scholarly journals In vitro assembly of an early spliceosome defining both splice sites

2018 ◽  
Author(s):  
Kaushik Saha ◽  
Mike Minh Fernandez ◽  
Tapan Biswas ◽  
Charles Leonard Mallari Lumba ◽  
Gourisankar Ghosh
Keyword(s):  
Splice Site ◽  
Splicing Factors ◽  
Hnrnp A1 ◽  
Splice Sites ◽  
Complex Assembly ◽  
In Vitro Assembly ◽  
U1 Snrnp ◽  
Site Recognition ◽  
Intron Definition

ABSTRACTFor splicing of a metazoan pre-mRNA, the four major splice signals – 5′ and 3′ splice sites (SS), branch-point site (BS), and a poly-pyrimidine tract (PPT) – are initially bound by splicing factors U1 snRNP, U2AF35, SF1, and U2AF65, respectively, leading up to an early spliceosomal complex, the E-complex. The E-complex consists of additional components and the mechanism of its assembly is unclear. Hence, how splice signals are organized within E-complex defining the exon-intron boundaries remains elusive. Here we present in vitro stepwise reconstitution of an early spliceosome, assembled by cooperative actions of U1 snRNP, SRSF1, SF1, U2AF65, U2AF35, and hnRNP A1, termed here the recognition (R) complex, within which both splice sites are recognized. The R-complex assembly indicates that the SRSF1:pre-mRNA complex initially defines a substrate for U1 snRNP, engaging exons at both ends of an intron. Subsequent 5′SS-dependent U1 snRNP binding enables recognition of the remaining splice signals, defining the intron. This R-complex assembly indicates the minimal constituents for intron definition revealing mechanistic principles behind the splice site recognition.

scholarly journals An intron element modulating 5' splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1.

1997 ◽  
Vol 17 (4) ◽  
pp. 1776-1786 ◽  
Author(s):  
B Chabot ◽  
M Blanchette ◽  
I Lapierre ◽  
H La Branche
Keyword(s):  
Splice Site ◽  
Site Selection ◽  
Donor Site ◽  
Rnase H ◽  
Hnrnp A1 ◽  
Splice Sites ◽  
Splice Site Selection ◽  
Direct Role ◽  
U1 Snrnp

The hnRNP A1 pre-mRNA is alternatively spliced to yield the A1 and A1b mRNAs, which encode proteins differing in their ability to modulate 5' splice site selection. Sequencing a genomic portion of the murine A1 gene revealed that the intron separating exon 7 and the alternative exon 7B is highly conserved between mouse and human. In vitro splicing assays indicate that a conserved element (CE1) from the central portion of the intron shifts selection toward the distal donor site when positioned in between the 5' splice sites of exon 7 and 7B. In vivo, the CE1 element promotes exon 7B skipping. A 17-nucleotide sequence within CE1 (CE1a) is sufficient to activate the distal 5' splice site. RNase T1 protection/immunoprecipitation assays indicate that hnRNP A1 binds to CE1a, which contains the sequence UAGAGU, a close match to the reported optimal A1 binding site, UAGGGU. Replacing CE1a by different oligonucleotides carrying the sequence UAGAGU or UAGGGU maintains the preference for the distal 5' splice site. In contrast, mutations in the AUGAGU sequence activate the proximal 5' splice site. In support of a direct role of the A1-CE1 interaction in 5'-splice-site selection, we observed that the amplitude of the shift correlates with the efficiency of A1 binding. Whereas addition of SR proteins abrogates the effect of CE1, the presence of CE1 does not modify U1 snRNP binding to competing 5' splice sites, as judged by oligonucleotide-targeted RNase H protection assays. Our results suggest that hnRNP A1 modulates splice site selection on its own pre-mRNA without changing the binding of U1 snRNP to competing 5' splice sites.

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 Intron definition in splicing of small Drosophila introns.

