Analysis of spliceosome assembly and the structure of a regulated intron in<i>Drosophila in vitro</i>splicing extracts

Spikes, D; Bingham, Paul M.

doi:10.1093/nar/20.21.5719

Cited by 16 publications

(13 citation statements)

References 36 publications

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“…6A). Wild-type mle precursor RNA assembles multiple ATPdependent complexes (19), similar to the assembly patterns of vertebrate precursor RNAs and more standard Drosophila introns containing consensus pyrimidine tracts, such as the single intron from the furishi taramazu gene (17). Mutation of the upstream pyrimidine tracts of the mle intron permitted assembly of complex A but arrested assembly at that point such that assembly of higher-order complexes did not occur.…”

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confidence: 76%

Pyrimidine Tracts between the 5′ Splice Site and Branch Point Facilitate Splicing and Recognition of a Small Drosophila Intron

Kennedy¹,

Berget²

1997

Molecular and Cellular Biology

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The minimum size for splicing of a vertebrate intron is approximately 70 nucleotides. In Drosophila melanogaster, more than half of the introns are significantly below this minimum yet function well. Such short introns often lack the pyrimidine tract located between the branch point and 3 splice site common to metazoan introns. To investigate if small introns contain special sequences that facilitate their recognition, the sequences and factors required for the splicing of a 59-nucleotide intron from the D. melanogaster mle gene have been examined. This intron contains only a minimal region of interrupted pyrimidines downstream of the branch point. Instead, two longer, uninterrupted C-rich tracts are located between the 5 splice site and branch point. Both of these sequences are required for maximal in vivo and in vitro splicing. The upstream sequences are also required for maximal binding of factors to the 5 splice site, cross-linking of U2AF to precursor RNA, and assembly of the active spliceosome, suggesting that sequences upstream of the branch point influence events at both ends of the small mle intron. Thus, a very short intron lacking a classical pyrimidine tract between the branch point and 3 splice site requires accessory pyrimidine sequences in the short region between the 5 splice site and branch point.Intron/exon architecture varies considerably across the eucaryotic kingdom (8). In vertebrates, introns are larger than exons, with an average size over 1 kb. In lower eucaryotes, introns are often smaller than exons. In fact, lower eucaryotes often have introns smaller than the vertebrate minimum. Drosophila melanogaster contains a mixture of intron sizes, with both very small introns characteristic of lower eucaryotes and larger introns similar to those found in vertebrates. A number of small introns in Drosophila cluster in the 51-to 80-nucleotide (nt) range, with a median of 79 nt (12), a length below the vertebrate limit. It is unclear how such small Drosophila introns function in splicing given the fact that identified splicing factors in Drosophila are similar in size to their vertebrate counterparts.One of the prominent vertebrate splicing consensus sequences is the pyrimidine tract located between the branch point and 3Ј splice site (for a recent review of splicing, see reference 11). This sequence is the binding site for the required splicing factor U2 snRNP auxiliary splicing factor, U2AF (22). In vertebrates, a functional pyrimidine tract has been defined as at least five consecutive uridines or nine consecutive pyrimidines (15), although weaker tracts do exist in known constitutively spliced genes. Large Drosophila introns often possess similar pyrimidine tracts. It has been noted, however, that short Drosophila introns statistically lack such pyrimidine tracts (12), and in this characteristic they resemble introns from Saccharomyces cerevisiae and Schizosaccharomyces pombe (13). Specifically, 51% of large (81-to 5,392-nt) Drosophila introns have a pyrimidine tract downstream of the branch ...

