ReviewTowards understanding pre-mRNA splicing mechanisms and the role of SR proteins
Introduction
Pre-mRNA splicing plays a crucial role in gene expression in the eukaryotic cell nucleus. For instance, it has been reported recently that pre-mRNA splicing is responsible for regulation of gene expression in temperature signaling (Capovilla et al., 2015) and response to abiotic stress in plants (Cui and Xiong, 2015). Protein-coding genes are disrupted by intervening intronic sequences. The intervening sequences (introns) in most pre-mRNAs are removed from the snRNAs, thereby linking all exon sequences within the same reading frame.
RNA polymerase II transcribes eukaryotic pre-mRNAs and is also responsible for multiple post-transcriptional modifications. Although the majority of constitutive events that occur in the nuclei of higher eukaryotes are precise and efficient, different mRNA isoforms can be produced through alternative mRNA splicing. Hence, the precise removal of introns via pre-mRNA splicing is an indispensable step in the regulation of gene expression. The spliceosome is a macromolecular machine consisting of five snRNAs – U1, U2, U4, U5, and U6 – and many different proteins such as small nuclear ribonucleic proteins (snRNPs). This machine catalyzes mRNA splicing in eukaryotic cells by removing the introns and ligating the exons (Wahl et al., 2009).
The canonical stepwise assembly of the spliceosome occurs twice on each molecule of heterogeneous nuclear RNA (hnRNA). Specific sequence elements contained within the 5′ and 3′ splice sites include the polypyrimidine tract (PPT) and the branch point sequence (BPS), which are both involved throughout spliceosome assembly. The 3′ splice site of hnRNA is rich in AG nucleotides and called the polypyrimidine tract (PPT); it contains a variable number of polypyrimidines. This polypyrimidine tract provides a suitable substrate for the recruitment of different factors to the 3′ splice sites and potentially facilitates their recruitment to the branch point sequence (BPS) of the hnRNA. The branch point sequence includes the conserved adenosine that is required to initiate the first splicing step. The canonical mRNA splicing pathway starts with the U1 snRNP binding the 5′ splice site, followed by the U2 snRNP binding the branch point sequence. Subsequently, they bind the U4/U5/U6 tri-snRNP to produce the mature spliceosome, which is required for catalyzing RNA-based sequences (Matlin and Moore, 2008, Smith et al., 2008).
Briefly, two main steps known as SN2-type transesterification reactions are involved in occurring pre-mRNA splicing (Fica et al., 2013). The 2′-OH group of “conserved RNA adenine nucleotide” in BPS of an individual intron attacks at the 5′ end of the intron to the phosphorous of guanine nucleotide (first nucleophilic attack) in the first step of pre-mRNA splicing. This step of pre-mRNA splicing causes to release 5′-exon and form an “intronlariat-3′-exon intermediate”. Following the first step, the 3′-OH group of RNA nucleotide on 3′ end of the 5′-exon attacks at the 5′ end of the 3′-exon on the phosphorous of the RNA guanine nucleotide (second nucleophilic attack), resulting in joining two exons and releasing the intron lariat.
The important features of pre-mRNA splicing are related to: i) the enhanced functional diversity of the proteome and ii) the regulation of gene expression by introducing additional regulatory layers; different mRNA isoforms have distinct coding capacities and stabilities (Bindereif, 2015).
The nature of spliceosome assembly steps has been distinctly explained for decades, but it is still enigmatic that how the spliceosome assists these reactions? How is a reacting piece placed into close of one another in a stringent temporal arrangement? With the different lengths of the introns and exons, how the spliceosome hold the 5′-exon in two reaction steps and accommodate pre-mRNA?
Several in vitro studies have been performed to understand the chemical mechanisms of pre-mRNA splicing, primarily that of pre-mRNAs poised at two exons and their intervening intron (Brow, 2002, Will and Lührmann, 2011). But the function of the protein components involved in the spliceosome have not understood yet. A growing number of studies have explored the structures of the components and domains of the spliceosome (Stefl et al., 2005, Auweter et al., 2006, Cléry et al., 2008), and the literature describes how alternative mRNA splicing is influenced by extracellular signals through different signal transduction pathways (Stamm, 2002, Lynch, 2004, Shin and Manley, 2004). This review briefly addresses the splicing process, discusses the structure of the pre-mRNA splicing machine, and reviews the effect of extracellular signal transduction pathways on the regulation of alternative mRNA splicing.
