and is the binding site for the known bridging mammalian splicing factor 1 (SF1). The enhancer and its bound SF1 act to increase recognition of the upstream exon during exon definition, such that competition of in vitro reactions with RNAs containing the GGGGCUG repeated sequence depress splicing of the upstream intron, assembly of the spliceosome on the 3′ splice site of the exon, and cross-linking of SF1. These results suggest a model in which SF1 bridges the small exon during initial assembly, thereby effectively extending the domain of the exon.

The natural 5' splice site of simian virus 40 large T antigen can be improved by increasing the base complementarity to U1 RNA

1987 ◽  
Vol 7 (8) ◽  
pp. 3018-3020
Author(s):  
Y Zhuang ◽  
H Leung ◽  
A M Weiner
Keyword(s):  
Splice Site ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Splice Sites ◽  
Small Nuclear Ribonucleoprotein ◽  
Antigen 5

The use of alternative 5' splice sites in the simian virus 40 early-transcription unit controls the ratio of large T to small t antigen during viral infection. To study the regulation of these alternative 5' splice sites, we made two mutants which improve the match of the large-T-antigen 5' splice site to the 5' splice site consensus sequence. Whether these mutants were assayed in vitro or in vivo, we found that the efficiency of large-T splicing is increased by improving the match of the large-T-antigen 5' splice site to the consensus. We conclude that the match of a 5' splice site is an important determinant of 5' splice site utilization and that the simian virus 40 large-T-antigen 5' splice site is almost certainly recognized by the U1 small nuclear RNA component of the U1 small nuclear ribonucleoprotein particle.

scholarly journals The natural 5' splice site of simian virus 40 large T antigen can be improved by increasing the base complementarity to U1 RNA.

1987 ◽  
Vol 7 (8) ◽  
pp. 3018-3020 ◽  
Author(s):  
Y Zhuang ◽  
H Leung ◽  
A M Weiner
Keyword(s):  
Splice Site ◽  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Splice Sites ◽  
Small Nuclear Ribonucleoprotein ◽  
Antigen 5

The use of alternative 5' splice sites in the simian virus 40 early-transcription unit controls the ratio of large T to small t antigen during viral infection. To study the regulation of these alternative 5' splice sites, we made two mutants which improve the match of the large-T-antigen 5' splice site to the 5' splice site consensus sequence. Whether these mutants were assayed in vitro or in vivo, we found that the efficiency of large-T splicing is increased by improving the match of the large-T-antigen 5' splice site to the consensus. We conclude that the match of a 5' splice site is an important determinant of 5' splice site utilization and that the simian virus 40 large-T-antigen 5' splice site is almost certainly recognized by the U1 small nuclear RNA component of the U1 small nuclear ribonucleoprotein particle.

scholarly journals The Allele-Specific Suppressor sup-39 Alters Use of Cryptic Splice Sites in Caenorhabditis elegans

Genetics ◽  
2000 ◽  
Vol 154 (3) ◽  
pp. 1169-1179
Author(s):  
A Brock Roller ◽  
David C Hoffman ◽  
Alan M Zahler
Keyword(s):  
Caenorhabditis Elegans ◽  
Splice Site ◽  
Consensus Sequence ◽  
Splice Junction ◽  
Splice Sites ◽  
Wild Type ◽  
Splice Donor ◽  
Nonsense Mediated Decay ◽  
Allele Specific ◽  
Cryptic Splice Sites

Abstract Mutations in the Caenorhabditis elegans sup-39 gene cause allele-specific suppression of the uncoordination defect of unc-73(e936). e936 is a point mutation that changes the canonical G at the 5′ end of intron 16 to a U. This mutation activates three splice donors, two of which define introns beginning with the canonical GU. Use of these two cryptic splice sites causes loss of reading frame; interestingly these messages are not substrates for nonsense-mediated decay. The third splice donor, used in 10% of steady-state e936 messages, is the mutated splice donor at the wild-type position, which defines an intron beginning with UU. In the presence of a sup-39 mutation, these same three splice donors are used, but the ratio of messages produced by splicing at these sites changes. The percentage of unc-73(e936) messages containing the wild-type splice junction is increased to 33% with a corresponding increase in the level of UNC-73 protein. This sup-39-induced change was also observed when the e936 mutant intron region was inserted into a heterologous splicing reporter construct transfected into worms. Experiments with splicing reporter constructs showed that the degree of 5′ splice site match to the splicing consensus sequence can strongly influence cryptic splice site choice. We propose that mutant SUP-39 is a new type of informational suppressor that alters the use of weak splice donors.

