Differential GC Content between Exons and Introns Establishes Distinct Strategies of Splice-Site Recognition

Amit, Maayan; Donyo, Maya; Hollander, Dror; Goren, Amir; Kim, Eddo; Gelfman, Sahar; Lev-Maor, Galit; Burstein, David; Schwartz, Schraga; Postolsky, Benny; Pupko, Tal; Ast, Gil

doi:10.1016/j.celrep.2012.03.013

Cited by 272 publications

(310 citation statements)

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“…5E,F). These alternate intron-exon architectures share characteristics with those proposed by Amit et al (2012) to segregate exons according to splicing by either intron-or exon-definition mechanisms.…”

Section: Branchpoint Motifsmentioning

confidence: 69%

“…5d-f, 6b-d). It has been proposed these alternative architectures correspond to intron-and exon-defined splicing mechanisms (Amit et al 2012). Therefore, B-box motifs contribute a further distinction between these two alternative architectures.…”

Section: Discussionmentioning

confidence: 99%

“…Uracil-rich PPTs are ancestral with a shift to cytosine enrichment appearing in birds and increasingly in mammals in association with high GC content introns (Amit et al 2012). U2AF, which binds PPTs cooperatively with the branchpoint binding SF1, has highest affinity for uracil-rich sequences (Coolidge et al 1997), suggesting that cytosine-rich PPTs, and hence cytosine B-boxes and associated exons, may require additional splicing enhancer sequences to mediate efficient splicing.…”

Section: Discussionmentioning

confidence: 99%

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Genome-wide discovery of human splicing branchpoints

et al. 2015

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During the splicing reaction, the 59 intron end is joined to the branchpoint nucleotide, selecting the next exon to incorporate into the mature RNA and forming an intron lariat, which is excised. Despite a critical role in gene splicing, the locations and features of human splicing branchpoints are largely unknown. We use exoribonuclease digestion and targeted RNA-sequencing to enrich for sequences that traverse the lariat junction and, by split and inverted alignment, reveal the branchpoint. We identify 59,359 high-confidence human branchpoints in >10,000 genes, providing a first map of splicing branchpoints in the human genome. Branchpoints are predominantly adenosine, highly conserved, and closely distributed to the 39 splice site. Analysis of human branchpoints reveals numerous novel features, including distinct features of branchpoints for alternatively spliced exons and a family of conserved sequence motifs overlapping branchpoints we term B-boxes, which exhibit maximal nucleotide diversity while maintaining interactions with the keto-rich U2 snRNA. Different B-box motifs exhibit divergent usage in vertebrate lineages and associate with other splicing elements and distinct intron-exon architectures, suggesting integration within a broader regulatory splicing code. Lastly, although branchpoints are refractory to common mutational processes and genetic variation, mutations occurring at branchpoint nucleotides are enriched for disease associations.[Supplemental material is available for this article.]The majority of human genes are spliced, a process whereby introns are removed from the nascent RNA and the remaining exonic sequence joined together into a mature RNA transcript. In addition, alternative splicing generates complex networks of isoforms from human gene loci and plays a major role in shaping the diversity of the transcriptome (Kapranov et al. 2005;Gerstein et al. 2007;Djebali et al. 2012).Splicing occurs in the spliceosome, a large ribonucleoprotein complex that recognizes at least three genetic elements within each intron: the 59 splice site (59SS), the 39 splice site (39SS), and the branchpoint (Will and L€ uhrmann 2011). RNU2-1, the U2 spliceosomal RNA (snRNA) base pairs to the sequence surrounding the unpaired branchpoint nucleotide, which then undergoes transesterification with the 59 end of the intron to form a closed lariat structure. The spliceosome then scans for the downstream 39 splice site, which undergoes a second trans-esterification reaction to join together the two exon ends and excise the intron lariat (Fig.

