FIGURE 3.

Splicing of adenovirus pre-mRNA in Xenopus oocytes. (A) Pre-mRNA containing a single ³²P at the branch point adenosine (5′ of the nucleotide) was injected into (lane 4) the nuclei of intact Xenopus oocytes or (lanes 1–3) U2-depleted Xenopus oocytes, which had been pre-injected with (lane 4) no guide RNA, (lanes 2,3) a box C/D guide RNA containing random guide sequences, or (lane 1) a box C/D guide RNA targeting the branch point adenosine. Injected oocytes were then supplemented (lanes 1,2) with or (lanes 3,4) without in vitro synthesized U2. (Lane 5) Un-injected pre-mRNA. The positions of pre-mRNA and lariat intron product are also indicated. (B) The lariat intron and unspliced pre-mRNA were recovered from lane 1 of A (lanes 1,2) and lane 2 of A (lanes 3,4). After debranching, (lanes 2,4) the debranched intron (released from the lariat structure) as well as (lanes 1,3) the unspliced pre-mRNA were treated with nuclease P1 and analyzed on a TLC plate. (Lane 6) 5′[³²P]-pA (pA) and (lane 5) 2′-O-methylated 5′[³²P]-pA (pAm) were analyzed in parallel.