Introduction

The inconsistency between adenovirus messenger RNA (mRNA) and its DNA transcription template has allowed researchers to understand that the genetic information transmission from DNA to RNA needs to undergo the removal of invalid information and the splicing of valid information. And such processes are called alternative splicing (AS) that enriches the repertoire of both transcriptome and proteome. It is estimated that 95% of human genes express more than one transcript isoform. Abnormal AS has been proven to be related to the onset and development of infectious diseases.

Infectious diseases are one kind of the most formidable and ubiquitous threats to public health worldwide. In the 21st century, human immunodeficiency virus (HIV) infection, malaria, and tuberculosis (TB) remain the three deadliest infectious diseases, while the outbreak of emerging infections such as coronavirus disease 2019, hits the global public health system hard and causes a devastating influence on lives. Decoding the pathogen–host interactions is critical to control the spread of infectious diseases. Remodeling at the transcriptomic level is considered the main way that pathogens and hosts fight each other, and AS has been confirmed as a fast and convenient approach to realize such remodeling. The importance of many AS events in pathogen–host interactions has been documented.

In this review, we focused on AS alternations from the perspective of pathogens, the hosts, and their interactions. We first compared the similarities and differences between pathogen and host splicing mechanisms. Then, we collated all reported regulators and discussed the potential regulatory mechanisms in infectious diseases. We also summarized aberrant AS events in infectious diseases and illustrated their biological effects. Finally, targeted drugs were also described. This work aimed to decode the host–pathogen interactions from the angle of AS, and guide the development of targeted therapies to a certain extent.

Occurrence of AS

AS can be divided into different types including exon skipping, intron retention, alternative 3′ splice site (3′SS), alternative 5′SS, and mutually exclusive exon, as well as alternative polyadenylation and exitron [Figure 1]. Regardless of splicing type, the essence of AS is a two-step SN2-type transesterification reaction.[1] First, the 2′ hydroxyl group of the branch point (BP) adenine in the midstream and downstream sequence of the intron attacks its 5′SS, to generate a lariat structure. Then, the exposed 3′-OH at the 3′ end of the upstream exon attacks the 3′SS of the intron, then the upstream and downstream exons are ligated, and the lariat intron is released [Figure 2A].

Figure 1:

Different types of alternative splicing. (A) Five major types of AS. (B) Schematic diagram of exitron splicing. (C) Schematic diagram of alternative polyadenylation splicing. (D) Four types of alternative polyadenylation splicing. APA: Alternative polyadenylation; AS: Alternative splicing; CDS: Coding sequence; mRNAs: Messenger RNAs; UTR: Untranslated region.

Figure 2:

Splicing reactions of cis-splicing and trans-splicing. (A) The transesterification reactions in cis-splicing. (B) Schematic diagram of genic trans-splicing. Genic trans-splicing can occur among pre-mRNAs from different genes (top right) or the same genes (middle right and bottom right). (C) Schematic diagram of SL trans-splicing. SL exons with caps from SL RNAs are ligated to different structural genes, generating mature mRNA with the same 5′ end. BPS: Branch point site; mRNAs: Messenger RNAs; SL: Spliced leader; SS: Splice site.

In eukaryotes, the above reactions are catalyzed by a spliceosome. The spliceosomes of most fungi are similar to those of mammals, while the spliceosomes of parasites behave quite differently from those of mammals. Viruses and bacteria realize their splicing in a host-dependent manner, that is, utilizing the splicing machinery of their hosts. In the following part, we focus on the spliceosome behaviors in infectious diseases, to offer more mechanistic insights.

