scholarly journals Factors affecting authentic 5' splice site selection in plant nuclei.

1993 ◽  
Vol 13 (3) ◽  
pp. 1323-1331 ◽  
Author(s):  
A J McCullough ◽  
H Lou ◽  
M A Schuler
Keyword(s):  
Splice Site ◽  
Transition Point ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Splice Sites ◽  
Factors Affecting ◽  
Plant Expression Vector ◽  
Normal Site ◽  
Intron Boundary

To define elements critical for 5' splice selection in dicot plant nuclei, wild-type and mutant transcripts containing the first intron of the pea rbcS3A gene were expressed in vivo by using an autonomously replicating plant expression vector. Mutations within the normal 5' splice site (+1) of this intron demonstrate that 5' splice sites at the normal exon-intron boundary having only limited agreement with a 5' splice site consensus sequence can be spliced quite effectively in dicot nuclei. Inactivation of the normal 5' splice site occurs only by point mutations of the G at position +1 of the intron (+1G) or +2U or by multiple mutations at other positions and results in the activation of three cryptic 5' splice sites in the adjacent exon and intron. cis competition of cryptic sites having consensus 5' splice site sequences with the normal 5' splice site demonstrates that cryptic splice sites in the exon, but not the intron, can compete to some extent with the normal site. Replacement of the sequences between the cryptic and normal 5' splice sites with heterologous exon or intron sequences demonstrates that the 5' boundary of this plant intron is defined by its position relative to the AU transition point between exon and intron. These results suggest that potential 5' splice sites upstream of the AU transition point are accessible for recognition by the plant pre-mRNA splicing machinery and that those downstream in the AU-rich intron are masked from recognition.

Factors affecting authentic 5' splice site selection in plant nuclei

1993 ◽  
Vol 13 (3) ◽  
pp. 1323-1331
Author(s):  
A J McCullough ◽  
H Lou ◽  
M A Schuler
Keyword(s):  
Splice Site ◽  
Transition Point ◽  
Consensus Sequence ◽  
Point Mutations ◽  
Splice Sites ◽  
Factors Affecting ◽  
Plant Expression Vector ◽  
Normal Site ◽  
Intron Boundary

To define elements critical for 5' splice selection in dicot plant nuclei, wild-type and mutant transcripts containing the first intron of the pea rbcS3A gene were expressed in vivo by using an autonomously replicating plant expression vector. Mutations within the normal 5' splice site (+1) of this intron demonstrate that 5' splice sites at the normal exon-intron boundary having only limited agreement with a 5' splice site consensus sequence can be spliced quite effectively in dicot nuclei. Inactivation of the normal 5' splice site occurs only by point mutations of the G at position +1 of the intron (+1G) or +2U or by multiple mutations at other positions and results in the activation of three cryptic 5' splice sites in the adjacent exon and intron. cis competition of cryptic sites having consensus 5' splice site sequences with the normal 5' splice site demonstrates that cryptic splice sites in the exon, but not the intron, can compete to some extent with the normal site. Replacement of the sequences between the cryptic and normal 5' splice sites with heterologous exon or intron sequences demonstrates that the 5' boundary of this plant intron is defined by its position relative to the AU transition point between exon and intron. These results suggest that potential 5' splice sites upstream of the AU transition point are accessible for recognition by the plant pre-mRNA splicing machinery and that those downstream in the AU-rich intron are masked from recognition.

CHARACTERIZATION OF THE SPLICE SITES IN GT–AG AND GC–AG INTRONS IN HIGHER EUKARYOTES USING FULL-LENGTH cDNAs

2004 ◽  
Vol 02 (02) ◽  
pp. 309-331 ◽  
Author(s):  
SUMIE KITAMURA–ABE ◽  
HITOMI ITOH ◽  
TAKANORI WASHIO ◽  
AKIHIRO TSUTSUMI ◽  
MASARU TOMITA
Keyword(s):  
Alternative Splice ◽  
Splice Site ◽  
Consensus Sequence ◽  
Full Length ◽  
Splice Sites ◽  
Consensus Sequences ◽  
Full Length Cdna ◽  
Strong Consensus ◽  
Higher Eukaryotes

For the purpose of analyzing the relation between the splice sites and the order of introns, we conducted the following analysis for the GT–AG and GC–AG splice site groups. First, the pre-mRNAs of H. sapiens, M. musculus, D. melanogaster, A. thaliana and O. sativa were sampled by mapping the full-length cDNA to the genomes. Next, the consensus sequences at different regions of pre-mRNAs were analyzed in the five species. We also investigated the mononucleotide and dinucleotide frequencies in the extensive regions around the 5' splice sites (5'ss) and 3' splice sites (3'ss). As a result, differential frequencies of nucleotides at the first 5'ss in both the GT–AG and GC–AG splice site groups were observed in A. thaliana and O. sativa pre-mRNAs. The trend, which indicates that GC 5'ss possess strong consensus sequences, was observed not only in mammalian pre-mRNAs but also in the pre-mRNAs of D. melanogaster, A. thaliana and O. sativa. Furthermore, we examined the consensus sequences of the constitutive and alternative splice sites. It was suggested that in the case of the alternative GC–AG introns, the tendency to have a weak consensus sequence at 5'ss is different between H. sapiens and M. musculus pre-mRNAs.

scholarly journals In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit.