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confidence: 76%

Pyrimidine Tracts between the 5′ Splice Site and Branch Point Facilitate Splicing and Recognition of a Small Drosophila Intron

Kennedy¹,

Berget²

1997

Molecular and Cellular Biology

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“…The Drosophila zeste gene that we characterized has two small introns and three large exons. Mutation of the 5' splice site bordering the internal exon resulted in 100% intron inclusion in vivo, with no observable exon skipping or activation of a cryptic 5' (32), intact intron (9,28,32), and expand intron (in vivo) (12). dine tract within the zeste 3' splice site.…”

Section: Discussionmentioning

confidence: 99%

“…Presumably, this complex is the Drosophila analog of the vertebrate A complex. Recent evidence by Spikes and Bingham (32) demonstrated that the complex A that is formed in Drosophila embryo extract contains U2 small nuclear RNA and unspliced precursor RNA, similarly to complex A in vertebrates. Indeed, in our assay the Drosophila complex showed migration on gels similar to that of the A complex assembled on an adenovirus precursor RNA in Drosophila S2 extract (data not shown).…”

Section: Methodsmentioning

confidence: 99%

Intron definition in splicing of small Drosophila introns.

Talerico¹,

Berget²

1994

Mol. Cell. Biol.

126

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Approximately half of the introns in Drosophila melanogaster are too small to function in a vertebrate and often lack the pyrimidine tract associated with vertebrate 3' splice sites. Here, we report the splicing and spliceosome assembly properties of two such introns: one with a pyrimidine-poor 3' splice site and one with a pyrimidine-rich 3' splice site. The pyrimidine-poor intron was absolutely dependent on its small size for in vivo and in vitro splicing and assembly. As such, it had properties reminiscent of those of yeast introns. The pyrimidine-rich intron had properties intermediate between those of yeasts and vertebrates. This 3' splice site directed assembly of ATP-dependent complexes when present as either an intron or exon and supported low levels of in vivo splicing of a moderate-length intron. We propose that splice sites can be recognized as pairs across either exons or introns, depending on which distance is shorter, and that a pyrimidine-rich region upstream of the 3' splice site facilitates the exon mode.Pairing of individual splice sites during early recognition of pre-mRNAs is an attractive model for the orchestration of splicing in genes with multiple introns. Indeed, in Saccharomyces cerevisiae, direct support exists for the pairing of splice sites across introns during the first step of spliceosome assembly (4,8,30,31). In vertebrate pre-mRNAs, however, initial interaction between factors recognizing both ends of an intron is problematic because of the large size of many vertebrate introns. We have recently suggested that the exon is the unit of recognition during early spliceosome assembly in vertebrates with the concomitant pairing of splice sites across exons (29,34). Interactive binding of factors across exons is feasible in vertebrates because internal exons are quite small, rarely exceeding 300 nucleotides (10).Exon-intron architecture varies widely among eukaryotes. Although vertebrate exons are quite small and cannot be expanded without loss of recognition (29), many genes in lower eukaryotes have large exons (10). Most of these genes with large exons have small introns; however, some have large introns. If splice sites are paired, these genes have an awkward array of large and small distances between pairs of sites, making the mechanism of splice site recognition in these pre-mRNAs unclear. To address this problem, we have turned to the study of splice site recognition in Drosophila melanogaster. The majority of Drosophila exons are 100 to 180 nucleotides in length; however, 15% are more than 550 nucleotides, which is predicted to be too large to function in a vertebrate (10). This difference suggests that pairing of splice sites across exons will not work for recognition of these large exons.A comparison of intron sizes between vertebrates and Drosophila species also reveals another obvious difference from vertebrates. More First, Drosophila introns tend not to have a G in the position preceding the branched nucleotide (24). G is the most common nucleotide at this position in ma...

