scholarly journals G triplets located throughout a class of small vertebrate introns enforce intron borders and regulate splice site selection.

1997 ◽  
Vol 17 (8) ◽  
pp. 4562-4571 ◽  
Author(s):  
A J McCullough ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Globin Gene ◽  
Exon Skipping ◽  
Sequence Element ◽  
Exon Definition ◽  
Common Component ◽  
Alpha Globin ◽  
Splicing Efficiency ◽  
Small Vertebrate

Splicing of small introns in lower eucaryotes can be distinguished from vertebrate splicing by the inability of such introns to be expanded and by the inability of splice site mutations to cause exon skipping-properties suggesting that the intron rather than the exon is the unit of recognition. Vertebrates do contain small introns. To see if they possess properties similar to small introns in lower eucaryotes, we studied the small second intron from the human alpha-globin gene. Mutation of the 5' splice site of this intron resulted in in vivo intron inclusion, not exon skipping, suggesting the presence of intron bridging interactions. The intron had an unusual base composition reflective of a sequence bias present in a collection of small human introns in which multiple G triplets stud the interior of the introns. Each G triplet represented a minimal sequence element additively contributing to maximal splicing efficiency and spliceosome assembly. More importantly, G triplets proximal to a duplicated splice site caused preferential utilization of the 5' splice site upstream of the triplets or the 3' splice site downstream of the triplets; i.e., sequences containing G triplets were preferentially used as introns when a choice was possible. Thus, G triplets internal to a small intron have the ability to affect splice site decisions at both ends of the intron. Each G triplet additively contributed to splice site selectivity. We suggest that G triplets are a common component of human 5' splice sites and aid in the definition of exon-intron borders as well as overall splicing efficiency. In addition, our data suggest that such intronic elements may be characteristic of small introns and represent an intronic equivalent to the exon enhancers that facilitate recognition of both ends of an exon during exon definition.

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.

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 Base pairing at the 5' splice site with U1 small nuclear RNA promotes splicing of the upstream intron but may be dispensable for slicing of the downstream intron.

1996 ◽  
Vol 16 (6) ◽  
pp. 3012-3022 ◽  
Author(s):  
D Y Hwang ◽  
J B Cohen
Keyword(s):  
Splice Site ◽  
Point Mutations ◽  
Exon Skipping ◽  
Small Nuclear Rna ◽  
Base Pairing ◽  
Enhancer Activity ◽  
S1 Nuclease ◽  
Exon Definition ◽  
Small Nuclear Rnas

We previously reported that exon skipping in vivo due to point mutations in the 5' splice site (5'ss) signal of an internal mammalian exon can be prevented by coexpression of U1 small nuclear RNAs, termed shift-U1s, with complementarity to sequence upstream or downstream of the mutated site. We now show by S1 nuclease protection experiments that a typical shift-U1 restores splicing of the upstream intron, but not necessarily of the down stream intron. This indicates that the normal 5'ss sequence acts as an enhancer for splicing of the upstream intron, that it owes this activity to base pairing with U1, and that the enhancer activity is reproduced by base pairing of U1 with other sequences in the area. Shift-U1s are dispensable when the 3'ss sequence of the upstream intron is improved, which suggests that base pairing of U1 with sequences at or near the downstream end of the exon normally functions by compensating for a weakness in the upstream 3'ss. Accordingly, U1 appears to be involved in communication across the exon, but our data indicate at the same time that extensive base pairing between U1 and the 5'ss sequence is not necessary for accurate splicing of the downstream intron. These findings are discussed in relation to the coordinate selection exon termini proposed by the exon definition model.

scholarly journals Exonic Sequences in the 5′ Untranslated Region of α-Tubulin mRNA Modulate trans Splicing inTrypanosoma brucei

1998 ◽  
Vol 18 (8) ◽  
pp. 4620-4628 ◽  
Author(s):  
Carlos López-Estraño ◽  
Christian Tschudi ◽  
Elisabetta Ullu
Keyword(s):  
Splice Site ◽  
Competition Assay ◽  
Splice Site Selection ◽  
Tubulin Gene ◽  
Branch Site ◽  
Trans Splicing ◽  
Tubulin Mrna ◽  
Splicing Efficiency ◽  
Β Tubulin

ABSTRACT Previous studies have identified a conserved AG dinucleotide at the 3′ splice site (3′SS) and a polypyrimidine (pPy) tract that are required for trans splicing of polycistronic pre-mRNAs in trypanosomatids. Furthermore, the pPy tract of the Trypanosoma brucei α-tubulin 3′SS region is required to specify accurate 3′-end formation of the upstream β-tubulin gene and transsplicing of the downstream α-tubulin gene. Here, we employed an in vivo cis competition assay to determine whether sequences other than those of the AG dinucleotide and the pPy tract were required for 3′SS identification. Our results indicate that a minimal α-tubulin 3′SS, from the putative branch site region to the AG dinucleotide, is not sufficient for recognition by thetrans-splicing machinery and that polyadenylation is strictly dependent on downstream trans splicing. We show that efficient use of the α-tubulin 3′SS is dependent upon the presence of exon sequences. Furthermore, β-tubulin, but not actin exon sequences or unrelated plasmid sequences, can replace α-tubulin exon sequences for accurate trans-splice-site selection. Taken together, these results support a model in which the informational content required for efficient trans splicing of the α-tubulin pre-mRNA includes exon sequences which are involved in modulation of trans-splicing efficiency. Sequences that positively regulate trans splicing might be similar tocis-splicing enhancers described in other systems.

