Repositioning of an alternative exon sequence of mouse IgM pre-mRNA activates splicing of the preceding intron

Abstract

Using a transient expression system of mouse IgM mini-gene constructs in mouse B-cell lines and in fibroblast L cell, we investigated splicing of the IgM transcript. We observed that the efficiency of splicing between exons C4 and M1 (C4-to-M1 splicing), the splicing reaction leading to the production of membrane-bound form (microns) mRNA, was drastically affected by mutations in a specific portion of the downstream exon (M2). The results show that the specific exon M2 sequence activates the C4-to-M1 splicing. This activation was not observed when splicing between exons M1 and M2 was abolished by base substitutions at the splice sites. These results indicate that positioning of the downstream exon is crucial for efficient splicing of the preceding intron.

Figure 1

Schematic representation of the hybrid μ gene construct pμW and the probe used for S1 nuclease analysis. A. The hybrid Mo-LTR-μ, gene construct, pμW. Solid lines represent introns. Open, filled, and hatched boxes represent exons. The dotted box represents the Moloney murine sarcoma virus genome fragment containing the LTR promoter (Mo-LTR). Filled triangles indicate polyadenylation sites: μs, secreted form polyadenylation site; μm, membrane-bound form polyadenylation site. Δ denotes the portion of the C4-M1 intron deleted in pμW; A, Acc I site; B, BamH I; C(K), Cla I linker inserted into the original Kpn I site; P, Pst I; Sa(Sp), Sal I linker inserted into the original Spe I site; X, Xho I. B. Diagram of the probe used for S1 nuclease analysis. The sizes (in nucleotides) of the full-length probe and protected fragments are indicated.

Figure 2

Structures and expression of μ gene deletion mutant plasmids. A. The 3′ terminal structures of the deletion mutant plasmids, ΔA, ΔB, and ΔC. The symbols are the same as in Figure 1. The stepwise deletions start from the Sal I site in exon M2 in the 3′-to-5′ direction. In ΔA, the deletion removes 166-bp of exon M2 and 29-bp of the 3′ end of M1-M2 intron. In ΔB, the deletion removes 166-bp of exon M2 and the entire M1-M2 intron sequences, as well as 3-bp of exon Ml. In ΔC, 36-bp of the 3′ end of exon M1 is deleted, along with the M1-M2 intron and exon M2 sequences. B. S1 nuclease analysis of RNA from M12 and P3U1 transfected with the plasmids indicated on the top of each lane. Lane M shows ³²P-labeled pBR322 DNA digested with Hpa II used as size markers. The probe used is the same as in Figure 1B. The positions of the bands corresponding to the probe, unprocessed, μs, and μm mRNAs are indicated. The gel pieces corresponding to these RNAs were cut out, and their radioactivity was measured by Cerenkov counting. The radioactivity of the bands corresponding to μm mRNA relative to that of μs mRNA is indicated under each lane (μm/μs).

Figure 3

Structure and expression of double deletion mutants of the μ gene plasmid. A. The 3′ terminal structures of the deletion mutants, (μs⁻)/W, (μs⁻)/ΔA, and (μs⁻)/ΔC. A portion of C4-M1 intron containing the ns polyadenylation site is deleted in these constructs (denoted by A and break lines). The deletions in the Ml-M2 region of (μs⁻)/ΔA and (μs⁻)/ΔC are the same as those of AA and AC in Figure 2. B. The diagram of the S1 probe and the protected fragments. C. S1 nuclease analysis of RNA from M12 and P3U1 transfected with the plasmids indicated on the top of each lane. Lane M is the same as that in Figure 2. The positions of the bands corresponding to the probe, the μ RNA which was not spliced between exons C4 and Ml (Unspliced), and the μ RNA Which was spliced (Spliced) are indicated. The gel pieces corresponding to these RNAs were cut out, and their radioactivity was measured by Cerenkov counting. The radioactivity of the bands corresponding to the spliced RNA relative to the total μ RNA is indicated under each lane.

Figure 4

Structure and expression of μ gene mutant plasmids. A. The 3′ terminal structures of the mutant plasmids, MD and ML. MD contains base substitutions at both 5′ and 3′ splice sites (*): /GT to/GA, at the 5′ splice site; AG; to AC/, at the 3′ splice site. In ML, M1-M2 intron is deleted. B. S1 nuclease analysis of RNA from M12 transfected with the plasmids indicated on the top of each lane. The positions of the bands corresponding to the probe, unprocessed, μs, and μm mRNAs are indicated.

Figure 5

Structures and expression of the mutant μ genes in L cells. A. The 3′ terminal structures of the mutant μ genes, ΔB, ΔC, and MD. B. S1 nuclease analysis of RNA from mouse fibroblast L cells transfected with the plasmids indicated on the top of each lane. The positions of the bands corresponding to the probe, unprocessed, μs, and μm mRNAs are indicated.