1993 ◽  
Vol 13 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
D A Sterner ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Troponin I ◽  
Exon Skipping ◽  
Splice Sites ◽  
Small Vertebrate ◽  
Upstream Exon ◽  
I Gene

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

scholarly journals An Intronic Splicing Enhancer Binds U1 snRNPs To Enhance Splicing and Select 5′ Splice Sites

2000 ◽  
Vol 20 (24) ◽  
pp. 9225-9235 ◽  
Author(s):  
Andrew J. McCullough ◽  
Susan M. Berget
Keyword(s):  
Splice Site ◽  
Maximal Activity ◽  
Splice Sites ◽  
Base Pairing ◽  
Rich Region ◽  
U1 Snrnp ◽  
Splicing Efficiency ◽  
Site Recognition ◽  
Splicing Enhancer

ABSTRACT Intronic G triplets are frequently located adjacent to 5′ splice sites in vertebrate pre-mRNAs and have been correlated with splicing efficiency and specificity via a mechanism that activates upstream 5′ splice sites in exons containing duplicated sites (26). Using an intron dependent upon G triplets for maximal activity and 5′ splice site specificity, we determined that these elements bind U1 snRNPs via base pairing with U1 RNA. This interaction is novel in that it uses nucleotides 8 to 10 of U1 RNA and is independent of nucleotides 1 to 7. In vivo functionality of base pairing was documented by restoring activity and specificity to mutated G triplets through compensating U1 RNA mutations. We suggest that the G-rich region near vertebrate 5′ splice sites promotes accurate splice site recognition by recruiting the U1 snRNP.

scholarly journals Factors influencing alternative splice site utilization in vivo.

1987 ◽  
Vol 7 (2) ◽  
pp. 738-748 ◽  
Author(s):  
X Y Fu ◽  
J L Manley
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Culture Cells ◽  
293 Cells

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA.

scholarly journals Comparison of in vitro and in vivo splice site selection in kappa-immunoglobulin precursor mRNA.

1988 ◽  
Vol 8 (6) ◽  
pp. 2610-2619 ◽  
Author(s):  
D E Lowery ◽  
B G Van Ness
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Donor Site ◽  
Splice Donor Site ◽  
Splice Sites ◽  
Splice Donor ◽  
Splice Site Selection ◽  
Nuclear Extracts

The processing of a number of kappa-immunoglobulin primary mRNA (pre-mRNA) constructs has been examined both in vitro and in vivo. When a kappa-immunoglobulin pre-mRNA containing multiple J segment splice sites is processed in vitro, the splice sites are used with equal frequency. The presence of signal exon, S-V intron, or variable (V) region has no effect on splice site selection in vitro. Nuclear extracts prepared from a lymphoid cell line do not restore correct splice site selection. Splice site selection in vitro can be altered by changing the position or sequence of J splice donor sites. These results differ from the processing of similar pre-mRNAs expressed in vivo by transient transfection. The 5'-most J splice donor site was exclusively selected in vivo, even in nonlymphoid cells, and even in transcripts where in vitro splicing favored a 3' J splice site. The in vitro results are consistent with a model proposing that splice site selection is influenced by splice site strength and proximity; however, our in vivo results demonstrate a number of discrepancies with such a model and suggest that splice site selection may be coupled to transcription or a higher-order nuclear structure.

Factors influencing alternative splice site utilization in vivo

1987 ◽  
Vol 7 (2) ◽  
pp. 738-748
Author(s):  
X Y Fu ◽  
J L Manley
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Simian Virus 40 ◽  
Cell Types ◽  
Simian Virus ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Culture Cells ◽  
293 Cells

To study factors that influence the choice of alternative pre-mRNA splicing pathways, we introduced plasmids expressing either wild-type or mutated simian virus 40 (SV40) early regions into tissue culture cells and then measured the quantities of small-t and large-T RNAs produced. One important element controlling splice site selection was found to be the size of the intron removed in the production of small-t mRNA; expansion of this intron (from 66 to 77 or more nucleotides) resulted in a substantial increase in the amount of small-t mRNA produced relative to large-T mRNA. This suggests that in the normal course of SV40 early pre-mRNA processing, large-T splicing is at a competitive advantage relative to small-t splicing because of the small size of the latter intron. Several additional features of the pre-mRNA that can influence splice site selection were also identified by analyzing the effects of mutations containing splice site duplications. These include the strengths of competing 5' splice sites and the relative positions of splice sites in the pre-mRNA. Finally, we showed that the ratio of small-t to large-T mRNA was 10 to 15-fold greater in human 293 cells than in HeLa cells or other mammalian cell types. These results suggest the existence of cell-specific trans-acting factors that can dramatically alter the pattern of splice site selection in a pre-mRNA.