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“…Here we compared the composition of spliceosomal complexes formed on the 62-nt Zeste intron with that of complexes formed on the 147-nt-long Ftz intron. Interestingly, the results of previous studies indicate that the sequence requirements for A complex formation differ between Ftz and Zeste62 in that the latter requires both the 3Ј and 5Ј ss for complex formation (52), whereas, similar to vertebrate introns, an A-like complex forms on the isolated Ftz 3Ј exon plus flanking sequences, including the BPS and polypyrimidine tract (50). As both introns have similar 3Ј ss sequences and uninterrupted polypyrimidine tracts of similar length, i.e., 12 (Zeste) versus 11 (Ftz), the mechanistic basis for this difference is not clear, but this region of Zeste62 apparently does not support stable U2 snRNP association on its own.…”

Section: Discussionmentioning

confidence: 99%

Conservation of the Protein Composition and Electron Microscopy Structure of Drosophila melanogaster and Human Spliceosomal Complexes

Herold

Will

Wolf

et al. 2009

Molecular and Cellular Biology

170

195

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Comprehensive proteomics analyses of spliceosomal complexes are currently limited to those in humans, and thus, it is unclear to what extent the spliceosome's highly complex composition and compositional dynamics are conserved among metazoans. Here we affinity purified Drosophila melanogaster spliceosomal B and C complexes formed in Kc cell nuclear extract. Mass spectrometry revealed that their composition is highly similar to that of human B and C complexes. Nonetheless, a number of Drosophila-specific proteins were identified, suggesting that there may be novel factors contributing specifically to splicing in flies. Protein recruitment and release events during the B-to-C transition were also very similar in both organisms. Electron microscopy of Drosophila B complexes revealed a high degree of structural similarity with human B complexes, indicating that higher-order interactions are also largely conserved. A comparison of Drosophila spliceosomes formed on a short versus long intron revealed only small differences in protein composition but, nonetheless, clear structural differences under the electron microscope. Finally, the characterization of affinity-purified Drosophila mRNPs indicated that exon junction complex proteins are recruited in a splicing-dependent manner during C complex formation. These studies provide insights into the evolutionarily conserved composition and structure of the metazoan spliceosome, as well as its compositional dynamics during catalytic activation.Pre-mRNA splicing is catalyzed by the spliceosome, a large RNP molecular machine consisting of the U1, U2, U4/U6, and U5 snRNPs plus a multitude of non-snRNP proteins (reviewed in reference 59). Spliceosome assembly is an ordered, highly dynamic process that leads to the stepwise formation of the catalytic site(s) responsible for catalyzing the excision of an intron of a pre-mRNA and ligation of its flanking exons (reviewed in reference 59). Initially, the U1 snRNP interacts with the conserved 5Ј splice site (5Ј ss) of the pre-mRNA, forming the spliceosomal E complex, and in the next step the U2 snRNP stably interacts with the pre-mRNA's branch site, leading to the A complex (i.e., prespliceosome). The preformed U4/U6.U5 tri-snRNP particle then interacts with the A complex to generate the precatalytic B complex. The latter is converted to the catalytically activated B* complex, a process involving major RNP rearrangements, including destabilization or loss of the U1 and U4 snRNPs. The first step of splicing subsequently ensues and involves a nucleophilic attack of the branch point adenosine at the 5Ј ss, generating a cleaved 5Ј exon and intron-3Ј exon lariat intermediate. The spliceosomal C complex is formed at this time and subsequently catalyzes the second step of splicing, which entails intron excision and concomitant ligation of the 5Ј and 3Ј exons to form mRNA. The mRNA, in the form of an RNP, is then exported to the cytoplasm for translation by the ribosome.During spliceosome assembly, a complex RNA-RNA network involving the snR...

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Analysis of spliceosome assembly and the structure of a regulated intron inDrosophila in vitrosplicing extracts

Cited by 16 publications

References 36 publications

Pyrimidine Tracts between the 5′ Splice Site and Branch Point Facilitate Splicing and Recognition of a Small Drosophila Intron

Pyrimidine Tracts between the 5′ Splice Site and Branch Point Facilitate Splicing and Recognition of a Small Drosophila Intron

Intron definition in splicing of small Drosophila introns.

Conservation of the Protein Composition and Electron Microscopy Structure of Drosophila melanogaster and Human Spliceosomal Complexes

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