Molecular mechanism of AS in mammals

Spliceosome is a massive ribonucleoprotein (RNP) complex, including nearly 100 distinct proteins and five small nuclear RNPs (snRNPs). These snRNPs are composed of corresponding uridine-rich small nuclear RNA (snRNA) and some specific proteins. All snRNAs except U6 snRNA are first exported to the cytoplasm and bound to a series of heptameric Sm proteins to complete assembly, and then the generated snRNPs are transported to the nucleus to perform their respective roles.[2] U6 snRNA directly combines with the Lsm complex in the nucleus to form U6 snRNP,[2] a process that does not involve changes in subcellular localization. Spliceosomes are further classified into two main types according to their composition: U2- and U12-type. U2-type spliceosome splices most introns by recognizing the sequence of “CURACU”[3] while U12-type spliceosome is only responsible for the removal of <0.5% introns by recognizing the 5′SS and 3′SS (AU-AC or GU-AG).[4]

Each splicing step performed by the spliceosome is accompanied by repeated dissociation and assembly. In each splicing reaction, the spliceosome undergoes the sequential state transformation from early complex (E), pre-spliceosome (A), pre-B complex, pre-catalytic spliceosome (B), activated spliceosome (Bact), catalytically activated spliceosome (B∗), step I complex (C), step II activated complex (C∗), post-catalytic complex (P) to intron lariat spliceosome (ILS).[5] First, U1 snRNP recognizes the 5′SS through the complementary base pairing, and then E complex is generated. Non-snRNP factors such as splicing factor 1 (SF1) and U2 auxiliary factor bind to the BP and 3′SS, respectively. U2 snRNP replaces SF1 through base complementary pairing, and aggregates at the BP, yielding A complex. Next, U4, U6, and U5 snRNPs come together to form a triple snRNPs that interact with A complex and thus urge the formation of pre-B complex. Subsequently, U1 snRNP dissociates, exposed 5′SS transfers to U6 snRNP, and U4 snRNP is unwound. With such conformational transitions, B, Bact, and B∗ complexes are formed in turn. The first transesterification reaction occurs, and C and C∗ complexes are generated. Then, exons are ligated by U5 snRNA and a P complex generates. As the mature mRNA is released, the ILS complex is formed. After the complex dissociation, released snRNPs proceed to the next round [Figure 3].

Figure 3:

Schematic diagram of splicing processes conducted by spliceosome in mammals. Complex A: Pre-spliceosome complex; Complex B∗: Catalytically activated spliceosome complex; Complex B: Pre-catalytic spliceosome complex; Complex Bact: Activated spliceosome complex; Complex C∗: Step II activated complex; Complex C: Step I complex; Complex E: Early complex; Complex P: Post-catalytic complex; ILS: Intron lariat spliceosome; mRNAs: Messenger RNAs; snRNP: Small nuclear ribonucleoprotein; SS: Splice site.

Molecular mechanism of AS in pathogens

High conservation between fungal and host spliceosomes, and limited evidence of bacterial splicing mechanism lead us to focus on the molecular splicing mechanism of parasites and viruses.

Parasites

Parasites can perform splicing by ligating exons from two separate pre-mRNAs (known as trans-splicing). In essence, trans-splicing is also a two-step transesterification reaction; however, it does not generate the lariat in the first step of cis-splicing but rather a Y structure intermediate.[6] There are two kinds of trans-splicing: genic trans-splicing and spliced leader (SL) trans-splicing [Figures 2B and 2C]. The former ligates exons from different pre-mRNAs of the same gene, from transcripts of different genes or intergenic regions, or from transcript products of different chromosomes.[7] Genic trans-splicing is observed in Drosophila, Caenorhabditis elegans, and mosquitoes.[7] The later connects the SL exon of SL RNA to various pre-mRNAs, and then generates mature mRNAs with the same common sequence on the 5′ end.[8] In contrast to snRNAs, SL RNAs dedicate SL exons to transcripts, triggering the consumption of themselves, while snRNAs are relatively constant and circulate in splicing rounds. Such splicing is reported in Euglenozoa, Dinoflagellates, and Ctenophores.[7]

Viruses

Human immunodeficiency virus-1 (HIV-1) has three types of transcripts: the un-spliced (US), partially spliced (PS), and multiply spliced (MS) transcripts. After the invasion, HIV-1 RNA is reverse-transcribed and integrated into the host genome. Through utilizing host RNA polymerase II (RNAPII), integrated HIV-1 provirus generates a single full-length RNA,[9] and initially, only MS transcripts are produced to encode the regulatory proteins. Then, PS and MS transcripts increase and produce the structural and accessory proteins. To improve the splicing efficiency, HIV-1 occupies multiple alternative 5′SS and 3′SS to disturb the normal recognition of spliceosome and thus cleaves at GT and AG dinucleotides to produce viral spliced mRNAs.[10] HIV-1 also employs its own cis-acting elements and host trans-acting elements to regulate splicing, thereby modulating viral protein ratio and infectivity.[11]

