DNA methylation, resulting in 5’ methylation of cytosine (5mC), is a conserved and heritable DNA modification that affects gene expression in a genome-wide manner (Li and Zhang, 2014). The impact of DNA methylation on gene expression varies depending on its genomic contexts. The role of promoter DNA methylation in gene expression has been well investigated, which is widely believed to cause transcriptional inhibition of downstream genes (Law and Jacobsen, 2010). Interestingly, recent studies in model plant Arabidopsis revealed that two SU(VAR)3–9 homologs, SUVH1 and SUVH3, bind to methylated DNA and recruit the DNAJ proteins to enhance proximal gene expression, thereby counteracting the repressive effects of transposon insertion near genes (Harris et al., 2018; Xiao et al., 2019; Zhao et al., 2019). Compared to DNA methylation in promoter regions, the function of genic DNA methylation remains elusive (Ball et al., 2009). During the last decade, several studies indicate that genic DNA methylation has a positive effect on the expression of associated genes and prevents spurious transcription initiation, and it is present within a number of cancer-related genes and has been regarded as a hallmark of human cancer (Baylin and Jones, 2011; Yang et al., 2014; Neri et al., 2017).

Recent studies reveal a strong correlation between DNA methylation and alternative splicing. Yang et al. (2014) showed that gene body DNA demethylation mediated by DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine results in reduced efficiencies of transcription elongation or splicing. In human cells, Shukla et al. (2011) reported that a DNA-binding protein, called CCCTC-binding factor (CTCF), can promote inclusion of weak upstream exons by mediating local RNA polymerase II pausing. In this case, DNA methylation inhibits CTCF binding to CD45 exon 5, which enables Pol II to transcribe more rapidly, giving rise to an exon 5 exclusion (Ong and Corces, 2014). More recently, Nanavaty et al. (2020) further revealed that CTCF is a bifunctional regulator which influences both alternative splicing and alternative polyadenylation. Removal of DNA methylation enables CTCF binding and recruitment of the cohesin complex, which in turn form chromatin loops to promote proximal polyadenylation site usage. These works clearly demonstrate that DNA methylation has an important participation in RNA processing regulation. While, limited information is currently available regarding how DNA binding proteins disturb the elongation of Pol II. It reminded us that there maybe are other factors influencing Pol II elongation in CTCF-mediated AS regulation, like the cohesin complex.

Unlike CTCF protein which binds to unmethylated DNA, a growing number of studies have shown that the methyl cytosine-guanine dinucleotide (CpG) binding protein 2 (MeCP2) binds to methylated regions to influence AS. MeCP2 is the earliest reported multifunctional protein that contains both methyl-CpG-binding domains and transcriptional repressor domains (Nan et al., 1997). Acting as a chromatin adaptor, MeCP2 is attracted to 5mC on alternative exons, triggering its interaction with histone deacetylases (HDACs), which modulate alternative splicing (Maunakea et al., 2013). As we delve deeper into the function of MeCP2, it is becoming clear that MeCP2 recruiting splicing factors to regulate mRNA splicing is also a nearly ubiquitous mechanism in animals (Cheng et al., 2017; Wong et al., 2017).

In plants, the available information regarding whether gene body DNA methylation affects AS and the extent of this mediation is currently limited. The first study of DNA methylation-related functions in splicing was reported in maize (Regulski et al., 2013). More recently, the cytosine methyltransferase OsMET1 was found to affect global AS events in rice, in which a total of 6319 more events were identified with the met1 mutant compared with those associated with the wild-type strain (Wang et al., 2016). However, deeper research combining DNA methylation and AS/APA in plant is lacking. Whether it has the similar regulatory mechanism with mammals needs to be further elucidated.

Chromatin structure is dominated by nucleosome density and positioning, as well as by histone modifications and DNA methylation (Duan et al., 2018). In contrast to DNA methylation, more than 50 diverse modifications have been identified on histone tails. Different modifications are linked with distinct functions, such as transcriptional activation or inhibition (Henikoff and Shilatifard, 2011). Recent reports indicate that histone modifications are also involved in the regulation of RNA processing. In fact, the involvement of histone modification in regulation of RNA processing was found earlier than DNA methylation (Luco et al., 2010). Similar to DNA methylation, absence of histone marks results in chromatin structure changes, immediately affecting Pol II elongation and alternative RNA processing.

