Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing

@article{Sraphin1989IdentificationOF,
  title={Identification of functional U1 snRNA-pre-mRNA complexes committed to spliceosome assembly and splicing},
  author={Bertrand S{\'e}raphin and Michael Rosbash},
  journal={Cell},
  year={1989},
  volume={59},
  pages={349-358}
}
Although both U1 and U2 snRNPs have been implicated in the splicing process, their respective roles in the earliest stages of intron recognition and spliceosome assembly are uncertain. [...] Key Result Complementation analyses and chase experiments show that a stable complex, committed to the splicing pathway, forms in the absence of U2 snRNP. U1 snRNP and a substrate containing both a 5' splice site and a branchpoint sequence are required for optimal formation of this commitment complex.Expand
Arrested yeast splicing complexes indicate stepwise snRNP recruitment during in vivo spliceosome assembly.
TLDR
In vivo depletions of U1, U2, or U5 snRNAs with chromatin immunoprecipitation analysis of other splicing snRNPs along an intron-containing gene allowed the characterization of in vivo assembled higher-order splicing complexes and indicated that snRNP recruitment to nascent pre-mRNA predominantly proceeds via the canonical three-step assembly pathway.
Composition and functional characterization of the yeast spliceosomal penta-snRNP.
TLDR
This work characterized a 45S yeast penta-snRNP which contains all five spliceosomal snRNAs and over 60 pre-mRNA splicing factors and proposes that the spliceo-snRNPs associate prior to binding of a pre- mRNA substrate rather than with pre-MRNA via stepwise addition of discrete snRNPs.
Targeted snRNP depletion reveals an additional role for mammalian U1 snRNP in spliceosome assembly
TLDR
HeLa cell nuclear splicing extracts have been prepared that are specifically and efficiently depleted of U1, U2, or U4/U6 snRNPs by antisense affinity chromatography using biotinylated 2'-OMe RNA oligonucleotides to promote stable preslicing complex formation.
SC35-mediated reconstitution of splicing in U2AF-depleted nuclear extract.
TLDR
Surprisingly, the SR protein SC35 can functionally substitute for U2AF65 in the reconstitution of pre-mRNA splicing in U2 AF-depleted extracts, suggesting that there are at least three distinguishable mechanisms for the binding of U2 snRNP to the pre- mRNA, including U2af-dependent and -independent pathways.
Interaction of the U1 snRNP with nonconserved intronic sequences affects 5' splice site selection.
TLDR
Nam8p is involved in a novel mechanism by which a snRNP component can affect splice site choice and regulate intron removal through its interaction with a nonconserved sequence, which supports a model where early 5' splice recognition results from a network of interactions established by the splicing machinery with various regions of the pre-mRNA.
Cotranscriptional spliceosome assembly dynamics and the role of U1 snRNA:5'ss base pairing in yeast.
TLDR
ChIP analysis of U1, U2, and U5 small nuclear ribonucleoprotein particles to intron-containing yeast genes suggests that pairing between U1 and the 5'ss occurs after U1 recruitment and contributes to a specific U1:substrate conformation required for efficient U2 and tri-snRNP recruitment.
Novel roles of U1 snRNP in alternative splicing regulation
TLDR
An account of the latest functional properties of U1snRNP as a splicing factor, its role in transcriptional and mRNA degradation processes, and how these properties can be exploited to act as prospective therapeutic or gene silencing strategies are provided.
Efficient association of U2 snRNPs with pre-mRNA requires an essential U2 RNA structural element.
TLDR
It is proposed that U2 stem-loop IIa is recognized by an assembly factor that assists U2 snRNP binding to pre-mRNA and that the cold sensitivity is due to a critical deficiency of correctly folded U2 for spliceosome assembly at low temperatures.
Cotranscriptional spliceosome assembly occurs in a stepwise fashion and requires the cap binding complex.
TLDR
The demonstration of an essential link between CBC and spliceosome assembly in vivo indicates that 5' end capping couples pre-mRNA splicing to transcription, and shows that the cap binding complex (CBC) is necessary, but not sufficient, for cotranscriptional splicesome assembly.
Commitment of yeast pre-mRNA to the splicing pathway requires a novel U1 small nuclear ribonucleoprotein polypeptide, Prp39p.
TLDR
A novel 75-kDa polypeptide of Saccharomyces cerevisiae, Prp39p, is identified, necessary for the stable interaction of mRNA precursors with the snRNP components of the pre-mRNA splicing machinery.
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TLDR
The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.
An ordered pathway of snRNP binding during mammalian pre‐mRNA splicing complex assembly.
TLDR
It is found that U1, U2, U4, U5 and U6 snRNPs associate with the pre‐mRNA and are in the mature, functional complex.
A factor, U2AF, is required for U2 snRNP binding and splicing complex assembly
TLDR
This work identifies a previously uncharacterized activity, U2AF, that is required for the U2 snRNP-branch point interaction and splicing complex formation and provides an explanation for the necessity of the 3' splice site region in U2SnRNP binding and, hence, the first step of splicing.
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TLDR
The results indicate that pairing between U1 snRNA and the highly conserved position 5 (GTATGT) of the intron occurs early in spliceosome assembly in vitro, and pairing at position 5 does not appear to influence 5′ splice site selection in vivo, indicating that the previously described U1SnR19:5′splice junction base pairing interaction is not sufficient to define the 5′ cleavage site.
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TLDR
Surprisingly, the analysis of double mutants, with sequence alterations at both the 5' and 3' splice sites, revealed that the 5- splice site genotype was important for the efficient formation of a U2 snRNA-containing alpha complex at the 3'splice site.
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TLDR
A pathway of spliceosome assembly was deduced that has at least three stages and U2 small nuclear ribonucleoprotein alone binds to sequences of mRNA upstream of the 3' splice site.
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TLDR
In order to analyze spliceosome structure, a stringent approach was developed to obtain splicing complexes free of cellular contaminants and this approach is a form of affinity chromatography based on the high specificity of the biotin-streptavidin interaction.
Stepwise assembly of a pre-mRNA splicing complex requires U-snRNPs and specific intron sequences
TLDR
Analysis with mutant substrate RNAs demonstrated that a polypyrimidine stretch near the 3' splice site and an intact 5' splicing site are absolutely required for splicing complex formation.
An essential yeast snRNA with a U5-like domain is required for splicing in vivo
TLDR
It is shown that mRNA splicing in yeast requires the function of a U5-like snRNA, and a 70 nucleotide domain within snR7 is identified with limited sequence-specific but striking structural homology to the mammalian snRNA U5.
U1 small nuclear ribonucleoproteins are required early during spliceosome assembly.
TLDR
The mechanism of association of U1 snRNPs with the spliceosome is investigated, and Mutual dependence of 5' and 3' recognition factors suggests significant snRNP-snRNP communication during early assembly.
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