Human papillomavirus (HPV) has an approximately 8 kb DNA sequence that includes an early transcription region (E), a late region, and a long control region. In HPV-16, transcription initiates from an early promoter located at P97, and polycistronic mRNAs that encode E6 and E7 E1, E2, E8 E4, and E5 are formed.[12] The E6E7 region is the most frequently spliced region and more than four potential isoforms have been found. Splice sites in the E6 open reading frames lead to the generation of a full-length E6 and several truncated E6∗ isoforms.[13] The splicing within E6 also regulates the generation of E7 mRNA due to the close position between E6 and E7.[14,15] The splicing in the E1E2 region is also discovered.[16] It is a pity that rare evidence reveals the molecular mechanism by which HPV takes advantage of the host splicing system. It is established that HPV-16 can drive host trans-acting factors to regulate its splicing.[17]

Regulation of AS

Splicing regulators are shown in Figure 4 and relevant evidence is presented in Table 1.

Figure 4:

Regulator mechanism of alternative splicing. A: Adenine; ESE: Exon splicing enhancer; ESS: Exon splicing silencer; G: Guanine; hnRNP: Heterogeneous nuclear ribonucleoproteins; ISE: Intron splicing enhancer; ISS: Intron splicing silencer; m6A: N6 position of adenosine; mRNA: Messenger RNA; RNA Pol II: RNA polymerase II; snRNA: Small nuclear RNA; snRNP: Small nuclear ribonucleoprotein; SRSF: Serine- and arginine-rich splicing factor; U: Uracil.

Table 1 - Infectious disease-related splicing regulators. Regulatory factors Pathogen Pathogen component Host target Action pattern∗ Biological effect∗ Reference Spliceosome  SnRNP HSV-1 ICP27 SnRNPs Changing cellular localization Inhibiting host splicing [19]  SnRNA IAV NS1 U6 Inhibiting the formation of U6-U2 and U6-U4 complexes Inhibiting host splicing [72]  SnRNP-related proteins HIV-1 Vpr SAP145 Inhibiting the formation of SAP145-SAP49 complex Inhibiting the spliceosome assembly and host splicing [73] MRV μ2 EFTUD2, PRPF8, and SnRNP200 Inhibiting expression Altering the splicing of specific genes [18] Trans-acting factors  SRSFs and related proteins HBV NA SRSF2 Inhibiting expression Promoting the splicing of viral pre-genomic RNA [24] Promoting the generation of viral HpZ/P′ HIV-1 NA SC35 and 9G8 Promoting expression Promoting splicing at HIV-1 A3 site [25] NA SRSF1 Promoting expression Promoting the splicing at viral A1 and A2 site [25] Tat protein SRSF2 Promoting phosphorylation Promoting TAU-exon10 skipping [74] HSV-1 ICP27 SRSF2 Changing cellular localization Inhibiting host pre-mRNA splicing [19] MTB NA SRSF2 and SRSF3 Inhibiting expression Promoting IL-4-exon2 skipping [26] Inhibiting TLR4-exon2 or 3 skipping Reovirus μ2 protein SRSF2 Changing cellular localization Changing host splicing [27] Promoting viral replication  hnRNPs EBV EBER1 AUF1 and hnRNP D Inhibiting the binding of AUF1 to AU-rich elements Changing host splicing [75] IAV NS1 hnRNP K Changing cellular localization Changing host splicing; [33] Promoting viral replication  Other RBPs DV NS5 RBM10 Promoting degradation Increasing SAT1-exon4 inclusion [36] Promoting viral replication Co-transcriptional regulation HCMV NA RNAPII Promoting phosphorylation Changing the splicing of viral RNAs [38] Changing cellular localization Epigenetic modifications  DNA level Escherichia coli NA Host genes Decreasing the methylation level Promoting LGR4-exon5 skipping [39]  RNA level Adenovirus NA m6A writing enzymes Changing cellular localization Increasing m6A level of viral mRNA [76] Increasing the splicing efficiency within adenovirus late transcriptional units DV
Zika virus
WNV
HCV NA CIRBP Decreasing the m6A level Decreasing the intron retention of CIRBP [40]  Protein level† HPV-31 E7 SETD2 Increasing the H3K36me3 level Regulating splice site selection and the generation of viral L1 RNAs [41] RNA structures HIV-1 NA NA Promoting the generation of the SL2 Promoting the exon 6D inclusion of viral mRNA [42] Phase separation EBV EBNA2 SRSF1 and SRSF7 Forming phase-separated droplets Promoting MPPE1-exon11 skipping [45]

