A minimal intron length but no specific internal sequence is required for splicing the large rabbit β-globin intron

Wieringa, B.; Hofer, Erhard; Weissmann, Charles

doi:10.1016/0092-8674(84)90426-4

Cited by 327 publications

(192 citation statements)

References 35 publications

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“…ALM is nonviable and is at least 200-fold deficient in late cytoplasmic mRNA accumulation but is unaffected in early gene expression (21). Restoration of leader length with unrelated sequences can restore viability (21 binding sites characterized as necessary for splicing can inhibit each others' usage, and support earlier speculation that juxtaposition of splice sites leads to RNA instability (7). Together these results support the notion that splicing complex formation is involved in nuclear pre-mRNA preservation, at least in the polyoma system.…”

Section: Introductionsupporting

confidence: 66%

“…More recently, a third site which shows some sequence requirements for splicing in vivo has been identified -the branch point, which lies about 30 nucleotides upstream of the 3' splice site (8). Results with deletion mutants and chimeric RNA's have indicated little or no sequence requirements for exon sequences and intron regions not already mentioned (7,9,10) -these other regions may only have a role in determining three-dimensional structures which facilitate the bringing together of companion splice sites as originally proposed (11)(12)(13).…”

Section: Introductionmentioning

confidence: 93%

“…Analyses of intron sequence requirements for in vivo mRNA splicing in higher eukaryotes originally indicated the importance of the 5' splice site, including the highly conserved GU dinucleotide at the 5' exon-intron boundary, and the 3' splice site, which consists of a similarly highly conserved dinucleotide, AG, at the 3' boundary, but which additionally includes an upstream stretch of pyrimidines (1)(2)(3)(4)(5)(6)(7). More recently, a third site which shows some sequence requirements for splicing in vivo has been identified -the branch point, which lies about 30 nucleotides upstream of the 3' splice site (8).…”

Section: Introductionmentioning

confidence: 99%

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The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability

Adami¹,

Carmichael

1987

Nucl Acids Res

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ABSTRACT'Polyoma virus late RNA processing provides a convenient model system in which to study the mechanics of splicing in vivo. In order to understand further the role of the untranslated "late leader" unit in late RNA processing we have constructed a group of polyoma viruses with deletions and substitutions in the leader exon. This has allowed us to determine that there is a minimum exon size required for both pre-mRNA splicing and stability in this system. We show here that the non-viability of a mutant (ALM) with a 9 base late leader unit is due to a general defect in late RNA splicing. In addition, ALM-infected cells show at least 40-fold depression in the accumulation of late nuclear RNA (spliced or unspliced). The ALM late promoter, however, functions nearly normally. Substituted leader variants with 51-to 96-base long exons of unrelated sequence are viable (G. Adami and G. Carmichael, J. Virol. 58, 417-425, 1986). We show here that late RNA from one of these substituted leader mutants (containing a 51-base leader exon) is spliced at wild type levels, with virtually no defect in accumulation. Thus, in the polyoma system, splice sites separated by only 9 bases can inhibit each others usage, presumably by steric interference. We suggest that this type of inhibition leads to extreme RNA instability. INTRODUCTION Much work in recent years has been directed towards further understanding the mechanism of splicing of mRNA precursors. Analyses of intron sequence requirements for in vivo mRNA splicing in higher eukaryotes originally indicated the importance of the 5' splice site, including the highly conserved GU dinucleotide at the 5' exon-intron boundary, and the 3' splice site, which consists of a similarly highly conserved dinucleotide, AG, at the 3' boundary, but which additionally includes an upstream stretch of pyrimidines (1-7). More recently, a third site which shows some sequence requirements for splicing in vivo has been identified -the branch point,

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Section: Introductionsupporting

confidence: 66%

Section: Introductionmentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

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The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability

Adami¹,

Carmichael

1987

Nucl Acids Res

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“…Con sistent with this, there is little evidence of an enriched polypyrimidine tract in the 3' region of most yeast in trons. One example of the importance of the polypyri midine tract in mammalian introns is that the efficiency of splicing in vivo of an intron of rabbit p-globin de creased > 13-fold when a deletion shortened the tract from 16 to 8 nucleotides (Wieringa et al 1984). Consis tent results have been reported with other mutations in the polypyrimidine tract (Ruskin and Green 1985).…”

Section: Discussionmentioning

confidence: 95%

Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns.

García-Blanco

Jamison

Sharp

1989

Genes & Development

345

296

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A protein of molecular size 62,000 daltons (p62) was detected in HeLa cell nuclear extracts by UV cross-linking to mRNA precursors. p62 binds specifically to the polypyrimidine tract of the 3' splice site region of introns. p62 purified to homogeneity binds the polypyrimidine tract of pre-mRNAs. This binding does not require the AG dinucleotide at the 3' splice site. Alterations in the polypyrimidine tract that reduce the binding of p62 yield a corresponding reduction in the efficiency of formation of a U2 snRNP/pre-mRNA complex and splicing. The p62 protein is retained in the spliceosome, where it remains bound to the pre-mRNA. This polypyrimidine tract binding protein (pPTB) is proposed to be a critical component in recognition of the 3' splice site during splicing.

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“…SA). This 3'-proximal region represents the socalled polypyrimidine tract (31); it appears that this sequence, together with the invariant terminal AG, constitutes the recognition signal which defines the 3' end of an intron and is important for initiation of the splicing event, including cleavage at the 5' splice site (12,39,40,52). A protein has recently been identified that has binding specificity for sequences in the polypyrimidine tract (13,48).…”

mentioning

confidence: 99%

Nuclear pre-mRNA processing in plants: distinct modes of 3'-splice-site selection in plants and animals.

Wiebauer¹,

Herrero²,

Filipowicz³

1988

Mol. Cell. Biol.

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, 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 I-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 ,-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.The splicing of nuclear pre-mRNAs in eucaryotes is a complex multistep process (for reviews, see references 16 and 33). The pre-mRNA is first assembled into a large ribonucleoprotein complex called the spliceosome. In mammalian systems its assembly requires, in addition to the appropriate signals in the RNA substrate, the heterogeneous nuclear ribonucleoprotein polypeptides, small nuclear ribonucleoprotein particles Ul, U2, U4 to U6, and US, and several other less-defined soluble proteins. Excision of an intron (IVS) then occurs in two stages. First, the pre-mRNA is cleaved at the intron 5' border to release exon 1 (Exl). An intron-Ex2 branched intermediate (a lariat) is formed simultaneously. In the second step, cleavage at the 3' splice site and ligation of the two exons occur, accompanied by the release of the lariat intron.Although much information is now available about spliceosome assembly and the importance of the splice junction and branch point consensus sequences in splicing (for reviews, see references 16 and 33), little is known about the structural features which distinguish introns from exons and which establish introns as the sequences to be removed from pre-mRNA. It is evident that the consensus nucleotides are in most instances not sufficient for this purpose; similar sequences are frequently present elsewhere in RNA but are not recognized. It has been proposed that the spatial structure of pre-mRNA may be an important factor in determining the choice of splice sites for processing (...

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A minimal intron length but no specific internal sequence is required for splicing the large rabbit β-globin intron

Cited by 327 publications

References 35 publications

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability

Identification and purification of a 62,000-dalton protein that binds specifically to the polypyrimidine tract of introns.

Nuclear pre-mRNA processing in plants: distinct modes of 3'-splice-site selection in plants and animals.

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