Features of 5′-splice-site efficiency derived from disease-causing mutations and comparative genomics

Roca, Xavier; Olson, Andrew; Rao, Atmakuri Ramakrishna; Enerly, Espen; Kristensen, Vessela N.; Børresen-Dale, Anne Lise; Andresen, Brage S.; Krainer, Adrian R.; Sachidanandam, Ravi

doi:10.1101/gr.6859308

Cited by 83 publications

(83 citation statements)

References 57 publications

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“…Using a model system of tandem 5Ј splice sites separated by 16 nucleotides, it was shown that the ability of U1 snRNA to base pair with competing splice sites is the major determinant of 5Ј splice site selection, in agreement with previous models (24). However, recent computational analyses indicate that pairwise dependencies between 5Ј splice site nucleotides are also significant in promoting splice site selection (23).…”

supporting

confidence: 81%

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

Hicks

Mueller

Shepard

et al. 2010

Molecular and Cellular Biology

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Alternative 5 splice site selection is one of the major pathways resulting in mRNA diversification. Regulation of this type of alternative splicing depends on the presence of regulatory elements that activate or repress the use of competing splice sites, usually leading to the preferential use of the proximal splice site. However, the mechanisms involved in proximal splice site selection and the thermodynamic advantage realized by proximal splice sites are not well understood. Here, we have carried out a systematic analysis of alternative 5 splice site usage using in vitro splicing assays. We show that observed rates of splicing correlate well with their U1 snRNA base pairing potential. Weak U1 snRNA interactions with the 5 splice site were significantly rescued by the proximity of the downstream exon, demonstrating that the intron definition mode of splice site recognition is highly efficient. In the context of competing splice sites, the proximity to the downstream 3 splice site was more influential in dictating splice site selection than the actual 5 splice site/U1 snRNA base pairing potential. Surprisingly, the kinetic analysis also demonstrated that an upstream competing 5 splice site enhances the rate of proximal splicing. These results reveal the discovery of a new splicing regulatory element, an upstream 5 splice site functioning as a splicing enhancer.The splicing of nuclear pre-mRNAs is a fundamental process required for the expression of most metazoan genes. It is carried out by the spliceosome, which recognizes splicing signals and catalyzes the removal of noncoding intronic sequences to assemble protein-coding sequences into mature mRNAs prior to export to the cytoplasm and translation (2). Of the approximately 25,000 genes carried by the human genome (16), more than 90% are believed to produce transcripts that are alternatively spliced, enriching the proteomic diversity of higher eukaryotic organisms (9, 29). Because regulation of this process can determine when and where a particular protein isoform is expressed, changes in alternative splicing patterns modulate many cellular activities.Exon recognition and the regulation of alternative splicing are complex processes. Splicing enhancers and silencers, either exonic or intronic, occur frequently and influence alternative splicing. Furthermore, it has been established that the strength of splice sites (31), the intron/exon architecture (i.e., the exon or intron definition modes of splice site recognition) (8), local RNA secondary structure (13,26), and the process of transcription by RNA polymerase II (19) influence splice site choice. Given the divergent sequence and architecture of genes, every exon is flanked by a unique pair of splice site signals and contains a unique group of splicing enhancers, silencers, and secondary structures. The sum of contributions from each of these elements then defines the overall recognition potential of an exon (11).When analyzing pre-mRNA sequences, it quickly becomes evident that multiple or cryptic splices site...

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supporting

confidence: 81%

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

Hicks

Mueller

Shepard

et al. 2010

Molecular and Cellular Biology

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“…From a set of 581 disease-causing mutations at 59ss (Roca et al 2008), we found that 24 (4.1%) mapped to bulge 59ss (Supplemental Table S2). As expected, the DG 1 values for the mutant 59ss are substantially smaller than those for the corresponding wild-type 59ss (mean difference of À1.7 kcal/mol).…”

Section: Implications For Disease Mutations and Snpsmentioning

confidence: 99%

“…From a set of 1,116 SNPs at human 59ss (Roca et al 2008), 57 (5.1%) mapped to bulge 59ss (Supplemental Table S3). The DG 1 values for either 59ss variant are similar (mean absolute difference [MAD] = À1.0 kcal/ mol), consistent with a generally neutral effect of the SNP on 59ss strength.…”

