Anything but Ordinary – Emerging Splicing Mechanisms in Eukaryotic Gene Regulation

Recent progress in sequencing methods and computational analyses have led to the discovery of novel splicing isoforms and noncanonical splicing mechanisms.Cotranscriptional splicing enables the fine tuning of gene expression by epigenetic and epitranscriptomics marks.Local condensates formed by intrinsically disordered domains of RNA polymerase II and splicing factors may optimize the splicing reaction.Recursive splicing and the inhibition of cryptic splice sites by the exon junction complex are two related processes with opposite outcome.Splicing of circular RNA is enhanced by the low efficiency splicing of the flanking introns and their ability to form secondary structures. Splicing of precursor mRNAs (pre-mRNA) is an important step during eukaryotic gene expression. The identification of the actual splice sites and the proper removal of introns are essential for the production of the desired mRNA isoforms and their encoded proteins. While the basic mechanisms of splicing regulation are well understood, recent work has uncovered a growing number of noncanonical splicing mechanisms that play key roles in the regulation of gene expression. In this review, we summarize the current principles of splicing regulation, including the impact of cis and trans regulatory elements, as well as the influence of chromatin structure, transcription, and RNA modifications. We further discuss the recent development of emerging splicing mechanisms, such as recursive and back splicing, and their impact on gene expression. Splicing of precursor mRNAs (pre-mRNA) is an important step during eukaryotic gene expression. The identification of the actual splice sites and the proper removal of introns are essential for the production of the desired mRNA isoforms and their encoded proteins. While the basic mechanisms of splicing regulation are well understood, recent work has uncovered a growing number of noncanonical splicing mechanisms that play key roles in the regulation of gene expression. In this review, we summarize the current principles of splicing regulation, including the impact of cis and trans regulatory elements, as well as the influence of chromatin structure, transcription, and RNA modifications. We further discuss the recent development of emerging splicing mechanisms, such as recursive and back splicing, and their impact on gene expression. The genomes of all eukaryotes contain introns, but their number, size, and distribution vary considerably between different species [1.Deutsch M. Long M. Intron-exon structures of eukaryotic model organisms.Nucleic Acids Res. 1999; 27: 3219-3228Crossref PubMed Scopus (304) Google Scholar]. In humans, for example, the average gene contains about eight introns, whereas genes in Drosophila melanogaster have fewer introns, which on average are also shorter (5.8 kb versus 1.5 kb) [1.Deutsch M. Long M. Intron-exon structures of eukaryotic model organisms.Nucleic Acids Res. 1999; 27: 3219-3228Crossref PubMed Scopus (304) Google Scholar, 2.Xiong H.Y. et al.RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease.Science. 2015; 3471254806Crossref PubMed Scopus (559) Google Scholar, 3.Zhu L. et al.Patterns of exon-intron architecture variation of genes in eukaryotic genomes.BMC Genomics. 2009; 10: 47Crossref PubMed Scopus (82) Google Scholar]. The correct identification and removal of introns by the splicing machinery is a central, conserved step during gene expression in all eukaryotes, and mutations that alter the sequence of splice sites or elicit splicing errors are often associated with disease [4.Wang G.S. Cooper T.A. Splicing in disease: disruption of the splicing code and the decoding machinery.Nat. Rev. Genet. 2007; 8: 749-761Crossref PubMed Scopus (667) Google Scholar,5.Anna A. Monika G. Splicing mutations in human genetic disorders: examples, detection, and confirmation.J. Appl. Genet. 2018; 59: 253-268Crossref PubMed Scopus (107) Google Scholar]. Furthermore, the noncontinuous exon–intron structure of eukaryotic genes allows the formation of alternative mRNA isoforms. During this process the exons of a pre-mRNA are assembled in different ways; for example, by skipping one or several exons or using alternative splice sites [6.Lee Y. Rio D.C. Mechanisms and regulation of alternative pre-mRNA splicing.Annu. Rev. Biochem. 2015; 84: 291-323Crossref PubMed Scopus (438) Google Scholar]. Alternative splice variants of one gene may encode different protein isoforms, which in extreme cases can have opposite functions [7.Schwerk C. Schulze-Osthoff K. Regulation of apoptosis by alternative pre-mRNA splicing.Mol. Cell. 2005; 19: 1-13Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar]. The occurrence of alternative splicing provides exciting new possibilities for gene regulation and is responsible for the remarkable transcriptome and proteome diversity in metazoans [8.Nilsen T.W. Graveley B.R. Expansion of the eukaryotic proteome by alternative splicing.Nature. 