Histone H3 lysine 36 trimethylation (H3K36me3) mark is an active mark and is abundant in actively transcribed gene bodies (Liu et al., 2010). It has been shown that dysfunction of SETD2, an H3K36me3 methyltransferase, induced changes in 186 AS events (Yuan et al., 2017). In humans, the MORF-related gene on chromosome 15 (MRG15) is a well-established model system to study the interplay between histone modifications and the splicing machinery. The H3K36me3 mark influences splicing by impacting the recruitment of splicing regulators through a chromatin-binding protein, that is, MRG15. In this mechanism, the H3K36me3 mark serves as anchors for MRG15 binding, which in turn recruits the splicing regulator polypyrimidine tract-binding (PTB) to pre-mRNA (Figure 1A). The H3K36me3–MRG15–PTB complex forms a chromatin-splicing adaptor system regulating numerous splicing events, including FGFR2 splicing, which is essential for tumor growth and invasion of lung cancer (Sanidas et al., 2014; Naftelberg et al., 2015).

In contrast to the H3K36me3–MRG15–PTB complex which favors exclusion of alternative exons, diverse histone modifications can lead to a diametrically opposite splicing pattern. Heterochromatin protein 1 (HP1), which has three isoforms in humans, HP1α, HP1β, and HP1γ, binds directly to histone H3 lysine 9 trimethylation (H3K9me3; Bannister et al., 2001). A previous study indicated that HP1γ forms an additional link with chromatin, binding to the coding region where it associates with pre-mRNA and favoring its transient retention on chromatin. The modification to the chromatin structures of the CD44 gene slows the elongation rate of Pol II, which in turn facilitates the recruitment of splicing factors such as U2AF65 and PRP8 to alternative exons, resulting in the inclusion of alternative exons (Saint-André et al., 2011; Yearim et al., 2015). Unsurprisingly, diverse adaptor proteins at H3K9me3 lead to distinct splicing patterns. HP1α and HP1β bind to methylated alternative exons and recruit the splicing factor serine/arginine-rich splicing factor 3 (SRSF3), thus enhancing the role of as a splicing silencer and reducing the number of induced alternative exons (Yearim et al., 2015).

In plants, Arabidopsis encodes two homologs of human MRG15, MRG1 and MRG2, which bind to H3K4me3/H3K36m3-modifying histone marks and trigger temperature-induced flowering via the florigen gene FT (Bu et al., 2014). However, it seems like that MRG1/2 have diversified from their animal homologs during evolution, yet they still maintain their conserved H3K36me3-binding molecular function (Xu et al., 2014; An et al., 2020; Guo et al., 2020). Recently, a protein complex in Arabidopsis, called anti-silencing 1 (ASI1)-ASI1 immunoprecipitated protein 1 (AIPP1)-enhanced downy mildew 2 (EDM2) (AAE) complex, was identified targeting genic heterochromatic elements to regulate APA (Duan et al., 2017). In this complex, ASI1, also named IBM2 and SG1 (Saze et al., 2013; Coustham et al., 2014), is a plant-specific chromatin regulator which bears chromatin- and RNA-binding capacity through its bromo-adjacent homology (BAH) and RNA recognition motif (RRM) domains, respectively (Wang et al., 2013). EDM2 is a multifunctional chromatin regulator containing two and half plant homeodomains (PHDs). Its PHD fingers have the binding capacity of H3K9me2 and other histone modifications (Lei et al., 2014). ASI1 and EDM2 associate in vivo through an RRM motif-containing bridge protein AIPP1 (also named EDM3; Duan et al., 2017). The AAE complex can bind to intronic heterochromatin, most of which come from insertions of epigenetically silenced transposable and repetitive elements, promoting the usage of distal polyadenylation site (Figure 1B). Dysfunctions of the AAE complex lead to ectopic accumulations of proximally polyadenylated short transcripts. Thus, the AAE complex is indispensable for the generation of full-length transcripts of genic heterochromatin-containing genes. Regarding the underlying mechanism, recent report indicates that EDM2 and AIPP1 mutations can slow down Pol II elongation rate at proximal polyadenylation site, leading to a promotion of proximal polyadenylation site usage (Lai et al., 2019). AAE complex-mediated polyadenylation regulation plays an important role in multiple biological processes, including modulating plant immunity by targeting innate immunity receptor gene RPP7 (Tsuchiya and Eulgem, 2013), epigenome regulation by targeting histone H3K9me2 demethylase gene IBM1, and T-DNA suppression (Saze et al., 2013; Wang et al., 2013). Similar mechanism may also exist in other plants, like bamboo and oil palm (Wang et al., 2017). For example, in oil palm, loss of Karma transposon methylation leads to ectopic splicing of the homeotic gene DEFICIENS, which accounts for the mantled soma clonal variant phenotype of oil palm (Ong-Abdullah et al., 2015). Interestingly, recent study indicates that FPA, a flowering time regulator in Arabidopsis, can antagonize ASI1 in the selection of polyadenylation site. In the double mutant of asi1 and fpa, the polyadenylation pattern phenocopies fpa but not asi1. While, this antagonistic control only occurs in specific target genes, indicating a complex regulation of AAE complex-mediated polyadenylation (Deremetz et al., 2019).

