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 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 U1 small nuclear RNAs with altered specificity can be stably expressed in mammalian cells and promote permanent changes in pre-mRNA splicing.

1993 ◽  
Vol 13 (5) ◽  
pp. 2666-2676 ◽  
Author(s):  
J B Cohen ◽  
S D Broz ◽  
A D Levinson
Keyword(s):  
Splice Site ◽  
Mammalian Cells ◽  
Mrna Processing ◽  
Mrna Splicing ◽  
Small Nuclear Rna ◽  
Splice Sites ◽  
Gene Complementation ◽  
Small Nuclear Rnas ◽  
Nuclear Rna ◽  
Mutant Genes

Pre-mRNA 5' splice site activity depends, at least in part, on base complementarity to U1 small nuclear RNA. In transient coexpression assays, defective 5' splice sites can regain activity in the presence of U1 carrying compensatory changes, but it is unclear whether such mutant U1 RNAs can be permanently expressed in mammalian cells. We have explored this issue to determine whether U1 small nuclear RNAs with altered specificity may be of value to rescue targeted mutant genes or alter pre-mRNA processing profiles. This effort was initiated following our observation that U1 with specificity for a splice site associated with an alternative H-ras exon substantially reduced the synthesis of the potentially oncogenic p21ras protein in transient assays. We describe the development of a mammalian complementation system that selects for removal of a splicing-defective intron placed within a drug resistance gene. Complementation was observed in proportion to the degree of complementarity between transfected mutant U1 genes and different defective splice sites, and all cells selected in this manner were found to express mutant U1 RNA. In addition, these cells showed specific activation of defective splice sites presented by an unlinked reporter gene. We discuss the prospects of this approach to permanently alter the expression of targeted genes in mammalian cells.

scholarly journals U1 small nuclear RNA-promoted exon selection requires a minimal distance between the position of U1 binding and the 3' splice site across the exon.

1997 ◽  
Vol 17 (12) ◽  
pp. 7099-7107 ◽  
Author(s):  
D Y Hwang ◽  
J B Cohen
Keyword(s):  
Splice Site ◽  
Minimum Size ◽  
Minimal Distance ◽  
Small Nuclear Rna ◽  
Small Nuclear Ribonucleoprotein ◽  
Optimal Distance ◽  
Small Nuclear Rnas ◽  
Constitutive Splicing ◽  
Nuclear Ribonucleoprotein ◽  
Lower Size

Both experimental work and surveys of the lengths of internal exons in nature have suggested that vertebrate internal exons require a minimum size of approximately 50 nucleotides for efficient inclusion in mature mRNA. This phenomenon has been ascribed to steric interference between complexes involved in recognition of the splicing signals at the two ends of short internal exons. To determine whether U1 small nuclear ribonucleoprotein, a multicomponent splicing factor that is involved in the first recognition of splice sites, contributes to the lower size limit of vertebrate internal exons, we have taken advantage of our previous observation that U1 small nuclear RNAs (snRNAs) which bind upstream or downstream of the 5' splice site (5'SS) stimulate splicing of the upstream intron. By varying the position of U1 binding relative to the 3'SS, we show that U1-dependent splicing of the upstream intron becomes inefficient when U1 is positioned 48 nucleotides or less downstream of the 3'SS, suggesting a minimal distance between U1 and the 3'SS of approximately 50 nucleotides. This distance corresponds well to the suggested minimum size of internal exons. The results of experiments in which the 3'SS region of the reporter was duplicated suggest an optimal distance of greater than 72 nucleotides. We have also found that inclusion of a 24-nucleotide miniexon is promoted by the binding of U1 to the downstream intron but not by binding to the 5'SS. Our results are discussed in the context of models to explain constitutive splicing of small exons in nature.

scholarly journals Structural analyses of the 7SK ribonucleoprotein (RNP), the most abundant human small RNP of unknown function.

1991 ◽  
Vol 11 (7) ◽  
pp. 3432-3445 ◽  
Author(s):  
D A Wassarman ◽  
J A Steitz
Keyword(s):  
Secondary Structure ◽  
Rnase H ◽  
Small Nuclear Rna ◽  
Base Pairing ◽  
Structural Determinants ◽  
Nuclear Rna ◽  
Sequence Complementarity ◽  
Associated Proteins ◽  
Stem Structure

The human 7SK ribonucleoprotein (RNP) has been analyzed to determine its RNA secondary structure and protein constituents. HeLa cell 7SK RNA alone and within its RNP have been probed by chemical modification and enzymatic cleavage, and sites of modification or cleavage have been mapped by primer extension. The resulting secondary structure suggests that structural determinants necessary for capping (a 5' stem followed by the sequence AUPuUPuC) and nuclear migration (the sequence AUPuUPuC) of 7SK RNA may be similar to those for U6 small nuclear RNA (snRNA). It also supports existence of a 3' stem structure which could serve to self-prime cDNA synthesis during pseudogene formation. Oligonucleotide-directed RNase H digestion indicated regions of 7SK RNA capable of base pairing with other nucleic acids. Antisense 2'-O-methyl RNA oligonucleotides were used to affinity select the 7SK RNP from an in vivo 35S-labeled cell sonic extract and identify eight associated proteins of 83, 48, 45, 43, 42, 21, 18, and 13 kDa. 7SK RNA has extensive sequence complementarity to U4 snRNA, within the U4/U6 base pairing domain, and also to U11 snRNA. The possibility that the 7SK RNP is an unrecognized component of the pre-mRNA processing machinery is discussed.

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 Developmental and tissue-specific expression of U4 small nuclear RNA genes.

1988 ◽  
Vol 8 (12) ◽  
pp. 5566-5569 ◽  
Author(s):  
G M Korf ◽  
I W Botros ◽  
W E Stumph
Keyword(s):  
Domestic Chicken ◽  
Sequence Variant ◽  
Small Nuclear Rna ◽  
Specific Expression ◽  
Small Nuclear Rnas ◽  
Specific Manner ◽  
Nuclear Rna ◽  
U4 Rna ◽  
Rna Genes

U4 RNA is one of several small nuclear RNAs involved in the splicing of mRNA precursors. The domestic chicken has two genes per haploid genome that are capable of encoding U4 RNA. The U4X RNA gene (which encodes a sequence variant of U4 RNA that was unknown prior to the cloning of the gene) and the U4B RNA gene were both expressed in vivo in each of seven adult and three embryonic chicken tissues examined. However, the ratio of U4B RNA to U4X RNA can vary more than sevenfold in both a tissue- and stage-specific manner.

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.

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