Regulation of alternative RNA splicing by exon definition and exon sequences in viral and mammalian gene expression

Abstract

Intron removal from a pre-mRNA by RNA splicing was once thought to be controlled mainly by intron splicing signals. However, viral and other eukaryotic RNA exon sequences have recently been found to regulate RNA splicing, polyadenylation, export, and nonsense-mediated RNA decay in addition to their coding function. Regulation of alternative RNA splicing by exon sequences is largely attributable to the presence of two major cis-acting elements in the regulated exons, the exonic splicing enhancer (ESE) and the suppressor or silencer (ESS). Two types of ESEs have been verified from more than 50 genes or exons: purine-rich ESEs, which are the more common, and non-purine-rich ESEs. In contrast, the sequences of ESSs identified in approximately 20 genes or exons are highly diverse and show little similarity to each other. Through interactions with cellular splicing factors, an ESE or ESS determines whether or not a regulated splice site, usually an upstream 3' splice site, will be used for RNA splicing. However, how these elements function precisely in selecting a regulated splice site is only partially understood. The balance between positive and negative regulation of splice site selection likely depends on the cis-element's identity and changes in cellular splicing factors under physiological or pathological conditions.

Fig. 1

Viral and eukaryotic gene expression. Exons (boxes) and introns (lines) are indicated. The diagram illustrates the processing steps that occur before an mRNA is exported from the nucleus for translation. Alternative RNA splicing leads to the production of two isoforms of the message and consequently two different proteins. N, nucleus; C, cytoplasm.

Fig. 2

Types of viral and mammalian alternative RNA splicing. Examples of splice types occurring naturally in viral and mammalian RNA are shown. Exons (boxes) and introns (lines) are illustrated for each species of RNA. Alternative splice sites are indicated as vertical dashed lines. Red dots show stop codon locations on spliced RNAs. The names of the alternate spliced RNAs are indicated at the right. Drawings are not to scale. SV40 T antigen, simian virus 40 large T antigen RNA; HPV16 E6, human papillomavirus type 16 E6 bicistronic RNA; AAV2 Rep, adeno-associated virus type 2 Rep RNA [132]; mouse c-src RNA [136].

Fig. 3

Alternative splicing of Kaposi’s sarcoma-associated herpesvirus K8 RNA. KSHV K8 exon 3 has three alternative 5′ ss that share a single 3′ ss. See other descriptions in Fig. 2. It has been noted that removal of K8 intron 2 requires the selection of nt 75838 5′ ss and exon definition. An incomplete removal of intron 2 leads to production of K8β. Intron 2 retention is most common in the mRNAs with the use of the other two 5′ ss because selection of either one makes exon 3 larger than 500 nts, which restrains exon definition for recognition of upstream 3′ ss [153]. Drawings are not to scale.

Fig. 4. Regulation of alternative splice site selection by ESE and ESS

A. SR and non-SR proteins (vertical arrows) regulate alternative 3′ ss selection on bovine papillomavirus (BPV-1) late pre-mRNA. BPV-1 late pre-mRNA has three exons and two introns, and exon 2 has two alternative 3′ ss. There are five cis-elements, three ESEs [two purine-rich (green boxes) and one AC-rich (grey boxes)] and two ESSs (red boxes), that control individual 3′ ss switching by interacting with cellular splicing factors, as indicated by the curved arrows. Two purine-rich ESEs (SE1 and SE2) between the two alternative 3′ ss synergistically promote the selection of the proximal 3′ ss over the suppression by the ESS1 positioned immediately downstream of the SE1. Similarly, an AC-rich ESE (SE4) coordinates with the downstream 5′ ss to overcome inhibition by another ESS (ESS2), also positioned immediately downstream of the AC-rich SE4, and to stimulate the distal 3′ ss selection. The alternative 3′ ss selection leads to production of L2 (proximal 3′ ss usage) or L1 (distal 3′ ss usage) mRNAs [106]. B. SR and non-SR proteins regulate alternative 5′ ss selection on ad2 E1A pre-mRNA. Ad2 E1A pre-mRNA has three alternative 5′ ss and one major (M) and one minor (m) 3′ ss whose modulation occurs during virus infection. Several SR and non-SR proteins have been associated with the regulation of the alternative 5′ ss switch as indicated by vertical arrows. The green box immediate upstream of 12S 5′ ss indicates a purine-rich ESE that functions bi-directionally as a splicing enhancer for selection of both the 12S 5′ss and the upstream, minor 3′ ss [11].

Fig. 5

Regulation of alternative SMN2 RNA splicing by ESE and ESS. Two identical SMN (survival motor neuron) genes, telomeric SMN1 or SMNt and centromeric SMN2 or SMNc, are located in a 20-kb region on Chromosome 5q13 and each contains 9 exons and encodes a 38 kDa protein with 294 aa expressed at various levels in most tissues with high level in spinal cord [7;30;97]. SMN1 is a disease gene for proximal spinal muscular atrophy (SMA), a relatively common, neurodegenerative disorder in childhood, since 96% of SMA patients show homozygous absence of SMN1 caused by deletions or point mutations, in particular within exon 6, 7 and 8 of SMN1 [65;96;162]. SMN2 determines SMA severity and its expression level correlates with three types (I, severe; II, intermediate; and III, mild) of SMA [12;51] as SMN2 expression is capable of compensating for loss of SMN1. Although DNA and cDNA sequencing of the SMN1 and the SMN2 reveal only 5 nucleotide substitutions that do not alter the protein sequence, a single nt change, C to T, at codon 280 in exon 7 of the SMN2 (A) has been found to cause the exon to be skipped in majority of SMN2 mRNAs due to this mutation converting an ASF/SF2-binding site [20] into an ESS [85] that counteracts with an existing purine-rich ESE downstream [72;108], resulting in a spliced isoform (SMN2∆7) (B) that encodes a truncated, nonfunctional protein missing the C-terminal 16 residues. Underlines in panel A show an ASF/SF2-binding site (ESE) in SMN1 and an ESS in SMN2 due to a C to U mutation at position +6 in exon 7 (capitalized letters).