scholarly journals Alternate trans splicing in Trypanosoma equiperdum: implications for splice site selection.

1988 ◽  
Vol 8 (3) ◽  
pp. 1352-1360 ◽  
Author(s):  
R E Layden ◽  
H Eisen
Keyword(s):  
Site Selection ◽  
Surface Glycoprotein ◽  
Splice Sites ◽  
Splice Acceptor ◽  
Splice Site Selection ◽  
Primary Transcript ◽  
Spliced Leader ◽  
Trans Splicing ◽  
Multiple Messages ◽  
Trypanosoma Equiperdum

We examined the structures of the 5' ends of mRNAs encoding variant surface glycoprotein 78 (VSG-78) and VSG-1(78) in Trypanosoma equiperdum. Several mRNA species were found for each gene, and all contained the 35-base miniexon (or spliced leader) sequence attached at different positions on their 5' ends. Thus, the generation of multiple messages for each VSG occurred by attachment of the miniexon at one of several 3' splice acceptor sites. The frequency with which individual splice sites were used varied from less than 1 to 95% of the RNA produced from a particular gene. We propose that the miniexon RNA and RNA from the VSG genes may interact via base pairing and that this in part specifies the use of particular acceptor sites. Sequences complementary to the miniexon primary transcript, termed the "med-comp site," were found in both genes and in several published sequences. Splice sites were most often used if they were the first site 3' of the med-comp site and contained a high pyrimidine content in the bases preceding the AG acceptor signal.

Alternate trans splicing in Trypanosoma equiperdum: implications for splice site selection

1988 ◽  
Vol 8 (3) ◽  
pp. 1352-1360
Author(s):  
R E Layden ◽  
H Eisen
Keyword(s):  
Site Selection ◽  
Surface Glycoprotein ◽  
Splice Sites ◽  
Splice Acceptor ◽  
Splice Site Selection ◽  
Primary Transcript ◽  
Spliced Leader ◽  
Trans Splicing ◽  
Multiple Messages ◽  
Trypanosoma Equiperdum

We examined the structures of the 5' ends of mRNAs encoding variant surface glycoprotein 78 (VSG-78) and VSG-1(78) in Trypanosoma equiperdum. Several mRNA species were found for each gene, and all contained the 35-base miniexon (or spliced leader) sequence attached at different positions on their 5' ends. Thus, the generation of multiple messages for each VSG occurred by attachment of the miniexon at one of several 3' splice acceptor sites. The frequency with which individual splice sites were used varied from less than 1 to 95% of the RNA produced from a particular gene. We propose that the miniexon RNA and RNA from the VSG genes may interact via base pairing and that this in part specifies the use of particular acceptor sites. Sequences complementary to the miniexon primary transcript, termed the "med-comp site," were found in both genes and in several published sequences. Splice sites were most often used if they were the first site 3' of the med-comp site and contained a high pyrimidine content in the bases preceding the AG acceptor signal.

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.

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.

scholarly journals Temporal order of RNA-processing reactions in trypanosomes: rapid trans splicing precedes polyadenylation of newly synthesized tubulin transcripts.

1993 ◽  
Vol 13 (1) ◽  
pp. 720-725 ◽  
Author(s):  
E Ullu ◽  
K R Matthews ◽  
C Tschudi
Keyword(s):  
Rna Processing ◽  
Temporal Order ◽  
Kinetic Studies ◽  
Splice Acceptor ◽  
Mrna Synthesis ◽  
Alpha Tubulin ◽  
Spliced Leader ◽  
Polycistronic Transcription ◽  
Trans Splicing

Many trypanosome genes are expressed as part of large polycistronic transcription units. This finding suggests that regulation of mRNA biogenesis may emphasize RNA-processing reactions more so than in other organisms. This study was undertaken to understand the temporal order of two RNA-processing reactions, trans splicing and polyadenylation, in the maturation of trypanosome mRNAs in vivo. Kinetic studies revealed rapid trans splicing of alpha-tubulin, beta-tubulin, and actin pre-mRNAs within 1 to 2 min after synthesis of the 3' splice site. Furthermore, following blockage of pre-mRNA synthesis, newly synthesized spliced leader RNA cannot be used for trans splicing, suggesting that trypanosomes do not accumulate substantial amounts of pre-mRNA which can provide splice acceptor sites. Thus, trans splicing is cotranscriptional. In addition, we show that trans splicing precedes polyadenylation in the processing of trypanosome tubulin pre-mRNAs.