1994 ◽  
Vol 14 (5) ◽  
pp. 3434-3445 ◽  
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Directed Assembly ◽  
Splice Sites ◽  
Rich Region ◽  
In Vitro Splicing ◽  
Low Levels ◽  
Intron Definition ◽  
Moderate Length

Approximately half of the introns in Drosophila melanogaster are too small to function in a vertebrate and often lack the pyrimidine tract associated with vertebrate 3' splice sites. Here, we report the splicing and spliceosome assembly properties of two such introns: one with a pyrimidine-poor 3' splice site and one with a pyrimidine-rich 3' splice site. The pyrimidine-poor intron was absolutely dependent on its small size for in vivo and in vitro splicing and assembly. As such, it had properties reminiscent of those of yeast introns. The pyrimidine-rich intron had properties intermediate between those of yeasts and vertebrates. This 3' splice site directed assembly of ATP-dependent complexes when present as either an intron or exon and supported low levels of in vivo splicing of a moderate-length intron. We propose that splice sites can be recognized as pairs across either exons or introns, depending on which distance is shorter, and that a pyrimidine-rich region upstream of the 3' splice site facilitates the exon mode.

scholarly journals Intron definition in splicing of small Drosophila introns

1994 ◽  
Vol 14 (5) ◽  
pp. 3434-3445
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Directed Assembly ◽  
Splice Sites ◽  
Rich Region ◽  
In Vitro Splicing ◽  
Low Levels ◽  
Intron Definition ◽  
Moderate Length

Approximately half of the introns in Drosophila melanogaster are too small to function in a vertebrate and often lack the pyrimidine tract associated with vertebrate 3' splice sites. Here, we report the splicing and spliceosome assembly properties of two such introns: one with a pyrimidine-poor 3' splice site and one with a pyrimidine-rich 3' splice site. The pyrimidine-poor intron was absolutely dependent on its small size for in vivo and in vitro splicing and assembly. As such, it had properties reminiscent of those of yeast introns. The pyrimidine-rich intron had properties intermediate between those of yeasts and vertebrates. This 3' splice site directed assembly of ATP-dependent complexes when present as either an intron or exon and supported low levels of in vivo splicing of a moderate-length intron. We propose that splice sites can be recognized as pairs across either exons or introns, depending on which distance is shorter, and that a pyrimidine-rich region upstream of the 3' splice site facilitates the exon mode.

scholarly journals Selection of Alternative 5′ Splice Sites: Role of U1 snRNP and Models for the Antagonistic Effects of SF2/ASF and hnRNP A1

2000 ◽  
Vol 20 (22) ◽  
pp. 8303-8318 ◽  
Author(s):  
Ian C. Eperon ◽  
Olga V. Makarova ◽  
Akila Mayeda ◽  
Stephen H. Munroe ◽  
Javier F. Cáceres ◽  
...  
Keyword(s):  
Splicing Factors ◽  
Hnrnp A1 ◽  
Splice Sites ◽  
Downstream Site ◽  
Sr Protein ◽  
Proximal Site ◽  
U1 Snrnp ◽  
The Individual ◽  
Dose Dependent

ABSTRACT The first component known to recognize and discriminate among potential 5′ splice sites (5′SSs) in pre-mRNA is the U1 snRNP. However, the relative levels of U1 snRNP binding to alternative 5′SSs do not necessarily determine the splicing outcome. Strikingly, SF2/ASF, one of the essential SR protein-splicing factors, causes a dose-dependent shift in splicing to a downstream (intron-proximal) site, and yet it increases U1 snRNP binding at upstream and downstream sites simultaneously. We show here that hnRNP A1, which shifts splicing towards an upstream 5′SS, causes reduced U1 snRNP binding at both sites. Nonetheless, the importance of U1 snRNP binding is shown by proportionality between the level of U1 snRNP binding to the downstream site and its use in splicing. With purified components, hnRNP A1 reduces U1 snRNP binding to 5′SSs by binding cooperatively and indiscriminately to the pre-mRNA. Mutations in hnRNP A1 and SF2/ASF show that the opposite effects of the proteins on 5′SS choice are correlated with their effects on U1 snRNP binding. Cross-linking experiments show that SF2/ASF and hnRNP A1 compete to bind pre-mRNA, and we conclude that this competition is the basis of their functional antagonism; SF2/ASF enhances U1 snRNP binding at all 5′SSs, the rise in simultaneous occupancy causing a shift in splicing towards the downstream site, whereas hnRNP A1 interferes with U1 snRNP binding such that 5′SS occupancy is lower and the affinities of U1 snRNP for the individual sites determine the site of splicing.