2010; 463: 457-463Crossref PubMed Scopus (1119) Google Scholar,9.Liu Y. et al.Impact of alternative splicing on the human proteome.Cell Rep. 2017; 20: 1229-1241Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar]. Of all human genes, only 5% consist of a single exon [10.Hube F. Francastel C. Mammalian introns: when the junk generates molecular diversity.Int. J. Mol. Sci. 2015; 16: 4429-4452Crossref PubMed Scopus (0) Google Scholar] and >90% are alternatively spliced [11.Wang E.T. et al.Alternative isoform regulation in human tissue transcriptomes.Nature. 2008; 456: 470-476Crossref PubMed Scopus (3033) Google Scholar, 12.Pan Q. et al.Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.Nat. Genet. 2008; 40: 1413-1415Crossref PubMed Scopus (2148) Google Scholar, 13.Sultan M. et al.A global view of gene activity and alternative splicing by deep sequencing of the human transcriptome.Science. 2008; 321: 956-960Crossref PubMed Scopus (927) Google Scholar]. Alternative splicing is regulated by interactions of RNA-binding proteins (RBPs; see Glossary) and splicing factors with sequences in the pre-mRNA, and by base-pairing between complementary RNA sequences in cis and in trans [6.Lee Y. Rio D.C. Mechanisms and regulation of alternative pre-mRNA splicing.Annu. Rev. Biochem. 2015; 84: 291-323Crossref PubMed Scopus (438) Google Scholar,14.Fu X.D. Ares Jr., M. Context-dependent control of alternative splicing by RNA-binding proteins.Nat. Rev. Genet. 2014; 15: 689-701Crossref PubMed Scopus (431) Google Scholar]. Since most splicing decisions occur concomitantly to transcription, the interaction of RBPs with pre-mRNAs can be impacted by chromatin structure, transcription factors as well as RNA modifications. In this review, we first summarize the general mechanisms of splicing regulation that integrate all these parameters, and in the second part, we discuss the emergence of new splicing mechanisms, also referred to as noncanonical splicing. Pre-mRNA splicing in eukaryotes is carried out by the spliceosome [15.Wahl M.C. et al.The spliceosome: design principles of a dynamic RNP machine.Cell. 2009; 136: 701-718Abstract Full Text Full Text PDF PubMed Scopus (1568) Google Scholar, 16.Wilkinson M.E. et al.RNA splicing by the spliceosome.Annu. Rev. Biochem. 2020; 89: 359-388Crossref PubMed Scopus (27) Google Scholar, 17.Shi Y. Mechanistic insights into precursor messenger RNA splicing by the spliceosome.Nat. Rev. Mol. Cell Biol. 2017; 18: 655-670Crossref PubMed Scopus (121) Google Scholar]. This large RNA–protein complex consists of five small nuclear ribonucleoproteins (snRNPs) as main components, which occur and function individually (U1 and U2), in heterodimers (U4/U6), and heterotrimers (U4/U6.U5). In addition, a substantial number of proteins that are not part of the core snRNPs associate with the spliceosome and are required for efficient and proper splicing. During splicing, the spliceosome follows a strict assembly and rearrangement choreography and each snRNP and splicing factor assumes a specific function and/or position within the spliceosome (Figure 1A , Key Figure). The initial detection of the splice sites is achieved by binding of the U1 snRNP to the 5′ splice site (5′ ss) and interaction of splicing factor 1 (SF1, or mammalian branch point binding protein, mBBP) and U2 auxiliary factor (U2AF) with the polypyrimidine tract and the branchpoint. Next, the U2 snRNP displaces SF1 and binds itself to the branchpoint. This prespliceosomal complex is referred to as the A complex. Subsequently, the U4/U6.U5 tri-snRNP joins the other two snRNPs to form a large and complex intermediate of the spliceosome, the precatalytic B complex. After several rearrangements and protein exchanges and the removal of the U1 and U4 snRNPs, the active spliceosome (B*) is able to carry out the first transesterification splicing reaction. The resulting C complex catalyzes the second splicing step after a few more rearrangements, leading to excision of the lariat and ligation of the exons. Major advances in the field of cryogenic electron microscopy (cryo-EM) in the last 5 years have enabled us to shed light on the organization and rearrangement of the spliceosome during the different steps of the splicing reaction in yeast and humans [16.Wilkinson M.E. et al.RNA splicing by the spliceosome.Annu. Rev. Biochem. 