scholarly journals An Upstream AG Determines Whether a Downstream AG Is Selected during Catalytic Step II of Splicing

2001 ◽  
Vol 21 (5) ◽  
pp. 1509-1514 ◽  
Author(s):  
Katrin Chua ◽  
Robin Reed
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Optimal Location ◽  
Transesterification Reaction ◽  
Precise Localization ◽  
Splice Sites ◽  
Precise Location ◽  
Branch Point Sequence ◽  
General Localization

ABSTRACT Specific mechanisms must exist to ensure fidelity in selecting the AG dinucleotide that functions as the 3′ splice site during the second transesterification step of splicing. Here we show that the optimal location for this AG is within a narrow distance (19 to 23 nucleotides [nt]) downstream from the branch point sequence (BPS). Contrary to previous expectations, AGs located less than 23 nt from the BPS are always recognized, even when a second AG located more optimally downstream is used in the transesterification reaction. Indeed, the AG closest to the BPS actually dictates the precise location of the AG that engages in the reaction. This mechanism, in which the AG is identified by a general localization step followed by a precise localization step, may be used to achieve fidelity while allowing flexibility in the location of 3′ splice sites.

Control of adenovirus E1B mRNA synthesis by a shift in the activities of RNA splice sites

1984 ◽  
Vol 4 (5) ◽  
pp. 966-972
Author(s):  
C Montell ◽  
E F Fisher ◽  
M H Caruthers ◽  
A J Berk
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Early Phase ◽  
Late Phase ◽  
Productive Infection ◽  
Splice Sites ◽  
Mrna Synthesis ◽  
Adenovirus E1b ◽  
Cryptic Splice Sites ◽  
Rna Splice Sites

The primary transcript from adenovirus 2 early region 1B (E1B) is processed by differential RNA splicing into two overlapping mRNAs, 13S and 22S. The 22S mRNA is the major E1B mRNA during the early phase of infection, whereas the 13S mRNA predominates during the late phase. In previous work, it has been shown that this shift in proportions of the E1B mRNAs is influenced by increased cytoplasmic stability of the 13S mRNA at late times in infection. Two observations presented here demonstrate that the increase in proportion of the 13S mRNA at late times is also regulated by a change in the specificity of RNA splicing. First, the relative concentrations of the 13S to 22S nuclear RNAs were not constant throughout infection but increased at late times. Secondly, studies with the mutant, adenovirus 2 pm2250 , provided evidence that there was an increased propensity to utilize a 5' splice in the region of the 13S 5' splice site at late times in infection. Adenovirus 2 pm2250 has a G----C transversion in the first base of E1B 13S mRNA intron preventing splicing of the 13S mRNA but not of the 22S mRNA. During the early phase of a pm2250 infection, the E1B primary transcripts were processed into the 22S mRNA only. However, during the late phase, when the 13S mRNA normally predominates, E1B primary transcripts were also processed by RNA splicing at two formerly unused or cryptic 5' splice sites. Both cryptic splice sites were located much closer to the disrupted 13S 5' splice site than to the 22S 5' splice site. Thus, the temporal increase in proportion of the 13S mRNA to the 22S mRNA is regulated by two processes, an increase in cytoplasmic stability of the 13S mRNA and an increased propensity to utilize the 13S 5' splice site during the late phase of infection. Adenovirus 2 pm2250 was not defective for productive infection of HeLa cells or for transformation of rat cells.

A novel protein factor is required for use of distal alternative 5' splice sites in vitro

1991 ◽  
Vol 11 (12) ◽  
pp. 5945-5953
Author(s):  
J E Harper ◽  
J L Manley
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Deae Cellulose ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Small Nuclear Ribonucleoprotein

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.

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