Virus deletion mutants that affect a 3' splice site in the E3 transcription unit of adenovirus 2

1985 ◽  
Vol 5 (9) ◽  
pp. 2405-2413
Author(s):  
B M Bhat ◽  
H A Brady ◽  
W S Wold
Keyword(s):  
Steady State ◽  
Splice Site ◽  
Transcription Unit ◽  
Mrna Splicing ◽  
Deletion Mutants ◽  
Splice Sites ◽  
Rich Region ◽  
Specific Sequence ◽  
Exon Deletion

Five viable virus mutants were constructed with deletions near a 3' splice site located at nucleotide 2157 in the E3 transcription unit of adenovirus 2. The mutants were examined for splicing activity at the 2157 3' splice site in vivo by nuclease-gel analysis of steady-state cytoplasmic mRNA. Splicing was not prevented by an exon deletion (dl719) that leaves 16 5'-proximal exon nucleotides intact or by intron deletions that leave 34 (dl717, dl712) or 18 (dl716) 3'-proximal intron nucleotides intact. The sequences deleted in one of these intron mutants (dl716) include the putative branchpoint site used in lariat formation during splicing. Thus, a surrogate branchpoint site apparently can be used for splicing. Another intron mutant (dl714) has a deletion that leaves 15 3'-proximal intron nucleotides intact; remarkably, this deletion virtually abolished splicing, even though the deletion is only 3 nucleotides closer to the splice site than is the deletion in dl716 which splices normally. The three nucleotides deleted in dl714 that are retained by dl716 are the sequence TGT. The TGT sequence is located on the 5' boundary of the pyrimidine-rich region upstream of the nucleotide 2157 3' splice site. Such pyrimidine-rich regions are ubiquitous at 3' splice sites. Most likely, the TGT is required for splicing at the nucleotide 2157 3' splice site. The TGT may be important because of its specific sequence or because it forms the 5' boundary of the pyrimidine-rich region.

scholarly journals Factors Affecting Splicing Strength of Yeast Genes

2011 ◽  
Vol 2011 ◽  
pp. 1-13 ◽  
Author(s):  
Pinchao Ma ◽  
Xuhua Xia
Keyword(s):  
Screening Tool ◽  
Consensus Sequence ◽  
Fungal Species ◽  
Splicing Factors ◽  
Splicing Regulation ◽  
Splice Sites ◽  
Crucial Importance ◽  
Factors Affecting ◽  
Unicellular Eukaryotes ◽  
Yeast Genes

Accurate and efficient splicing is of crucial importance for highly-transcribed intron-containing genes (ICGs) in rapidly replicating unicellular eukaryotes such as the budding yeastSaccharomyces cerevisiae. We characterize the 5′ and 3′ splice sites (ss) by position weight matrix scores (PWMSs), which is the highest for the consensus sequence and the lowest for splice sites differing most from the consensus sequence and used PWMS as a proxy for splicing strength.HAC1, which is known to be spliced by a nonspliceosomal mechanism, has the most negative PWMS for both its 5′ ss and 3′ ss. Several genes under strong splicing regulation and requiring additional splicing factors for their splicing also have small or negative PWMS values. Splicing strength is higher for highly transcribed ICGs than for lowly transcribed ICGs and higher for transcripts that bind strongly to spliceosomes than those that bind weakly. The 3′ splice site features a prominent poly-U tract before the 3′AG. Our results suggest the potential of using PWMS as a screening tool for ICGs that are either spliced by a nonspliceosome mechanism or under strong splicing regulation in yeast and other fungal species.

scholarly journals Role of the 3′ Splice Site in U12-Dependent Intron Splicing

2001 ◽  
Vol 21 (6) ◽  
pp. 1942-1952 ◽  
Author(s):  
Rosemary C. Dietrich ◽  
Marian J. Peris ◽  
Andrew S. Seyboldt ◽  
Richard A. Padgett
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Intron Splicing ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Branch Site ◽  
Site Function

ABSTRACT U12-dependent introns containing alterations of the 3′ splice site AC dinucleotide or alterations in the spacing between the branch site and the 3′ splice site were examined for their effects on splice site selection in vivo and in vitro. Using an intron with a 5′ splice site AU dinucleotide, any nucleotide could serve as the 3′-terminal nucleotide, although a C residue was most active, while a U residue was least active. The penultimate A residue, by contrast, was essential for 3′ splice site function. A branch site-to-3′ splice site spacing of less than 10 or more than 20 nucleotides strongly activated alternative 3′ splice sites. A strong preference for a spacing of about 12 nucleotides was observed. The combined in vivo and in vitro results suggest that the branch site is recognized in the absence of an active 3′ splice site but that formation of the prespliceosomal complex A requires an active 3′ splice site. Furthermore, the U12-type spliceosome appears to be unable to scan for a distal 3′ splice site.

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