Efficient in vivo manipulation of alternative pre-mRNA splicing events using antisense morpholinos in mice

Abstract

Mammalian pre-mRNA alternative splicing mechanisms are typically studied using artificial minigenes in cultured cells, conditions that may not accurately reflect the physiological context of either the pre-mRNA or the splicing machinery. Here, we describe a strategy to investigate splicing of normal endogenous full-length pre-mRNAs under physiological conditions in live mice. This approach employs antisense vivo-morpholinos (vMOs) to mask cis-regulatory sequences or to disrupt splicing factor expression, allowing functional evaluation of splicing regulation in vivo. We applied this strategy to gain mechanistic insight into alternative splicing events involving exons 2 and 16 (E2 and E16) that control the structure and function of cytoskeletal protein 4.1R. In several mouse tissues, inclusion of E16 was substantially inhibited by interfering with a splicing enhancer mechanism using a target protector morpholino that blocked Fox2-dependent splicing enhancers in intron 16 or a splice-blocking morpholino that disrupted Fox2 expression directly. For E2, alternative 3'-splice site choice is coordinated with upstream promoter use across a long 5'-intron such that E1A splices almost exclusively to the distal acceptor (E2dis). vMOs were used to test the in vivo relevance of a deep intron element previously proposed to determine use of E2dis via a two-step intrasplicing model. Two independent vMOs designed against this intronic regulatory element inhibited intrasplicing, robustly switching E1A splicing to the proximal acceptor (E2prox). This finding strongly supports the in vivo physiological relevance of intrasplicing. vMOs represent a powerful tool for alternative splicing studies in vivo and may facilitate exploration of alternative splicing networks in vivo.

FIGURE 1.

In vivo exon skipping induced by a splice-blocking vivo-morpholino. Upper panels, diagram showing the normal splicing pattern in which 4.1R E16 is partially included in mouse kidney and liver (left panel) and the predominant exon skipping that occurs in mice treated with splice-blocking vivo-morpholino against the 5′-splice site of E16 (right panel). E14 and E15 are alternatively spliced in other tissues. Lower panel, RT-PCR analysis of E16 splicing patterns in mouse tissues treated with the following reagents: normal saline (lanes 1 and 5), control vMO against human globin (lanes 2 and 6), vMO against the E16 5′-splice site (lanes 3 and 7), and control vMO against a different region of 4.1R mRNA (intra-E1B 5′-splice site) (lanes 4 and 8). E16 splicing was quantitated by densitometry and is reported as percent inclusion (incl). All results were reproducible in at least two independent mice for each morpholino. Lane 9 represents a negative PCR control.

FIGURE 2.

In vivo regulation of alternative E16 splicing by morpholinos that disrupt Fox2-mediated splicing enhancer activity. A, strategy for disrupting Fox2-enhanced E16 splicing. Fox2 binding at three intronic UGCAUG motifs comprising the intron splicing enhancer (ISE) promotes E16 inclusion (incl). Blocking Fox2 enhancer activity by splice-blocking vMOs against Fox2 or target-blocking vMOs against the Fox2-dependent intron splicing enhancer should reduce E16 splicing efficiency. B, scheme for vivo-morpholino-induced disruption of Fox2 expression. Diagrams show inclusion of E5 (93 nucleotides) in normal mouse kidney and liver (left) and E5 skipping in tissues from mice treated with the indicated splice-blocking vivo-morpholino (right). The lower panel indicates the predicted Fox2 protein generated from E5-skipped RNA, lacking a critical part of the RNA-binding motif. Fox2^DN, dominant-negative Fox2. C, dose-dependent splicing changes induced in Fox2 RNA isolated from these tissues. Lanes 1, analysis in tissues treated with control morpholino at 13.5 mg/kg; lanes 2–5, Fox2 splicing analysis in tissues treated with splice-blocking vivo-morpholino at 1.75, 3.5, 7.0, and 13.5 mg/kg, respectively. D, time course of splicing changes induced in Fox2 transcripts. Lanes 1 and 4, Fox2 splicing analysis at 24 h after a single injection of vivo-morpholino at 13.5 mg/kg; lanes 2 and 5, analysis at 48 h after two injections at 13.5 mg/kg; lanes 3 and 6, analysis at 48 h after two injections of control morpholino. E, inhibition of 4.1R E16 splicing by morpholinos that disrupt Fox2 expression or block the Fox2-binding sites in intron 16. RT-PCR analysis shows E16 splicing behavior in mice treated with the following reagents: normal saline (lanes 1 and 5), control vMO (lanes 2 and 6), Fox2 splice-blocking vMO as in B (lanes 3 and 7), and intron splicing enhancer-blocking vMO (lanes 4 and 8).

FIGURE 3.

In vivo disruption of alternative E2 splicing with morpholinos against deep intron splicing motifs far upstream at the intraexon (originally annotated as E1B). A, the left panel shows the normal intrasplicing pathway by which E1A splices to the distal 3′-splice site at E2 (splicing event 2); this nested splicing pathway is initiated by splicing event 1. In contrast, E1C splices directly to E2 at its proximal 3′-splice site (splicing event 3). The right panel shows the aberrant splicing of E1A to the E2 proximal acceptor predicted to occur if intraexon (IE) function is inhibited by MOs against its 5′-splice site or branch point motifs. B, RT-PCR analysis of RNA isolated from kidney (left panel) and liver (right panel) using primers in E1A and E2. C, RT-PCR analysis of RNA isolated from kidney (left panel) and liver (right panel) using primers in E1C and E2. In B and C, mice were treated with the following reagents: normal saline (lanes 1), irrelevant MO versus globin (lanes 2), intraexon 5′-splice-blocking MO (lanes 3), intraexon branch point-blocking MO (lanes 4), and control MO versus a different region of 4.1R pre-mRNA (E16 5′-splice site blocker) (lanes 5). Lanes 6 represent negative control PCRs. D, upper panel, the normal 4.1R pre-mRNA is spliced by splicing event 1 to form an intermediate product that is subsequently processed by splicing event 2 to generate proper mature mRNA. vMO that produces aberrant product should cause reduction in the amount of intermediate product. Lower panel, RT-PCR analysis showing the amount of intermediate product (upper band) relative to steady-state mRNA (lower band). Lanes 1 and 4, no vMO; lanes 2 and 5, nonspecific control vMO; lanes 3 and 6, intraexon 5′-splice-blocking vMO. Values below each lane represent the relative amount of intermediate (int) RNA to mRNA, which is substantially reduced by the intraexon-directed vMO.