A multicomponent complex is involved in the splicing of messenger RNA precursors

Grabowski, Paula J.; Seiler, Sharon R.; Sharp, Phillip A.

doi:10.1016/s0092-8674(85)80130-6

Cited by 441 publications

(303 citation statements)

References 31 publications

Supporting

Mentioning

286

Contrasting

Unclassified

Order By: Relevance

“…Pre-mRNAs have been shown to interact with snRNPs and proteins in nuclear extracts to form splicing com plexes (Brody and Abelson 1985;Grabowski et al 1985) that can be separated by gel electrophoresis Sharp 1986, 1987;Pikielny et al 1986;Lamond et al 1988). Three complexes (A,B,C) are discernible using Tris-glycine native gels (Fig.…”

Section: P62 Is Bound To Pre-mrna In Splicing Complexesmentioning

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

345

296

View full text Add to dashboard Cite

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.

show abstract

Section: P62 Is Bound To Pre-mrna In Splicing Complexesmentioning

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

345

296

View full text Add to dashboard Cite

show abstract

“…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).…”

mentioning

confidence: 99%

“…The association of the Ul snRNPs with the 50S to 60S spliceosomes appears to be more labile than that of the other snRNPs (2,11,33 interact with the 5' splice site and intron branch point, respectively. The 50S to 60S spliceosomes contain the products of the first step in splicing, 5' exon and lariat-3' exon, indicating that these spliceosomes are functional structures (1,4,7,10,24).Two influenza virus mRNAs, the NS1 and Ml mRNAs, are spliced in the nucleus to form two smaller mRNAs, NS2 and M2, respectively (12,18,20). Of the pre-mRNAs that undergo splicing, the influenza virus NS1 and Ml mRNAs are the only known examples that are not DNA directed and that are not synthesized by RNA polymerase II.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

See 2 more Smart Citations

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.

View full text Add to dashboard Cite

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...

show abstract

RNA Splicing and Genes

Sharp¹

1988

JAMA

View full text Add to dashboard Cite

The splicing of long transcripts of RNA (copied from DNA in the cell nucleus) into smaller, specific mRNA (ready for export to the protein-producing machinery in the cytoplasm) is an important event in the regulation of gene expression in eukaryotic cells. The splicing reaction occurs as a late step in the nuclear pathway for synthesis of mRNAs. This pathway commences with initiation of transcription by RNA polymerase II and probably involves an integrated series of steps each dependent on previous events. Splicing of precursors to mRNAs involves the formation of a spliceosome complex containing the 5' and 3' splice sites. This complex contains the evolutionarily highly conserved small nuclear RNAs (snRNAs) U2, U4, U5, and U6. The most abundant snRNA, U1, is required to form the spliceosome and may be a part of the spliceosome. Analogues of these snRNAs have been identified in yeast. Assembly of the spliceosome probably involves the binding of a multi-snRNA complex containing U4, U5, and U6 snRNAs. Several observations suggest that the association of snRNAs in such complexes is quite dynamic. It is argued that the snRNAs in the spliceosome form a catalytic RNA structure that is responsible for the cleavage and ligation steps during splicing.

show abstract

A multicomponent complex is involved in the splicing of messenger RNA precursors

Cited by 441 publications

References 31 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.

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

RNA Splicing and Genes

Contact Info

Product

Resources

About