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Section: Branchpoint Motifsmentioning

confidence: 69%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Genome-wide discovery of human splicing branchpoints

et al. 2015

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“…In the process, it encounters the physical forces of DNA/DNA and RNA/DNA pairing that can vary depending on the local sequence composition. It has been shown [8][9][10] that the 5 0 -and 3 0 -untranslated regions (UTRs), introns and exons have characteristic guanine/cytosine (GC) content, which could affect RNA transcription and processing. Nucleotide composition could influence protein recruitment 9 , RNA secondary structure 10 , transcription rate 11,12 , DNA melting 13 or RNA/DNA and DNA/DNA duplex stability 14 .…”

mentioning

confidence: 99%

“…It has been shown [8][9][10] that the 5 0 -and 3 0 -untranslated regions (UTRs), introns and exons have characteristic guanine/cytosine (GC) content, which could affect RNA transcription and processing. Nucleotide composition could influence protein recruitment 9 , RNA secondary structure 10 , transcription rate 11,12 , DNA melting 13 or RNA/DNA and DNA/DNA duplex stability 14 . The free energy (DG) necessary to unwind polynucleotide duplexes with defined length can be calculated from the measured values of entropy (DS) and enthalpy (DH) for the 10 possible nearest-neighbour DNA/ DNA [15][16][17][18][19][20] interactions, and the 16 possible RNA/DNA 21,22 interactions.…”

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confidence: 99%

The thermodynamic patterns of eukaryotic genes suggest a mechanism for intron–exon recognition

et al. 2013

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The essential cis-and trans-acting elements required for RNA splicing have been defined, however, the detailed molecular mechanisms underlying intron-exon recognition are still unclear. Here we demonstrate that the ratio between stability of mRNA/DNA and DNA/DNA duplexes near 3 0 -spice sites is a characteristic feature that can contribute to intron-exon differentiation. Remarkably, throughout all transcripts, the most unstable mRNA/DNA duplexes, compared with the corresponding DNA/DNA duplexes, are situated upstream of the 3 0 -splice sites and include the polypyrimidine tracts. This characteristic instability is less pronounced in weak alternative splice sites and disease-associated cryptic 3 0 -splice sites. Our results suggest that this thermodynamic pattern can prevent the re-annealing of mRNA to the DNA template behind the RNA polymerase to ensure access of the splicing machinery to the polypyrimidine tract and the branch point. In support of this mechanism, we demonstrate that RNA/DNA duplex formation at this region prevents pre-spliceosome A complex assembly.

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Messenger RNA Splicing Signals

Cygan

Taggart

Fairbrother

et al. 2017

Encyclopedia of Life Sciences

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Splicing is a post‐transcriptional processing step in which intervening sequences (introns) are excised and coding sequences (exons) are ligated together to create the mature mRNA (messenger ribonucleic acid) molecule. It is a sequential process that is facilitated by the information in the RNA (ribonucleic acid) sequence (splicing regulatory elements/signals) and numerous RNA‐binding proteins (trans factors). Splicing occurs through two biochemical steps, and is catalysed by a large ribonucleoprotein known as the spliceosome. The spliceosomal machinery recognises the core splicing signals and assembles in a stepwise fashion on the premRNA molecule. These signals include the obligate 5′ splice site, 3′ splice site and branch site sequence, as well as enhancer and silencer sequences which functionally interact with RNA binding proteins. A current research application in the field uses computational approaches to study splicing signals and predict the effect of single nucleotide variations on message processing. Key Concepts Splicing is a post‐transcriptional process in which certain sections of RNA (known as introns) are removed and other sections (known as exons) are ligated together, producing the mature mRNA molecule. Splicing is catalysed by a large macromolecular machine known as the spliceosome. The major elements required for splicing include the 5′ splice site (5′ss), the 3′ splice site (3′ss), the polypyrimidine tract and the branch site. Most introns begin with a ‘GT’ sequence and end with an ‘AG’ sequence; however, there exists a class of intron with different boundary sequences that are recognised by a parallel spliceosome. Outside of these major elements, there are additional enhancer and suppressor signals that interact with RNA‐binding proteins (RBPs) and affect splicing outcome. Different combinations and positions of binding of RBPs can result in different splicing outcome. Scientists do not yet have a complete understanding of all of the different splicing signals, and this topic is an area of active research. Researchers are using computational machine learning approaches to predict the effects of sequence variation on splicing outcome. Researchers are also using new methods of sequencing to determine which RNA sequences the RBPs are recognising.

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Differential GC Content between Exons and Introns Establishes Distinct Strategies of Splice-Site Recognition

Cited by 272 publications

References 65 publications

Genome-wide discovery of human splicing branchpoints

Genome-wide discovery of human splicing branchpoints

The thermodynamic patterns of eukaryotic genes suggest a mechanism for intron–exon recognition

Messenger RNA Splicing Signals

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