Section: Implications For Disease Mutations and Snpsmentioning

confidence: 99%

“…We cross-analyzed the bulge 59ss with updated data sets of disease-causing mutations and SNPs at human 59ss that fall outside of the invariant dinucleotide at positions +1/+2 (Roca et al 2008). We assessed the conservation of the bulge structure by running UNAFold for the 59ss with either the mutated nucleotide or the SNP allele that is not in the reference genome sequence.…”

Section: In Silico Analysesmentioning

confidence: 99%

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Widespread recognition of 5′ splice sites by noncanonical base-pairing to U1 snRNA involving bulged nucleotides

Roca¹,

Akerman²,

Gaus³

et al. 2012

Genes Dev.

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137

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An established paradigm in pre-mRNA splicing is the recognition of the 59 splice site (59ss) by canonical basepairing to the 59 end of U1 small nuclear RNA (snRNA). We recently reported that a small subset of 59ss base-pair to U1 in an alternate register that is shifted by 1 nucleotide. Using genetic suppression experiments in human cells, we now demonstrate that many other 59ss are recognized via noncanonical base-pairing registers involving bulged nucleotides on either the 59ss or U1 RNA strand, which we term ''bulge registers.'' By combining experimental evidence with transcriptome-wide free-energy calculations of 59ss/U1 base-pairing, we estimate that 10,248 59ss (~5% of human 59ss) in 6577 genes use bulge registers. Several of these 59ss occur in genes with mutations causing genetic diseases and are often associated with alternative splicing. These results call for a redefinition of an essential element for gene expression that incorporates these registers, with important implications for the molecular classification of splicing mutations and for alternative splicing.[Keywords: 59 splice site; U1 small nuclear RNA; base-pairing register; bulged nucleotide; splicing mutation; alternative splicing] Supplemental material is available for this article. Pre-mRNA splicing is an essential processing step for the expression of ;90% of protein-coding human genes and relies on conserved sequence elements at both ends of introns, termed splice sites (Sheth et al. 2006;Wahl et al. 2009). These elements are highly diverse, considering that thousands of different sequences act as naturally occurring splice sites in the human transcriptome (Sahashi et al. 2007;Roca and Krainer 2009). The characterization of these sequence elements and the factors that recognize them has been essential for predicting exons in new genes, for the study of alternative splicing (Nilsen and Graveley 2010), and for classifying mutations in these elements that cause human genetic diseases (Buratti et al. 2007).Typically, the strength of a splice site (or splice site score) is estimated by algorithms that measure its concordance to matrices built using large collections of splice sites (Senapathy et al. 1990;Brunak et al. 1991;Yeo and Burge 2004;Sahashi et al. 2007;Hartmann et al. 2008). These methods implicitly assume that all of the sequences used to build the matrix are recognized by the same mechanism. However, there are cases in which the splice site score does not reflect the strength of the splice site determined experimentally (Roca and Krainer 2009), highlighting the limitations of these tools. Furthermore, the recognition mechanisms for many splice sites predicted to be weak are poorly understood.Splicing of >99% of pre-mRNA introns is catalyzed by the major spliceosome, a dynamic macromolecular machine composed of five small nuclear RNAs (snRNAs) and associated polypeptides, plus many other protein factors (Wahl et al. 2009). The U1 small nuclear ribonucleoprotein particle (snRNP), comprising the U1 snRNA and 10 polypeptides (Pomeranz K...

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“…Later PWMs were further processed using information content theory (Rogan and Schneider 1995). These methods assume independence between 59ss positions, yet there is now ample evidence for complex associations between 59ss positions (Burge 1998;Carmel et al 2004;Roca et al 2008). Other algorithms-like firstorder Markov models, decision trees, and maximum entropy models-take into consideration these associations (Yeo and Burge 2004).…”

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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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Features of 5′-splice-site efficiency derived from disease-causing mutations and comparative genomics

Cited by 83 publications

References 57 publications

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

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

Widespread recognition of 5′ splice sites by noncanonical base-pairing to U1 snRNA involving bulged nucleotides

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

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