2020; 89: 359-388Crossref PubMed Scopus (27) Google Scholar,18.Kastner B. et al.Structural insights into nuclear pre-mRNA splicing in higher eukaryotes.Cold Spring Harb. Perspect. Biol. 2019; 11: a032417Crossref PubMed Scopus (33) Google Scholar, 19.Yan C. et al.Molecular mechanisms of pre-mRNA splicing through structural biology of the spliceosome.Cold Spring Harb. Perspect. Biol. 2019; 11: a032409Crossref PubMed Scopus (30) Google Scholar, 20.Zhang L. et al.RNAs in the spliceosome: insight from cryoEM structures.Wiley Interdiscip. Rev RNA. 2019; 10e1523Crossref PubMed Scopus (7) Google Scholar, 21.Fica S.M. Cryo-EM snapshots of the human spliceosome reveal structural adaptions for splicing regulation.Curr. Opin. Struct. Biol. 2020; 65: 139-148Crossref PubMed Scopus (0) Google Scholar]. This work has rationalized decades of biochemical and functional studies, and provided a molecular understanding of the basic mechanisms of splicing, demonstrating how specific factors drive splice site recognition and catalysis. In addition to core splicing factors, additional RBPs modulate the recognition of exons and introns, which enhance or silence splicing dependent on the context of the bound sequence [14.Fu X.D. Ares Jr., M. Context-dependent control of alternative splicing by RNA-binding proteins.Nat. Rev. Genet. 2014; 15: 689-701Crossref PubMed Scopus (431) Google Scholar] (Figure 1B). All these events are perfectly orchestrated and mutations that alter sequence elements or trans-acting factors can lead to diseases. In fact, 35% of all disease-causing mutations are predicted to disrupt splicing, and abnormally expressed splicing factors can have oncogenic properties and are involved in several disorders [22.Sterne-Weiler T. Sanford J.R. Exon identity crisis: disease-causing mutations that disrupt the splicing code.Genome Biol. 2014; 15: 201Crossref PubMed Scopus (66) Google Scholar, 23.Sveen A. et al.Aberrant RNA splicing in cancer; expression changes and driver mutations of splicing factor genes.Oncogene. 2016; 35: 2413-2427Crossref PubMed Scopus (213) Google Scholar, 24.Scotti M.M. Swanson M.S. RNA mis-splicing in disease.Nat. Rev. Genet. 2016; 17: 19-32Crossref PubMed Scopus (378) Google Scholar]. Particularly well-studied examples are mutations in pre-mRNA processing factor 8 (PRPF8) that lead to retinitis pigmentosa and mutations in splicing factor 3B subunit 1 (SF3B1) in chronic lymphocytic leukemia (CLL) [25.Mordes D. et al.Pre-mRNA splicing and retinitis pigmentosa.Mol. 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Early evidence for cotranscriptional splicing came from electron microscopy preparation of Drosophila embryos depicting nascent RNA-bearing loops that corresponded to intermediate forms of the splicing reaction [29.Beyer A.L. Osheim Y.N. Splice site selection, rate of splicing, and alternative splicing on nascent transcripts.Genes Dev. 1988; 2: 754-765Crossref PubMed Google Scholar]. Cotranscriptional splicing was later confirmed in human tissues by next-generation sequencing of total RNA. The intronic sequence read coverage exhibited a saw tooth pattern as a result of nascent transcription concurrent with intron removal [30.Ameur A. et al.Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain.Nat. Struct. Mol. Biol. 2011; 18: 1435-1440Crossref PubMed Scopus (172) Google Scholar]. Further sequencing of nascent RNA estimated that cotranscriptional splicing is a widespread phenomenon that occurs at a frequency of 65–85% in human cells [31.Tilgner H. et al.Deep sequencing of subcellular RNA fractions shows splicing to be predominantly co-transcriptional in the human genome but inefficient for lncRNAs.Genome Res. 2012; 22: 1616-1625Crossref PubMed Scopus (266) Google Scholar, 32.Windhager L. et al.Ultrashort and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution.Genome Res. 2012; 22: 2031-2042Crossref PubMed Scopus (85) Google Scholar, 33.Neugebauer K.M. Nascent RNA and the coordination of splicing with transcription.Cold Spring Harb. Perspect. Biol. 2019; 11a032227Crossref PubMed Scopus (11) Google Scholar]. Moreover, single-molecule live imaging in human U2-OS and HEK 293 cells revealed that the time needed for removal of β-globin introns, after transcription has begun, was extremely short, in the order of tens of seconds [34.Martin R.M. et al.Live-cell visualization of pre-mRNA splicing with single-molecule sensitivity.Cell Rep. 2013; 4: 1144-1155Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar,35.Coulon A. et al.Kinetic competition during the transcription cycle results in stochastic RNA processing.eLife. 2014; 3e03939Crossref Google Scholar]. This was in stark contrast to the speed determined earlier from in vitro splicing reactions that could reach up to 45 min [36.Krainer A.R. et al.Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro.Cell. 1984; 36: 993-1005Abstract Full Text PDF PubMed Scopus (0) Google Scholar]. Collectively, these studies indicate that the process of transcription is strongly linked to splicing and it significantly increases its efficiency. More recent work allowing global determination of nascent RNA sequences relative to the progression of RNA polymerase II (RNAP II) on the transcript found that splicing is complete, nearly immediately, upon synthesis of the introns in yeast [37.Oesterreich F.C. et al.Splicing of nascent RNA coincides with intron exit from RNA polymerase II.Cell. 2016; 165: 372-381Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar,38.Herzel L. et al.Long-read sequencing of nascent RNA reveals coupling among RNA processing events.Genome Res. 2018; 28: 1008-1019Crossref PubMed Scopus (26) Google Scholar], while the precise timing is still under debate in mammals [39.Nojima T. et al.RNA polymerase II phosphorylated on CTD serine 5 interacts with the spliceosome during co-transcriptional splicing.Mol. Cell. 2018; 72 (e4): 369-379Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 40.Drexler H.L. et al.Splicing kinetics and coordination revealed by direct nascent RNA sequencing through nanopores.Mol. Cell. 2020; 77 (e8): 985-998Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 41.Reimer K.A. et al.Rapid and efficient co-transcriptional splicing enhances mammalian gene expression.bioRxiv. 2020; (Published online February 12, 2020. https://doi.org/10.1101/2020.02.11.944595)Google Scholar]. Since the rate of transcription can modulate splicing (see later), it is likely that in most cases the spliceosome assembles soon after introns are transcribed (splicing commitment), while the removal of introns may occur immediately or at a later time, depending on splice site strength and flanking cis-regulatory sequences. Two nonexclusive models regarding the impact of transcription on splicing have been proposed over the past decades [42.Bentley D.L. Coupling mRNA processing with transcription in time and space.Nat. Rev. Genet. 2014; 15: 163-175Crossref PubMed Scopus (373) Google Scholar]. First, the so-called recruitment model involved the ability of the C-terminal domain (CTD) of the large RNAP II subunit to recruit a wide range of RBPs to nascent transcripts, thereby influencing intron removal. Notably, this recruitment is modulated by the phosphorylation state of the CTD that evolves during the different stages of transcription, allowing sequential binding of RBPs as RNAP II progresses along the gene body. In particular, the phosphorylation of the CTD at the Ser5 position is strongly associated with intermediates of the splicing reaction and splicing factors, suggesting that this post-translational modification plays a key role in the splicing process [39.Nojima T. et al.RNA polymerase II phosphorylated on CTD serine 5 interacts with the spliceosome during co-transcriptional splicing.Mol. 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It is therefore conceivable that via this phase-separation property, the CTD mediates the assembly of RNAP II with splicing factors into local higher-order complexes, thereby optimizing the efficiency of the splicing reaction. The recruitment of RBPs can also occur via alternative roads, which are linked to the gene environment, through binding to specific histone marks or transcription elongation factors. For instance, trimethylation of H3K36 can induce exon skipping in human mesenchymal stem cells (hMSCs) by recruiting the splicing factor polypyrimidine tract-binding protein (PTB) via the adaptor MRG15 [46.Luco R.F. et al.Regulation of alternative splicing by histone modifications.Science. 2010; 327: 996-1000Crossref PubMed Scopus (677) Google Scholar] (Figure 2A ). Other RBPs, including the splicing regulator serine and arginine-rich splicing factor 1 (SRSF1) can also be recruited via binding of another adaptor, Rsip1, to the same mark [47.Pradeepa M.M. et al.Psip1/Ledgf p52 binds methylated histone H3K36 and splicing factors and contributes to the regulation of alternative splicing.PLoS Genet. 2012; 8e1002717Crossref PubMed Scopus (201) Google Scholar] (Figure 2A). RNAP II can influence splicing via a second mechanism referred to as kinetic coupling. According to the model, changes of elongation rates can regulate splicing by modulating the presence of competing splicing sites. Slow transcriptional rate has been traditionally associated with exon inclusion due to the absence of downstream competing splice sites, while a faster rate promotes exon exclusion since more favorable splicing sites may become available as the nascent pre-mRNA is being transcribed [48.de la Mata M. et al.A slow RNA polymerase II affects alternative splicing in vivo.Mol. 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Stimulatory effect of splicing factors on transcriptional elongation.Nature. 