U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing

Black, Douglas L.; Chabot, Benoît; Steitz, Joan A.

doi:10.1016/0092-8674(85)90270-3

Cited by 575 publications

(445 citation statements)

References 56 publications

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“…It has long been recognized that the formation of spe cific complexes containing Ul and U2 snRNPs, respec tively, bound to the 5' splice site, and the branch site is an early step in the splicing of pre-mRNAs (Mount et al 1983;Black et al 1985;Konarska and Sharp 1986;Ruby and Abelson 1988). Depletion of nuclear extracts of Ul snRNP abolishes the activity of the 5' splice site in pro moting the U2 snRNP/pre-mRNA association (Ruby and Abelson 1988;Seraphin et al 1988).…”

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confidence: 99%

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

344

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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mentioning

confidence: 99%

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

344

296

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“…In the yeast Saccharomyces cerevisiae, the branch point sequence UACUAAC is an indispensable element (25,37) which is recognized by a U2-like RNA (34). Although animal introns do not contain such strongly conserved sequences, a similar interaction between the branch point and the U2 RNA may also occur (2,20).It has recently been found that several higher-plant species introns can be faithfully processed in a HeLa cell extract (6,17). On the other hand, Barta et al (1) have reported that human growth hormone pre-mRNA, which contains four intervening sequences, does not undergo processing in transgenic plant tissues.…”

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confidence: 99%

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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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“…The Ul snRNP binds to the 5' splice site, almost certainly via base pairing (3,22,30,32). The U2 snRNP binds to branch point sequences (3,22), and this interaction apparently requires a factor designated U2AF that binds to the 3' splice site (28 (25).…”

Section: Methodsmentioning

confidence: 99%

A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins.

Agris¹,

Nemeroff²,

Krug³

1989

Mol. Cell. Biol.

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The assembly of mammalian pre-mRNAs into large 50S to 60S complexes, or spliceosomes, containing small nuclear ribonucleoproteins (snRNPs) leads to the production of splicing intermediates, 5' exon and lariat-3' exon, and the subsequent production of spliced products. Influenza virus NS1 mRNA, which encodes a virus-specific protein, is spliced in infected cells to form another viral mRNA (the NS2 mRNA), such that the ratio of unspliced to spliced mRNA is 10 to 1. NS1 mRNA was not detectably spliced in vitro with nuclear extracts from uninfected HeLa cells. Surprisingly, despite the almost total absence of splicing intermediates in the in vitro reaction, NS1 mRNA very efficiently formed ATP-dependent 55S complexes. The formation of 55S complexes with NS1 mRNA was compared with that obtained with an adenovirus pre-mRNA (pKT1 transcript) by using partially purified splicing fractions that restricted the splicing of the pKT1 transcript to the production of splicing intermediates. At RNA precursor levels that were considerably below saturation, approximately 10-fold more of the input NS1 mRNA than of the input pKT1 transcript formed 55S complexes at all time points examined. The pKT1 55S complexes contained splicing intermediates, whereas the NS1 55S complexes contained only precursor NS1 mRNA. Biotin-avidin affinity chromatography showed that the 55S complexes formed with either NS1 mRNA or the pKT1 transcript contained the Ul, U2, U4, U5, and U6 snRNPs. Consequently, the formation of 55S complexes containing these five snRNPs was not sufficient for the catalysis of the first step of splicing, indicating that some additional step(s) needs to occur subsequent to this binding. These results indicate that the 5' splice site, 3' splice site, and branchpoint of NS1 mRNA were capable of interacting with the five snRNPs to form 55S complexes, but apparently some other sequence element(s) in NS1 mRNA blocked the resolution of the 55S complexes that leads to the catalysis of splicing. On the basis of our results, we suggest mechanisms by which the splicing of NS1 mRNA is controlled in infected cells.Eucaryotic pre-mRNAs synthesized by RNA polymerase II usually contain intervening sequences (introns) that are removed by splicing. The first step is cleavage at the 5' splice site, to generate the 5' exon and a lariat form of the intron attached to the 3' exon. Subsequently, the 5' and 3' exons are ligated to form the mature mRNA and to release the intron lariat (23,27). During the initial phase of the reaction, the pre-mRNA substrate is assembled into ribonucleoprotein complexes, or spliceosomes, containing small nuclear ribonucleoproteins (snRNPs) (1,4,7,10,24). The largest complexes found in mammalian systems sediment at about 50S to 60S in sucrose gradients (1,7,10,24). Analysis of the snRNP composition of 50S to 60S spliceosomes by affinity selection of biotinylated pre-mRNA on streptavidin-agarose beads indicated that the Ul, U2, U4, U5, and U6 snRNPs are present in these spliceosomes (2, 11). The same snRNPs have been ide...

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U2 as well as U1 small nuclear ribonucleoproteins are involved in premessenger RNA splicing

Cited by 575 publications

References 56 publications

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

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.

A block in mammalian splicing occurring after formation of large complexes containing U1, U2, U4, U5, and U6 small nuclear ribonucleoproteins.

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