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.

Effect of 5' splice site mutations on splicing of the preceding intron

1990 ◽  
Vol 10 (12) ◽  
pp. 6299-6305
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factors ◽  
Exon 2 ◽  
Intron 1 ◽  
Direct Assembly ◽  
Internal Exon ◽  
Exon 1 ◽  
Lariat Rna

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

scholarly journals hnRNP A1 Regulates Alternative Splicing of Tau Exon 10 by Targeting 3′ Splice Sites

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 936 ◽  
Author(s):  
Yongchao Liu ◽  
Donggun Kim ◽  
Namjeong Choi ◽  
Jagyeong Oh ◽  
Jiyeon Ha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Ontology ◽  
Alternative Splicing ◽  
Splice Site ◽  
Hnrnp A1 ◽  
Gene Ontology Analysis ◽  
Splice Sites ◽  
Neuronal Function ◽  
Brain Functions ◽  
Exon 10

The ratio control of 4R-Tau/3R-Tau by alternative splicing of Tau exon 10 is important for maintaining brain functions. In this study, we show that hnRNP A1 knockdown induces inclusion of endogenous Tau exon 10, conversely, overexpression of hnRNP A1 promotes exon 10 skipping of Tau. In addition, hnRNP A1 inhibits splicing of intron 9, but not intron 10. Furthermore, hnRNP A1 directly interacts with the 3′ splice site of exon 10 to regulate its functions in alternative splicing. Finally, gene ontology analysis demonstrates that hnRNP A1-induced splicing and gene expression targets a subset of genes with neuronal function.

scholarly journals Regulation of Sex-Specific Selection offruitless 5′ Splice Sites by transformerand transformer-2

1998 ◽  
Vol 18 (1) ◽  
pp. 450-458 ◽  
Author(s):  
Volker Heinrichs ◽  
Lisa C. Ryner ◽  
Bruce S. Baker
Keyword(s):  
Splice Site ◽  
Courtship Behavior ◽  
Specific Pattern ◽  
Splice Sites ◽  
Repeat Elements ◽  
Specific Alternative ◽  
Female Specific ◽  
Male Specific ◽  
Splicing Enhancer

ABSTRACT In Drosophila melanogaster, the fruitless(fru) gene controls essentially all aspects of male courtship behavior. It does this through sex-specific alternative splicing of the fru pre-mRNA, leading to the production of male-specific fru mRNAs capable of expressing male-specificfru proteins. Sex-specific fru splicing involves the choice between alternative 5′ splice sites, one used exclusively in males and the other used only in females. Here we report that the Drosophila sex determination genestransformer (tra) and transformer-2(tra-2) switch fru splicing from the male-specific pattern to the female-specific pattern through activation of the female-specific fru 5′ splice site. Activation of female-specific fru splicing requirescis-acting tra and tra-2 repeat elements that are part of an exonic splicing enhancer located immediately upstream of the female-specific fru 5′ splice site and are recognized by the TRA and TRA-2 proteins in vitro. Thisfru splicing enhancer is sufficient to promote the activation by tra and tra-2 of both a 5′ splice site and the female-specific doublesex (dsx) 3′ splice site, suggesting that the mechanisms of 5′ splice site activation and 3′ splice site activation may be similar.

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.

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