scholarly journals Competing Upstream 5′ Splice Sites Enhance the Rate of Proximal Splicing

2010 ◽  
Vol 30 (8) ◽  
pp. 1878-1886 ◽  
Author(s):  
Martin J. Hicks ◽  
William F. Mueller ◽  
Peter J. Shepard ◽  
Klemens J. Hertel
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Splice Sites ◽  
Base Pairing ◽  
Splice Site Selection ◽  
U1 Snrna ◽  
Pairing Potential

ABSTRACT Alternative 5′ splice site selection is one of the major pathways resulting in mRNA diversification. Regulation of this type of alternative splicing depends on the presence of regulatory elements that activate or repress the use of competing splice sites, usually leading to the preferential use of the proximal splice site. However, the mechanisms involved in proximal splice site selection and the thermodynamic advantage realized by proximal splice sites are not well understood. Here, we have carried out a systematic analysis of alternative 5′ splice site usage using in vitro splicing assays. We show that observed rates of splicing correlate well with their U1 snRNA base pairing potential. Weak U1 snRNA interactions with the 5′ splice site were significantly rescued by the proximity of the downstream exon, demonstrating that the intron definition mode of splice site recognition is highly efficient. In the context of competing splice sites, the proximity to the downstream 3′ splice site was more influential in dictating splice site selection than the actual 5′ splice site/U1 snRNA base pairing potential. Surprisingly, the kinetic analysis also demonstrated that an upstream competing 5′ splice site enhances the rate of proximal splicing. These results reveal the discovery of a new splicing regulatory element, an upstream 5′ splice site functioning as a splicing enhancer.

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.

scholarly journals Interplay of primary sequence and RNA secondary structure in determining 5′ splice site choice

2018 ◽  
Author(s):  
Frances Anne Tosto ◽  
Asaf Shilo ◽  
Jason W. Rausch ◽  
Stuart F. J. Le Grice ◽  
Tom Misteli
Keyword(s):  
Secondary Structure ◽  
Splice Site ◽  
Site Selection ◽  
Mutational Analysis ◽  
Regulatory Elements ◽  
Premature Aging ◽  
Common Mechanism ◽  
Splice Sites ◽  
Primary Sequence ◽  
Splice Site Selection

AbstractSelective use of 5′ splice sites is a common mechanism by which pre-mRNAs are alternatively spliced. Whereas the sequence requirements of 5′ splice site choice have been well characterized, other important determinants remain poorly defined. Here we apply a combination of structural mapping by SHAPE-MaP and targeted mutational analysis in a cell-based system to comprehensively probe the interplay of primary sequence, secondary RNA structure, regulatory elements and linear splice site position to determine mechanisms of splice site choice in vivo. Using the disease-causing alternative 5′ splice site selection in LMNA in the premature aging disorder Hutchinson-Gilford Progeria Syndrome as a model system, we identify RNA secondary structural elements near the alternative 5′ splice sites. We show that splice site choice is significantly influenced by the structural context of the available splice sites. While local structure alone is not sufficient to account for splice site selection, the choice of 5′ splice sites depends on the structural stability of the 5′ splice site region which is conferred by downstream elements. In addition, relative positioning of the competing sites within the primary sequence of the pre-mRNA is a predictor of 5′ splice site usage, with the distal position favored over the proximal, regardless of sequence composition. Together, these results reveal an intricate interplay amongst RNA sequence, secondary structure and splice site position in determining 5′ splice site choice.

Exon definition may facilitate splice site selection in RNAs with multiple exons

1990 ◽  
Vol 10 (1) ◽  
pp. 84-94 ◽  
Author(s):  
B L Robberson ◽  
G J Cote ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Exon Skipping ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Spliceosome Assembly ◽  
Exon Definition ◽  
Exon 2 ◽  
Correct Orientation

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.

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.

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