<i>U11/U12-65K</i> transcripts with a long 3′UTR are associated with transcriptional read-through.

<p><b>(A)</b> Following transfection with mock or block morpholinos in HEK293 cells, RT-PCR was performed to detect both <i>65K</i> isoforms simultaneously, using a reverse primer binding at the canonical terminal exon (upper panel), or located in the IE (lower panel). Amplicons were separated on a 1% agarose gel. Endoporter transfection reagent was used for the (-) control sample. The marker (M) is Generuler (Thermo Scientific: #SM0331). <b>(B)</b> qRT-PCR analysis of IE expression level for morpholino transfected samples normalized against total <i>65K</i> expression. Error bars represent the standard deviation of 3 biological replicates and the asterisk indicates p-value < 0.05 in a two-tailed Student′s t-test in (B), (D), (F) and (G). <b>(C)</b> Upper panel. Schematics of the reporter plasmid construction. Shaded boxes depict <i>65K</i> sequences cloned into pGL4.13. Lower panel indicate mutations of the putative poly(A) sites (in bold and underlined) of the <i>65K</i> gene to generate the pAdel reporter construct. <b>(D)</b> CHO cells were transfected with wt and pAdel reporter constructs and construct <i>65K</i> long-3′UTR and short-3′UTR splicing isoforms were quantified by qPCR. Fold change is relative to the wt construct <i>65K</i> short-3′UTR splicing isoform. <b>(E)</b> Multiplex (upper panel) and normal RT-PCR of the samples analyzed in (D) were performed and amplicons separated on 1.3% agarose gel. Primers (shown as arrows) were designed to quantify construct <i>65K</i> 3′UTR splicing isoform ratio (upper panel) and read-through and IE splicing of construct <i>65K</i> short- and long-3′UTR splicing isoform transcripts. Dashed lines indicate an exon-spanning primer and an asterisk a construct-specific isoform (splicing event could not be found from endogenous <i>65K</i> gene) skipping the short-3′UTR specific exon and directly splicing towards to IE. <b>(F)</b> qPCR quantification of construct <i>65K</i> 3′UTR splicing isoform transcripts that undergo IE splicing from samples analyzed in (E). <b>(G)</b> RT-PCR analysis of expression of short- and long-3′UTR splicing isoform transcripts that read-through past the poly(A) site but do not splice to the IE (RT transcripts) from samples analyzed in (E). Fold change is relative to the wt RT transcript expression and samples were normalized against total construct <i>65K</i> short- or long-3′UTR splicing isoform levels.