∗Researches that do not provide detailed action patterns or biological effects are not listed.

†Some relevant evidence was listed in the part of “Trans-acting factors”.AU: Adenine and uracil; AUF1: AU-rich element binding factor 1; CIRBP: Cold-inducible RNA binding protein; DV: Dengue virus; EBER1: Epstein-Barr virus-encoded RNA 1; EBNA2: Epstein-Barr virus-encoded nuclear antigen 2; EBV: Epstein–Barr virus; EFTUD2: Elongation factor Tu GTP binding domain containing 2; HBV: Hepatitis B virus; HCMV: Human cytomegalovirus; HCV: Hepatitis C virus; HIV-1: Human immunodeficiency virus-1; HPV-31: Human papillomavirus 31; hnRNP D: Heterogeneous nuclear ribonucleoprotein D; hnRNPs: Heterogeneous nuclear ribonucleoproteins; HSV-1: Herpes simplex virus-1; IAV: Influenza A virus; IL-4: Interleukin 4; LGR4: Leucine rich repeat containing G protein-coupled receptor 4; MPPE1: Metallophosphoesterase 1; mRNA: Messenger RNA; m6A: N6 position of adenosine; MRV: Mammalian orthoreovirus; MTB: Mycobacterium tuberculosis; NA: Not available; NS: Nonstructural protein; PRPF8: Pre-mRNA processing factor 8; RBM10: RNA binding motif protein 10; RBPs: RNA-binding proteins; RNAPII: RNA polymerase II; RNP: Ribonucleoprotein; SAP145-SAP49: Splicing-associated protein 145-splicing-associated protein 49; SAT1: Spermidine/spermine N1-acetyltransferase 1; SETD2: Histone methyltransferase SET domain-containing 2; SL2: Stem loop 2; SnRNA: Small nuclear RNA; SnRNP: Small nuclear RNP; SRSF: Serine- and arginine-rich splicing factor; TAU: Translational andrology and urology; TLR4: Toll like receptor 4; Vpr: Viral protein R; WNV: West Nile virus.

Spliceosome

Pathogen-induced changes in the abundance and localization of both snRNAs and snRNP-related proteins can modulate AS by affecting the recognition of splice sites. In general, pathogens secrete some proteins to interact with host snRNP-related proteins to arrest the expression of these proteins. For instance, mammalian orthoreovirus (MRV) releases μ2 protein to interact with host U5 snRNP-related proteins and thus inhibit their expression.[18] Such alternations further affect the splicing of interleukin 34 (IL34), thereby influencing the immune response of the host. In contrast to abundance alternations, pathogen-induced re-localization is rarely observed.[19] While poliovirus has been reported to use its 2A(pro) to regulate the assembly of snRNAs and snRNP-related proteins, downstream biological effects have not been elucidated.[20]

Cis-acting elements

Cis-acting elements are the short-conserved sequence elements within the pre-mRNA. These elements regulate splicing by promoting or inhibiting the recognition of adjacent splice sites and the binding of trans-acting factors. Both mutations in cis-acting elements and impaired cis-acting element motifs in adjacent introns or exons can generate a certain impact on splicing.[21,22] It is a pity that we fail to collect relevant evidence on infectious diseases.

Trans-acting factors

Trans-acting factors are RNA-binding proteins (RBPs) that specifically bind the cis-acting elements to regulate splicing.