Nucleosome, consisting of 147-bp double-stranded DNA and a single histone octamer, is the basic unit of chromatin. Histone variants, which are transcribed from separate genes, have been shown playing key roles in the regulation of chromatin features. This finding reminds us that histone variants may also regulate co-transcriptional RNA processing. In mammals, five somatic H1 variants (H1.1 to H1.5) have been identified (Happel and Doenecke, 2009). More recently, Glaich et al. (2019) reported that H1.5 deposition is observed at the splicing sites of the short exons in human lung fibroblasts (IMR90 cells), and Pol II on H1.5-marked exons exhibits greater stalling than it does on unmarked exons. Deletion of H1.5 affects the inclusion of short exons with relatively long introns and reduces Pol II occupancy on these exons (Glaich et al., 2019). This finding clearly indicates that the linker histones participate in the regulation of alternative RNA processing, which has not been previously demonstrated (Glaich et al., 2019).

In addition to histone variants, chromatin remodeling factors also affect chromosome segregation and transcription (Clapier and Cairns, 2009). During the last two decades, a growing number of studies have indicated that chromatin remodeling factors also play a role in alternative splicing. Brahma (BRM), the core adenosine triphosphatase (ATPase) subunit of the switch/sucrose nonfermenting (SWI/SNF) chromatin-remodeling complex, was firstly shown to facilitate the inclusion of alternative exons by interacting with Pol II to induce its stalling (Figure 1A; Batsché et al., 2006; Jancewicz et al., 2019). Actually, chromatin remodeler mediated-regulation of AS is an evolutionarily conserved mechanism across most species, such as in maize. ZmCHB101, a SWI3D protein, has been shown controlling AS by altering chromatin status and transcriptional elongation rates under osmotic stress (Yu et al., 2019), although the mechanism by which chromatin remodeling factors interact with Pol II transcription to impact mRNA processing machinery remains unclear.

It is assumed that most snoRNAs, nearly 70 nt in length, are derived from excised introns through exonucleolytic processing (Watkins and Bohnsack, 2012). There are hundreds of different snoRNAs in vertebrates and have even been found in archaea (Terns and Terns, 2002). The first evidence of the participation of snoRNA in AS was snoRNA HBII-52, which regulates the serotonin receptor 2C and is associated with the congenital disease Prader–Willi syndrome (PWS). HBII-52 regulates AS of 5-HT_2CR by binding to a silencing element in exon Vb. PWS patients do not express HBII-52. They have different 5-HT_2CR messenger RNA (mRNA) isoforms than healthy individuals (Kishore and Stamm, 2006). Recently, a class of intronic lncRNAs named snoRNA-related lncRNAs (sno-lncRNAs) was identified in humans. The sno-lncRNAs generated from the PWS region associate strongly with Fox family splicing regulators, altering serotonin receptor 5-HT_2CR splicing (Figure 2A). In patients with PWS, the expression of some specific sno-lncRNAs is downregulated. As a result, these patients have different 5-HT_2CR mRNA isoforms than healthy individuals, which have been identified during early embryonic development and adulthood (Yin et al., 2012).

