Does steric interference between splice sites block the splicing of a short c-src neuron-specific exon in non-neuronal cells?

Black, Douglas L.

doi:10.1101/gad.5.3.389

Cited by 139 publications

(138 citation statements)

References 67 publications

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“…In general, small exons are thought to be poorly recognized by the splicing machinery, possibly as a result of steric hindrance between snRNPs and other splicing factors (22,43); presumably to compensate for this, small CS exons tend to be accompanied by stronger splice site signals and splicing enhancer motifs (22,44). Because AS exons tend to be small (21,23), it is important to consider the possibility that the increased sequence conservation in AS exons is due solely to their size.…”

Section: Discrimination Of Misclassified Exonsmentioning

confidence: 99%

Sequence conservation, relative isoform frequencies, and nonsense-mediated decay in evolutionarily conserved alternative splicing

Baek

Green

2005

Proc. Natl. Acad. Sci. U.S.A.

122

127

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Studies of expressed sequence tag data sets have revealed large numbers of splicing variants for human genes, but it remains challenging to distinguish functionally important variants from aberrant splicing, clarify the nature of the alternative functions, and understand the signals that regulate splicing choices. To help address these issues, we have constructed and analyzed a large data set of 1,478 exon-skipping alternative splicing (AS) variants evolutionarily conserved in human and mouse. In about one-fifth of cases, one isoform appears subject to nonsense-mediated mRNA decay (NMD), supporting the idea that a major role of AS is to regulate gene expression; one-quarter of these NMD-inducing cases involve a conserved exon whose apparent sole purpose is to mediate destruction of the message when included. We explore sequence conservation likely related to splicing regulation, using in part a measure of the overall amount of conserved information in a sequence, and find that the increased conservation that has been observed within AS exons primarily affects synonymous sites, suggesting that regulatory signals significantly constrain synonymous substitution rates. We show that a lower frequency of the inclusion isoform relative to the exclusion isoform tends to be associated with weaker splice site signals, smaller exon size, and higher intronic sequence conservation, and provide evidence that all of these factors are under selection to control relative isoform frequencies. Some conserved instances of AS appear to represent aberrant splicing events that by chance have occurred in both species, and we develop a nonparametric likelihood approach to identify these.T wo known roles for alternative splicing (AS) are to allow several proteins to be produced from a single gene, and to reduce gene expression by yielding isoforms that are degraded by nonsensemediated mRNA decay (NMD) or other mechanisms (1, 2). Genome-wide studies using cDNA and EST databases have suggested that 22-74% of mammalian genes are alternatively spliced, with perhaps 35% of AS isoforms predicted to be subject to NMD (2-8). However, the biological significance of these estimates remains unclear, because many database variants may represent aberrant splicing events lacking a functional role (ref. 9 and references therein). Given that the primary function of NMD is presumably to remove aberrant transcripts, it is particularly difficult to assess the significance of NMD isoforms. Filtering out variants that are supported by few ESTs (5, 6), that have been found only in tumor cells or cell lines (10), or other anomalies (9), is not guaranteed to catch all aberrant splicing cases, and may incorrectly eliminate functional rare variants. Determining which variants are biologically important consequently remains a major challenge.An important approach to identifying functionally important AS is to focus on isoforms common to orthologous genes in two species (11)(12)(13)(14). This method does not eliminate the possibility of false positives representing...

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Section: Discrimination Of Misclassified Exonsmentioning

confidence: 99%

Sequence conservation, relative isoform frequencies, and nonsense-mediated decay in evolutionarily conserved alternative splicing

Baek

Green

2005

Proc. Natl. Acad. Sci. U.S.A.

122

127

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“…Another group of potential splicing regulators is the heterogeneous nuclear ribonucleoprotein (hnRNP) fam ily of proteins, which bind to unspliced hnRNA in mam malian cells, but again these proteins are not highly tis-sue specific (Dreyfuss et al 1993). As yet there are no cell type-specific splicing factors identified in mammalian cells, and there are few systems where a regulated splic ing event has been reconstructed in vitro (Ge and Manley 1990;Black 1992).…”

mentioning

confidence: 99%

“…We are using the mouse c-src gene as a model system for study ing neural-specific splicing in mammalian cells (Black 1991(Black ,1992. c-src is a proto-oncogene encoding a protein tyrosine kinase (Cooper 1990;Bolen 1993).…”

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confidence: 99%

The generally expressed hnRNP F is involved in a neural-specific pre-mRNA splicing event.