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.

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.

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.

scholarly journals HMGA1a Trapping of U1 snRNP at an Authentic 5′ Splice Site Induces Aberrant Exon Skipping in Sporadic Alzheimer's Disease

2010 ◽  
Vol 30 (9) ◽  
pp. 2220-2228 ◽  
Author(s):  
Kenji Ohe ◽  
Akila Mayeda
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Splice Site ◽  
High Mobility ◽  
Exon Skipping ◽  
Mobility Shift ◽  
Sporadic Alzheimer’S Disease ◽  
Exon Definition ◽  
Small Nuclear Ribonucleoprotein ◽  
U1 Snrnp

ABSTRACT Overexpression of high-mobility group A protein 1a (HMGA1a) causes aberrant exon 5 skipping of the Presenilin-2 (PS2) pre-mRNA, which is almost exclusively detected in patients with sporadic Alzheimer's disease. An electrophoretic mobility shift assay confirmed aberrant U1 small nuclear ribonucleoprotein particle (snRNP)-HMGA1a complex formation (via the U1-70K component), with RNA containing a specific HMGA1a-binding site and an adjacent 5′ splice site. Psoralen cross-linking analysis demonstrated that the binding of HMGA1a adjacent to the 5′ splice site induces unusually extended association of U1 snRNP to the 5′ splice site. As a result, spliceosome assembly across either the intron or the exon is arrested at an early ATP-independent stage. We conclude that the HMGA1a-induced aberrant exon skipping is caused by impaired dissociation of U1 snRNP from the 5′ splice site, leading to a defect in exon definition. The proposed molecular mechanism has profound implications for other known posttranscriptional modulation strategies in various organisms, all of which are triggered by aberrant U1 snRNP binding.

scholarly journals Cooperation of pre-mRNA sequence elements in splice site selection

1992 ◽  
Vol 12 (5) ◽  
pp. 2108-2114
Author(s):  
Z Dominski ◽  
R Kole
Keyword(s):  
Splice Site ◽  
Hela Cells ◽  
Site Selection ◽  
Exon Skipping ◽  
Polypyrimidine Tract ◽  
Splice Site Selection ◽  
Branch Point Sequence ◽  
Sequence Elements

We have recently demonstrated that short internal exons in pre-mRNA transcripts with three exons and two introns are ignored by splicing machinery in vitro and in vivo, resulting in exon skipping. Exon skipping is reversed when the pyrimidine content of the polypyrimidine tract in the upstream intron is increased (Z. Dominski and R. Kole, Mol. Cell. Biol. 11:6075-6083, 1991). Here we show that skipping of the short internal exon can be partially reversed by mutations which modify the upstream branch point sequence of the 5' splice site at the end of the exon to their respective consensus sequences. When the modified elements are combined with one another in the same pre-mRNA, exon skipping is fully reversed. Full reversion of exon skipping is also observed when these elements are combined individually with the upstream polypyrimidine tract strengthened by three purine-to-pyrimidine mutations. The observed patterns of splice site selection are similar in vitro (in nuclear extracts from HeLa cells) and in vivo (in transfected HeLa cells). We also show that the length of the downstream intron plays a role in splice site selection. Our data indicate that the interplay between the sequence elements in pre-mRNA controls the outcome of each splicing event, providing the means for very subtle regulation of alternative splicing.

scholarly journals The exon junction complex regulates the release and phosphorylation of paused RNA polymerase II

2018 ◽  
Author(s):  
Junaid Akhtar ◽  
Nastasja Kreim ◽  
Federico Marini ◽  
Giriram Kumar Mohana ◽  
Daniel Brune ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Exon Skipping ◽  
Exon Junction Complex ◽  
Elongation Complex ◽  
Exon Definition ◽  
Pol Ii ◽  
Exon Junction ◽  
Transcriptional Regulatory

SUMMARYPromoter proximal pausing of RNA polymerase II (Pol II) is a widespread transcriptional regulatory step across metazoans. Here we find that the nuclear exon junction complex (pre-EJC) plays a critical and conserved role in this process. Depletion of pre-EJC subunits leads to a global decrease in Pol II pausing and to premature entry into elongation. This effect occurs, at least in part, via non-canonical recruitment of pre-EJC components at promoters. Failure to recruit the pre-EJC at promoters results in increased binding of the positive transcription elongation complex (P-TEFb) and in enhanced Pol II release. Notably, restoring pausing is sufficient to rescue exon skipping and the photoreceptor differentiation defect associated with depletion of pre-EJC components in vivo. We propose that the pre-EJC serves as an early transcriptional checkpoint to prevent premature entry into elongation, ensuring proper recruitment of RNA processing components that are necessary for exon definition.

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