2001; 414: 929-933Crossref PubMed Scopus (261) Google Scholar]. For instance, in mouse embryonic fibroblasts (MEFs), the recruitment of the splicing factor SRSF2 to nascent transcripts promotes the release of RNAP II at promoters, which ultimately may result in increased elongation rate [53.Ji X. et al.SR proteins collaborate with 7SK and promoter-associated nascent RNA to release paused polymerase.Cell. 2013; 153: 855-868Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar] (Figure 2B). Conversely, in Drosophila, the binding of the exon junction complex (EJC) to nascent RNAs augments RNAP II pausing at promoters, which indirectly impacts splice site choice [54.Akhtar J. et al.Promoter-proximal pausing mediated by the exon junction complex regulates splicing.Nat. Commun. 2019; 10: 521Crossref PubMed Scopus (5) Google Scholar] (Figure 2B). Mechanistically, SRSF2 exerts this function through recruitment of the positive transcription elongation factor b (P-TEFb) complex to gene loci, while the EJC has an opposite activity by restricting its binding. Whether these functions are constitutive or regulated awaits future investigations. Of note, every factor or process that affects P-TEFb activity is a potential regulator of splicing. For instance, the 7SK complex, which sequesters P-TEFb in an inactive form, has also been suggested to influence splicing through modulation of transcription elongation rates [55.Barboric M. et al.7SK snRNP/P-TEFb couples transcription elongation with alternative splicing and is essential for vertebrate development.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 7798-7803Crossref PubMed Scopus (113) Google Scholar] (Figure 2B). In addition to P-TEFb activity, elongation rate can be affected through additional means. For instance, nucleosomes can represent a physical obstacle that impedes the progression of RNAP II through the gene body [56.Hodges C. et al.Nucleosomal fluctuations govern the transcription dynamics of RNA polymerase II.Science. 2009; 325: 626-628Crossref PubMed Scopus (246) Google Scholar]. Since nucleosomes are globally enriched over exons versus introns, it is suggested that they could aid in the recognition of exons by stalling the polymerase [57.Schwartz S. et al.Chromatin organization marks exon-intron structure.Nat. Struct. Mol. Biol. 2009; 16: 990-995Crossref PubMed Scopus (414) Google Scholar, 58.Spies N. et al.Biased chromatin signatures around polyadenylation sites and exons.Mol. Cell. 2009; 36: 245-254Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 59.Tilgner H. et al.Nucleosome positioning as a determinant of exon recognition.Nat. Struct. Mol. Biol. 2009; 16: 996-1001Crossref PubMed Scopus (310) Google Scholar] (Figure 2C). Following the same logic, nucleosome remodeling complexes can impact splicing by altering the compaction of the chromatin [60.Batsche E. et al.The human SWI/SNF subunit Brm is a regulator of alternative splicing.Nat. Struct. Mol. Biol. 2006; 13: 22-29Crossref PubMed Scopus (0) Google Scholar], and the same applies to DNA methylation within the gene body that can alter nucleosome positioning as well as histone modification locally [61.Lorincz M.C. et al.Intragenic DNA methylation alters chromatin structure and elongation efficiency in mammalian cells.Nat. Struct. Mol. Biol. 2004; 11: 1068-1075Crossref PubMed Scopus (348) Google Scholar,62.Chodavarapu R.K. et al.Relationship between nucleosome positioning and DNA methylation.Nature. 2010; 466: 388-392Crossref PubMed Scopus (505) Google Scholar]. 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From all these examples, it is obvious that pre-mRNA splicing is not an isolated event and is intricately linked to the transcriptional and epigenetic environment of a given gene. Ultimately, the final splice site choice will be determined by the combined activity of multiple factors. A new layer of splicing regulation has been uncovered recently by the characterization of internal chemical modifications in eukaryotic mRNA that can alter the property of cis-regulatory sequences and thereby impact pre-mRNA splicing. So far, 13 distinct modifications have been described on mRNA, yet the most widespread and undisputed one is N6-methyladenosine (m6A) [66.Anreiter I. et al.New twists in detecting mRNA modification dynamics.Trends Biotechnol. 2020; (Published online July 1, 2020. https://doi.org/10.1016/j.tibtech.2020.06.002)Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. m6A is deposited cotranscriptionally by a dedicated methyltransferase complex with core components methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) [67.Knuckles P. et al.RNA fate determination through cotranscriptional adenosine methylation and microprocessor binding.Nat. Struct. Mol. Biol. 2017; 24: 561-569Crossref PubMed Scopus (59) Google Scholar, 68.Zaccara S. et al.Reading, writing and erasing mRNA methylation.Nat. Rev. Mol. 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