Intronic Alus Influence Alternative Splicing (original) (raw)
Figure 2
The effect of intronic _Alu_s on the splicing of a flanking exon.
(A) Schematic illustration of the RABL5 minigene containing three exons and two introns. The intronic _Alu_s (1 through 5) are marked by boxes with a point indicating the orientation of the Alu relative to the pre-mRNA. _Alu_4 is an _Alu_Sx inserted between the two arms of _Alu_Jo. (B) The indicated wild-type (wt) and mutant plasmids were transfected into 293T cells, total RNA was extracted, and splicing products were separated on a 2% agarose gel after RT-PCR analysis. Lane 1, splicing products of wt RABL5 minigene. Lanes 2–26, splicing products of the indicated mutants. The following abbreviations were used: Δ indicates deletion of the specified Alu element, X 1w3 indicates replacement of _Alu_1 with _Alu_3 (i.e., the sequence of _Alu_3 was inserted instead of that of _Alu_1 and in the same orientation as _Alu_3), and 1int specifies replacement of the _Alu_1 sequence with a non-Alu intronic fragment. The two mRNA isoforms are shown on the right. Numbers on top of the gel indicate percentage of exon inclusion as determined using ImageJ software. PCR products were sequenced.