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Intronic features that determine the selection of the 3′ splice site

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Abstract Most eukaryotic primary transcripts include segments, or introns, that will be accurately removed during RNA biogenesis. This process, known as pre‐messenger RNA splicing, is catalyzed by the spliceosome, accurately selecting a set of intronic marks from others apparently equivalent. This identification is critical, as incorrectly spliced RNAs can be toxic for the organism. One of these marks, the dinucleotide AG, signals the intronic 3′ end, or 3′ splice site (ss). In this review we will focus on those intronic features that have an impact on 3′ ss selection. These include the location and type of neighboring sequences, and their distance to the 3′ end. We will see that their interplay is needed to select the right intronic end, and that this can be modulated by additional intronic elements that contribute to alternative splicing, whereby diverse RNAs can be generated from identical precursors. This complexity, still poorly understood, is fundamental for the accuracy of gene expression. In addition, a clear knowledge of 3′ ss selection is needed to fully decipher the coding potential of genomes. WIREs RNA 2012 doi: 10.1002/wrna.1131 This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Processing > Splicing Regulation/Alternative Splicing Regulatory RNAs/RNAi/Riboswitches > Riboswitches

Pre‐mRNA splicing consists of two transesterification reactions. In the first (also known as first step of splicing), the 2′‐hydroxyl group from the branch‐site adenosine carries out a nucleophilic attack on the phosphate (green) at the intron 5′ end (known as 5′ ss). The products of this reaction are the exon 1 and exon 2–intron lariat. These are also the substrates for the second step, where the exon 1 3′‐hydroxyl group conducts a second nucleophilic attack on the phosphate (red) at the intron 3′ end (3′ ss). The products are the mature RNA and the intron lariat. In this example, a pre‐mRNA has been depicted, which after splicing will generate an mRNA. This process is catalyzed by the spliceosome, and the introns thus removed are termed spliceosomal introns. Transcripts without protein‐coding potential can also bear spliceosomal introns.

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3′ ss site definition in Saccharomyces cerevisiae introns. As the core components of the spliceosome are conserved in evolution, it is likely that the model in (a) is relevant to some metazoan introns. Numbers indicate the distance to the BS A in nucleotides (nt). (a) The end of the intron cannot be anywhere after the BS, and AG efficiency as 3′ ss is reduced beyond ∼45 nt. Because stems are not considered by the spliceosome when determining this distance, and consequently included AGs (red) are ignored, folding of this pre‐mRNA region is critical for 3′ ss choice. AGs preceded by a pyrimidine (green) are favored over AAG (orange), otherwise they are used efficiently. The spliceosome will both ignore AGs (red) and block the formation of stems in the first nine nucleotides after the BS. (b) The size and predicted folding of the stems are highly variable (for instance, UBC13 is estimated to have a fold including more than 100 nt23). This opens evolutionary possibilities, as documented by this example. Acting as a thermosensor that controls 3′ ss choice, a stem loop in the APE2 transcript promotes a 3′ ss switch depending on the temperature.23 At low temperatures (left) the BS‐distal CAG (green) prevails as 3′ ss over the proximal AAG (orange), which is also partially blocked by a stem. At higher temperatures (right) the stem partly opens, favoring AAG (green) and placing CAG (orange) outside the optimal range for 3′ ss selection.23 This illustrates the pivotal role that a relatively simple RNA structure can have on gene expression.

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Key intronic elements identified by the spliceosome, during assembly (a) and catalysis (b). (a) In a 5′ → 3′ direction, the indicated spliceosomal components identify the intron as follows. U1 snRNP (or U1 for short) associates with the 5′ ss, U2 with the BS and U2AF with the PPT and 3′ ss. This intermediate complex, referred as complex A, defines the intron and it can be isolated in vitro. At this stage there are no known RNA–RNA contacts between U1 and U2. (b) Scheme depicting the catalytic spliceosome before the second step (complex C). U1 and U2AF have been displaced prior to catalysis. U6 and U2 interact closely, including base‐pairing in several regions. They are thought to be forming the catalytic center. U6 is likely to be close to the 5′ ss, and it genetically interacts with the 3′ ss, which is identified again after complex A formation. U5 contacts both exons. Sizes, shapes, and distances are only illustrative. See Ref 6,20,32,33 for more details.

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RNA Processing
Regulatory RNAs/RNAi/Riboswitches > Riboswitches
RNA Processing > Splicing Regulation/Alternative Splicing
RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems

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