Competing Upstream 5′ Splice Sites Enhance the Rate of Proximal Splicing

Hicks, Martin; Mueller, William F.; Shepard, Peter J.; Hertel, Klemens J.

doi:10.1128/mcb.01071-09

Cited by 38 publications

(40 citation statements)

References 32 publications

(40 reference statements)

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“…6b). Previous reports show that competing splice sites can affect splicing potential [39], suggesting that the conserved yet cryptic alternative 3′ss may be functionally relevant in this event.…”

Section: Resultsmentioning

confidence: 99%

In silico to in vivo splicing analysis using splicing code models

Vaquero-Garcia

Lynch

Barash

2014

Methods

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With the growing appreciation of RNA splicing’s role in gene regulation, development, and disease, researchers from diverse fields find themselves investigating exons of interest. Commonly, researchers are interested in knowing if an exon is alternatively spliced, if it is differentially included in specific tissues or in developmental stages, and what regulatory elements control its inclusion. An important step towards the ability to perform such analysis in silico was made with the development of computational splicing code models. Aimed as a practical how-to guide, we demonstrate how researchers can now use these code models to analyze a gene of interest, focusing on Bin1 as a case study. Bridging integrator 1 (BIN1) is a nucleocytoplasmic adaptor protein known to be functionally regulated through alternative splicing in a tissue-specific manner. Specific Bin1 isoforms have been associated with muscular diseases and cancers, making the study of its splicing regulation of wide interest. Using AVISPA, a recently released web tool based on splicing code models, we show that many Bin1 tissue-dependent isoforms are correctly predicted, along with many of its known regulators. We review the best practices and constraints of using the tool, demonstrate how AVISPA is used to generate high confidence novel regulatory hypotheses, and experimentally validate predicted regulators of Bin1 alternative splicing.

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Section: Resultsmentioning

confidence: 99%

In silico to in vivo splicing analysis using splicing code models

Vaquero-Garcia

Lynch

Barash

2014

Methods

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“…In vitro, the rate of splicing can be altered by changing the strength of the 5 ′ splice site or the length of the intron, or by adding enhancer or silencer elements (Hertel and Maniatis 1998;Yu et al 2008;Hicks et al 2010). We did not observe correlations of either splice site strength or intron length with the rates of splicing (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…5 mRNA to be matured for export is not known Schmid and Jensen 2010;Rabani et al 2011). The kinetics of excision for individual introns have been described, but larger scale analyses are limited (Kessler et al 1993;Bauren and Wieslander 1994;Tennyson et al 1995;Singh and Padgett 2009;Hicks et al 2010). Nevertheless, understanding the sequence of events in mRNA production will be essential before we can accurately describe their mechanisms of regulation.…”

Section: Introductionmentioning

confidence: 99%

Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression

Pandya-Jones

Bhatt

Lin

et al. 2013

RNA

102

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The expression of eukaryotic mRNAs is achieved though an intricate series of molecular processes that provide many steps for regulating the production of a final gene product. However, the relationships between individual steps in mRNA biosynthesis and the rates at which they occur are poorly understood. By applying RNA-seq to chromatin-associated and soluble nucleoplasmic fractions of RNA from Lipid A-stimulated macrophages, we examined the timing of exon ligation and transcript release from chromatin relative to the induction of transcription. We find that for a subset of genes in the Lipid A response, the ligation of certain exon pairs is delayed relative to the synthesis of the complete transcript. In contrast, 3′ end cleavage and polyadenylation occur rapidly once transcription extends through the cleavage site. Our data indicate that these transcripts with delayed splicing are not released from the chromatin fraction until all the introns have been excised. These unusual kinetics result in a chromatin-associated pool of completely transcribed and 3 ′ -processed transcripts that are not yet fully spliced. We also find that long introns containing repressed exons that will be excluded from the final mRNA are excised particularly slowly relative to other introns in a transcript. These results indicate that the kinetics of splicing and transcript release contribute to the timing of expression for multiple genes of the inflammatory response.