Serine- and arginine-rich splicing factors (SRSFs)

SRSFs family members have certain structural similarities that share one or two N-terminal RNA recognition motif (RRM) domains and an arginine/serine-rich (RS) domain in the C-terminal region. The activity of SRSFs is controlled by the phosphorylation of serine residues within their RS domain. In general, SRSFs bind their RRM domains to exon splicing enhancer (ESE) or intron splicing enhancer and utilize their RS domains to recruit splicing components, then activate splicing. Such an action pattern is position-dependent, that is, SRSFs can also inhibit splicing when they bind to an intron.[23] The expression, modification, and cellular position of SRSFs all generate significant influence on splicing. Hepatitis B virus, HPV, and Mycobacterium tuberculosis (MTB) have been reported to manipulate host SRSF expression to regulate splicing and benefit themselves.[24–26] While HPV-16 exerts its E2 protein to enhance the SRSF1 phosphorylation, and then facilitates its own splicing and replication.[12,17] Unlike the aforementioned pathogens, MRV utilizes its μ2 protein to mediate the localization transition of SRSF2 from the nucleus to the cytoplasm, therefore changing host splicing and enhancing its own replication and pathogenicity.[27] Some special regulatory mechanisms have also been discovered.[28]

Heterogeneous nuclear ribonucleoproteins (hnRNPs)

hnRNPs family members typically harbor the RRM, K homology (KH), and glycine-rich domains. RRM and KH domains are responsible for binding to pre-mRNA sequence, while the glycine-rich domain interacts with other hnRNPs.[29] Likewise, the post-translational modification of hnRNPs partly determines the activity of these proteins. Upon activation, hnRNPs are more inclined to inhibit splicing, whereas this family can also act as a splicing activator in some special situations.

Changing the hnRNPs’ expression is one of the most common strategies employed by pathogens to regulate AS. For example, HPV-16 increases the host hnRNP A1 expression to promote the binding of hnRNP A1 and viral late regulatory element, thereby facilitating viral splicing and improving the expression efficiency of viral late gene.[30] The roles of hnRNPs’ modifications in some infectious diseases have also been reported.[31,32] Changes in the subcellular location of hnRNPs also influence splicing. Thompson et al[33] reported that influenza A viruses (IAVs) migrated host hnRNP H to nuclear speckles (NSs) and then changed the host splicing to influence viral replication.

Other RBPs

The RNA-binding FOX (RBFOX), neuro-oncological ventral antigen (NOVA), and RNA-binding motif (RBM) families also belong to trans-acting factors. The effect of the RBFOX family on exon splicing depends on the binding position, while NOVA proteins mainly target the intron splicing.[34,35] RBM family functions in a flexible way and their action pattern has not been fully summarized. A recent study reported that Dengue virus non-structural 5 protein increased the inclusion of spermidine/spermine N1-acetyltransferase 1-exon4 and thus favored viral replication by inducing the degradation of RBM10.[36]

Co-transcriptional regulation

Transcription that occurs simultaneously with splicing (termed as co-transcription splicing) can influence splicing and thus play a regulatory role. Two potential models have been proposed to explain the co-transcriptional regulation.[37] One is the recruitment model, referring to RNAPII using its own C-terminal domain to recruit trans-acting factors to pre-mRNA and then promote the recognition of splicing sites and splicing. Another one is the kinetic coupling model, indicating that the elongation rate of RNAPII alters the duration of spliceosome-splice site interactions and thus determines whether exons are skipped or retained. These two mechanisms are not mutually exclusive, and sometimes they can co-exist. Changes in RNAPII (expression, modification, etc.) can influence splicing significantly. For example, human cytomegalovirus infection leads to the increased phosphorylation level of RNAPII and changed RNAPII localization, which further regulates the splicing of viral RNA.[38]

Epigenetic modifications

In infectious diseases, the regulatory roles of DNA or RNA methylation in splicing are relatively less reported. Ju et al[39] found that Escherichia coli infection induced the methylation of leucine-rich-repeat-containing G-protein-coupled receptor 4-exon5, to affect the exon recognition and subsequent splicing. While Gokhale et al[40] documented that the Flaviviridae family decreased the N6 position of adenosine (m6A) modification of host cold-inducible RNA binding protein (CIRBP) mRNA, to inhibit the expression of long CIRBP isoform and thus promote viral replication. In addition to the modification of trans-acting factors, the modification of histones also plays a regulatory role. For instance, HPV-31 applies its E7 protein to augment the expression of histone methyltransferase SET domain-containing 2 (SETD2) and the level of H3K36me3 that is enriched in HPV-31 genome, further recruiting trans-acting factors and preventing the splicing defects in late viral RNAs brought by SETD2 depletion.[41]