Almost all eukaryotic pre-mRNAs and many ncRNAs are subject to cleavage/polyadenylation at the 3′ end, which takes place in macromolecular machinery called the mRNA 3′-processing complex (Tian and Manley, 2016). It has been shown that snoRNAs, which are classified as trans-acting RNAs, directly interact with Fip1, a component of the cleavage and polyadenylation specificity factor (CPSF) complex. Small Nucleolar RNA C.D Box 50A (SNORD50A), a U/A-rich C/D-box snoRNA, inhibits mRNA 3 processing by disturbing the Fip1-poly(A) site (PAS) interaction (Figure 2B). SNORD50A depletion leads to more frequent binding of Fip1 to PAS and increases the 3′ processing of target mRNAs containing U-rich sequences (Huang et al., 2017; Shi et al., 2017). Taken together, these studies strongly suggest that snoRNA is an important regulator of polyadenylation for specific genes by serving as an antagonistic RNA. An important question remains for future studies to address: how do ncRNAs bind to neighboring sequences and regulate the interactions between the core mRNA processing factors and processing sites?

Recently, lncRNAs have received increasing attention. In human, Metastasis-associated lung adenocarcinoma transcript 1 (Malat1) is the most widely studied lncRNA. Malat1 was first identified in human non-small cell lung cancer (NSCLC; Ji et al., 2003). A number of serine/arginine-rich (SR) proteins, including SRSF1, SRSF2, and SRSF3, associate with Malat1. Absence of Malat1 affects the localization of some splicing factors in the HeLa cell line and leads to changes AS pattern (Blencowe, 2006). However, the loss of Malat1 in normal mice rarely causes global changes in splicing factor levels and results only in the dysregulation of small mRNAs (Zhang et al., 2012). Meanwhile, deletion of Malat1 in mammary carcinoma mice leads to many AS events in genes essential for cell differentiation and tumorigenesis (Arun et al., 2016). It can therefore be proposed that Malat1 regulates AS in specific cells and tissues under particular conditions. In human cells, the lncRNA Gomafu, which is dynamically regulated by neuronal activation, directly binds to the splicing factors QKI and SRSF1 and inhibits their association with the schizophrenia disease-related gene transcripts, thereby affecting alternative splicing (Barry et al., 2014). In Arabidopsis, an lncRNA called alternative splicing competitor (ASCO) binds to the highly conserved spliceosome component PRP8a, thereby impairing the recognition of specific flagellin-related transcripts by PRP8a (Rigo et al., 2020). Actually, it has been shown that ASCO can binds to multiple splicing factors, indicating that lncRNAs may integrate a dynamic network to modulate transcriptome reprogramming, including alternative splicing.

In addition to the evidence we discussed above, some ncRNAs are directly or indirectly involved in RNA processing. It has been shown that piRNAs and piRNA biogenesis components affect mRNA splicing of P-transposable element transcripts in vivo, resulting in the production of a non-transposase-encoding mature mRNA isoform in Drosophila germ cells (Teixeira et al., 2017). In plants, there is a special family of ncRNAs that can confer de novo DNA methylation through the RNA-directed DNA methylation (RdDM) pathway, and thereby inducing global AS/APA events (Matzke and Mosher, 2014; Wang and Chekanova, 2016). As the non-coding transcriptome, ncRNAs are important components of the eukaryotic genome. There may be a large number of mechanisms by which ncRNAs enhance the plasticity of the proteome by interacting with mRNA-processing machinery. A deep understanding of this mechanism will open up broad prospects for gene therapy of various diseases, including cancer, and the application of biotechnology in agricultural and human health fields.