Section snippets
Transcription initiation via alternative promoters
The first layer of control in gene expression is related to transcription initiation during mRNA biogenesis (Sanyal et al., 2012, Consortium, F., 2014). Different transcripts result from the initiation of transcription at the first exon or within the upstream 5′-untranslated-region (UTR). Alternative first exons result in different transcripts with different open reading frames (ORFs), giving rise to diverse protein isoforms with alternative N-termini (Goossens et al., 2007). Transcripts
The mRNA splicing process
The RNA splicing process defines the removal of introns (internal sequences from the pre-messenger RNA), which are RNA sequences without encoding any proteins, and ligation of sequences (exons) that are parts of the mature mRNA. Pre-mRNA splicing is catalyzed by the spliceosome, which is a large complex consisting of small nuclear RNAs (snRNA) and their associated proteins. Precise splicing occurs when the spliceosome recognizes the sequence-specific splice sites on the intron–exon borders, and
Polyadenylation: 3′-end maturation
The polyadenylation process is an additional step in mRNA processing (Danckwardt et al., 2008). The APA site provides an additional regulatory layer for gene expression and results in transcripts with different 3′ ends. It has been shown that multiple mRNA isoforms with different 3′ ends obtained from 70% of eukaryotic genes through the alternative polyadenylation process (APA) (Di Giammartino et al., 2011, Shi, 2012, Elkon et al., 2013, Tian and Manley, 2013).
The coding regions of transcripts
Alternative translation initiation: from mRNA to protein
In addition to the regulation of transcriptional processing, translation is also tightly regulated. Although ‘translational regulation’ refers to protein abundance because translation can be initiated from alternative ORFs (aORFs) or uORFs, the term also encompasses the amino acid composition of the proteins, which results from the use of different start codons (Kochetov, 2008). Generally, changes in protein synthesis are measured based on total mRNA levels or via proteomic approaches (Lee and
The Sr protein family and the regulation of alternative splicing, constitutive splicing and post-splicing activities
Only a small percentage of a primary transcript (exons) is joined via splicing process to form a mature mRNA that is exported into the cytosol. Nearly all introns and other intervening sequences remain in the nucleus, where they are degraded (Sharp, 2005). The majority of pre-mRNAs contain exons that can be incorporated into the mature mRNA or can be excluded from it, and this is the basis of alternative splicing.
Often, transcripts incorporate many alternative exons in various combinations. In
The integration of alternative splicing with other gene expression mechanisms
The utilization of alternative exons is synchronized with other gene expression events, especially transcription (Pickrell et al., 2010). Two suggested models for this mechanism are the kinetics and recruitment models. The recruitment model assumes that splicing factor assembly occurs on the CTD of RNA polymerase II and that these assembled factors are then bound to the nascent pre-mRNA during transcription. These factors affect the splice sites independently of their concentration, meaning
Crucial functions of RNA in the alternative splicing process
Despite great advances in determining the model of exon regulation, a comprehensive mechanistic understanding is still lacking. A comparison between the regulation of exonic splicing and alternative splicing showed that modifications to alternative exons did not affect the cis-elements. Various regulatory factors have been reported to have a direct effect on cis-elements (Irimia et al., 2009, Wagner and Berglund, 2014, Wong et al., 2014). The splice site selection schematic shown in Fig. 3
The roles of co- and post-transcriptional RNA splicing
To understand splicing and transcription, it is necessary to focus on two important principles: i) the direct effect of transcriptional regulation on alternative splicing; both Pol II processivity and promoter identity apparently exert great influence on splicing (Cramer et al., 1999, de la Mata et al., 2003). This strategy also increases the effect of epigenetic regulation of RNA splicing. In fact, certain chromatin marks (Muñoz et al., 2009, Luco et al., 2010) and chromatin remodeling
Co-transcriptional RNA splicing
Researchers have concluded that most constitutive splicing is co-transcriptional. Spliced mRNA can be detected using biochemical fractionation of chromatin and mechanical dissection (Baurén and Wieslander, 1994, Pandya-Jones and Black, 2009). Looped RNAs can contain introns that are bound to chromatin, and they are generally removed from nascent RNA prior to the termination of transcription; this process may also facilitate mRNA splicing (Beyer and Osheim, 1991). Spliced mRNAs at different
Post-transcriptional RNA splicing
In many dendrites, pre-mRNAs appear to be directly exported into the cytoplasm, where their splicing is activated through Ca2 + signaling (Glanzer et al., 2005, Bell et al., 2010). The interleukin-1b pre-mRNA is another example of post-transcriptional processing; a portion of the pre-mRNA remains unspliced despite the cell being anucleate, and splicing may be completed in response to signaling in the anucleate platelets (Denis et al., 2005). The transition from a preliminary complex to a
Conclusions
Predicting the specific transcripts involved in a particular cell type is still a challenge. Multiple parameters, such as the strength of the splice site, the structure of the exon–intron junction, the presence of splicing regulators, and the transcription process are involved in selecting the splice site (Fig. 5). Despite the challenges created by its energetic nature, a sharper image of the order and nature of the complicated rearrangements inside the spliceosome, along with their
Conflict of interest
The authors declare that they have no conflict of interest.
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