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“…This is manifested as a strong preference for the intron-proximal 59ss if the pre-mRNA contains two or more 59ss with high affinity for U1 snRNP (Reed and Maniatis 1986;Cunningham et al 1991;Yu et al 2008;Hicks et al 2010) or the concentration of some SR proteins, such as SRSF1, is increased (see ''Extrinsic Factors Affecting 59ss Choices'' above). With high-affinity candidate 59ss, multiple sites on a molecule of pre-mRNA are occupied by U1 snRNPs at the same time (Eperon et al 1993(Eperon et al , 2000Hodson et al 2012), presumably because the higher affinities increase the independent chance that each site is occupied and therefore increase the proportion of molecules on which multiple sites are occupied.…”

Section: U1 Snrnps May Bind Independently To Multiple Candidate 59ssmentioning

confidence: 99%

Pick one, but be quick: 5′ splice sites and the problems of too many choices

2013

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Splice site selection is fundamental to pre-mRNA splicing and the expansion of genomic coding potential. 59 Splice sites (59ss) are the critical elements at the 59 end of introns and are extremely diverse, as thousands of different sequences act as bona fide 59ss in the human transcriptome. Most 59ss are recognized by base-pairing with the 59 end of the U1 small nuclear RNA (snRNA). Here we review the history of research on 59ss selection, highlighting the difficulties of establishing how basepairing strength determines splicing outcomes. We also discuss recent work demonstrating that U1 snRNA:59ss helices can accommodate noncanonical registers such as bulged duplexes. In addition, we describe the mechanisms by which other snRNAs, regulatory proteins, splicing enhancers, and the relative positions of alternative 59ss contribute to selection. Moreover, we discuss mechanisms by which the recognition of numerous candidate 59ss might lead to selection of a single 59ss and propose that protein complexes propagate along the exon, thereby changing its physical behavior so as to affect 59ss selection.Questions about the mechanisms by which 59 splice sites (59ss) are selected are deeply rooted in the history of research on pre-mRNA splicing. Identification of the sequences associated with 59ss triggered the first key insights into splicing mechanisms, efforts that are reflected now in the widespread use of genomic methods to quantify the contributions of other sequences and their cognate factors. The first factors shown to modulate alternative splicing affected 59ss selection, and the difficulties of working out the molecular mechanisms involved provided a foretaste of the complexities awaiting investigations into other regulatory proteins. Despite the many insights resulting from such studies over the years, it is clear that our conceptual frameworks are not yet adequate. New ideas and models are needed for studies on splice site selection. One purpose of this review is to emphasize that developing new ideas may involve first the challenge of uprooting commonsense but unsubstantiated preconceptions hidden in established models.The 59ss is involved in both steps of splicing. In the first step, the 29-hydroxyl group of the branchpoint adenosine attacks the phosphodiester bond at the 59ss and displaces the 59 exon; in the second step, the 39-hydroxyl group of the 59 exon attacks the phosphodiester bond at the 39 splice site (39ss) and displaces the lariat intron. Splicing was discovered just before new, gel-based DNA-sequencing methods transformed molecular biology. Thus, although the original discoveries were made without the benefit of sequence information (Berget et al. 1977;Chow et al. 1977), sequences emerged rapidly thereafter and revealed clear similarities among 59ss. Moreover, the ''consensus'' sequence (comprising, at each position, the nucleotide most commonly found there) was complementary to the sequence at the 59 end of U1 small nuclear RNA (snRNA), which immediately suggested a mechanism for recognition of t...

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Competing Upstream 5′ Splice Sites Enhance the Rate of Proximal Splicing

Cited by 38 publications

References 32 publications

In silico to in vivo splicing analysis using splicing code models

In silico to in vivo splicing analysis using splicing code models

Splicing kinetics and transcript release from the chromatin compartment limit the rate of Lipid A-induced gene expression

Pick one, but be quick: 5′ splice sites and the problems of too many choices

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