RNA structures

RNA structures act by determining the exposure degree of RNA sequence, recruiting trans-acting proteins, or adjusting the movement speed of RNAPII. Jablonski et al[42] documented that HIV-1 drove the generation of the stem loop 2 by inducing the T-to-C mutation and thus promoted the exon 6D inclusion, achieving its enhanced infectivity. In the host, the RNA structural transformation inhibits the hnRNP H activity and then reverses the inhibition effect of this trans-acting factor.[42]

Phase separation

Phase separation refers to the phenomenon that single-phase molecular complexes separate into two phases (a dense phase and a dilute phase) that then stably co-exist, mediating the formation of membrane-less organelles (NSs, etc).[43] Phase separation is emerging as a splicing regulator. At the molecular level, NSs contain abundant trans-acting factors and snRNPs, and such composition is strongly suggestive of the potential that NSs promote splicing by offering a separate space.[44] From a more microscopic perspective, exons are preferentially sequestered into NSs through binding to SRSFs, whereas introns tend to be excluded due to their binding to hnRNPs.[44] Space partition places the exon–intron boundaries at the NS interfaces, exposes splice sites located at these boundaries, and hence catalyzes the splicing reactions. EBV-encoded nuclear antigen 2 has been verified to harbor the potential to perform phase separation and aggregate SRSF1 and SRSF7 together into the same phase, inducing the cancer-associated splicing landscapes.[45] The exploration of phase separation is in its infancy and plenty of areas deserve to be investigated.

Specific AS Events of Infectious Diseases

Decoding these events and their biological roles is beneficial to further understanding host–pathogen interactions. Based on available evidence, we preferentially describe AS events related to viral and bacterial infections. Relevant evidence is shown in Table 2.

Table 2 - Infectious disease-related splicing events. Infectious disease Targets Isoforms Action pattern Biological effect∗ References Viral infections  EBV MPPE1 Isoforms with and without exon11 Increasing MPPE1-exon11 skipping Inducing EBV-related tumorigenesis [45] STAT1 STAT1α and STAT1β Increasing STAT1β expression Promoting cell proliferation [28,51]  HBV NA Viral spliced isoform SP1RNA Increasing SP1RNA expression Reducing the recruitment of inflammatory monocytes/macrophages [77]  HIV-1/AIDS CCNT1 Isoforms with and without exon7 Increasing CCNT1-exon7 Suppressing the transcriptional activation of HIV-1 and maintaining the latency of infected CD4+ T cells [46,48,49] HLA-1 HLA-A11 and HLA-A11svE4 Increasing HLA-1-exon4 skipping Inhibiting the activation of natural killer cells [78] RUNX1 RUNX1a, RUNX1b, and RUNX1c Inhibiting the expression of RUNX1b and RUNX1c Promoting HIV-1 replication and latency [50]  High-risk HPV E6 and E7 E6, E6∗I, E6∗II, E6∗III, E6∗IV, E6∗V, E6∗VI, E6^E7, E6^E7∗I and E6^E7∗II Increasing E6∗I expression Inhibiting E6-mediated degradation of p53 and promoting the DNA damage [79]  HSV-1 MxA MxA and varMxA Increasing the exon14–16 skipping of MxA Promoting HSV-1 infection [80]  IAV TP53-i9 P53α, p53β, and p53γ Increasing the expression of p53α and p53β Promoting viral replication [81] Bacterial infections  TB IL-4 IL-4 and IL-4 delta2 Increasing IL-4-exon2 skipping Inhibiting the biological effects of IL-4 [82] IL-7 IL-7 and IL-7δ5 Increasing IL-7-exon5 skipping Promoting STAT5 phosphorylation and prolonging T cells’ life [56] IL-7R sIL-7R and mIL-7R