Min¹,

Chan²,

Black³

1995

Genes Dev.

Self Cite

186

197

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The proteins and RNA regulatory elements that control tissue-specific pre-mRNA splicing in mammalian cells are mostly unknown. In this study, a set of proteins is identified that binds to a splicing regulatory element downstream of the neuron specific c-src Nl exon. This complex of proteins bound specifically to a short RNA containing the regulatory sequence in neuronal extracts that splice the Nl exon. It was not seen in non-neuronal cell extracts that fail to splice this exon. UV-cross-linking experiments identified a neuron-specific 75-kD protein and several nontissue-specific proteins, including the 53-kD heterogeneous nuclear ribonucleoprotein F (hnRNP F), as components of this complex. Although present in both extracts, hnRNP F binds tightly to the RNA only in the neuronal extracts. A mutation in the regulatory RNA sequence, that inhibits Nl splicing in vivo, abolished formation of the neuron-specific complex and the binding of the neuron-specific 75-kD protein. Competition experiments in the two extracts show that the binding of the neuronal protein complex to the src pre-mRNA is required to activate Nl exon splicing in vitro. Antibody inhibition experiments indicate that the hnRNP F protein is a functional part of this complex. The assembly of regulatory complexes from both constitutive and specific proteins is likely to be a general feature of tissue-specific splicing regulation.

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“…Lower efficiencies for the splicing of small exons (Black 1991;Dominski and Kole 1991;Hwang and Cohen 1997) and large exons (Peterson et al 1994;Sterner et al 1996;Borensztajn et al 2006) have been observed previously. Surprisingly, when we used different splice site sequences, we found a striking difference in exon size dependence.…”

Section: Discussionmentioning

confidence: 72%

Splicing of designer exons informs a biophysical model for exon definition

Arias¹,

Lubkin²,

Chasin³

2014

RNA

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Pre-mRNA molecules in humans contain mostly short internal exons flanked by longer introns. To explain the removal of such introns, exon recognition instead of intron recognition has been proposed. We studied this exon definition using designer exons (DEs) made up of three prototype modules of our own design: an exonic splicing enhancer (ESE), an exonic splicing silencer (ESS), and a Reference Sequence (R) predicted to be neither. Each DE was examined as the central exon in a three-exon minigene. DEs made of R modules showed a sharp size dependence, with exons shorter than 14 nt and longer than 174 nt splicing poorly. Changing the strengths of the splice sites improved longer exon splicing but worsened shorter exon splicing, effectively displacing the curve to the right. For the ESE we found, unexpectedly, that its enhancement efficiency was independent of its position within the exon. For the ESS we found a step-wise positional increase in its effects; it was most effective at the 3 ′ end of the exon. To apply these results quantitatively, we developed a biophysical model for exon definition of internal exons undergoing cotranscriptional splicing. This model features commitment to inclusion before the downstream exon is synthesized and competition between skipping and inclusion fates afterward. Collision of both exon ends to form an exon definition complex was incorporated to account for the effect of size; ESE/ESS effects were modeled on the basis of stabilization/destabilization. This model accurately predicted the outcome of independent experiments on more complex DEs that combined ESEs and ESSs.

show abstract

Does steric interference between splice sites block the splicing of a short c-src neuron-specific exon in non-neuronal cells?

Cited by 139 publications

References 67 publications

Sequence conservation, relative isoform frequencies, and nonsense-mediated decay in evolutionarily conserved alternative splicing

Sequence conservation, relative isoform frequencies, and nonsense-mediated decay in evolutionarily conserved alternative splicing

The generally expressed hnRNP F is involved in a neural-specific pre-mRNA splicing event.

Splicing of designer exons informs a biophysical model for exon definition

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