FIG. 2.
Nucleolar expression of the mouse ribosomal minigene tagged with human U6, U2, U3, and 5.8S RNA-specific sequences. (A) Schematic structure of the pW(Xb/Xh) mouse ribosomal minigene construct. pW(Xb/Xh) contains the mouse polymerase I (Pol I) promoter and terminator (term), some 5′ (hatched boxes) and 3′ (open box) external transcribed spacer (ETS) sequences, and a fragment of the chloramphenicol acetyltransferase (CAT) gene that carries the XbaI (Xb) and XhoI (Xh) restriction sites. The pW-U6-1, pW-U6-2, pW-U6-3, pW-U6-4, pW-U5.8S, pW-U3, and pW-U2 constructs were created by insertion of appropriate synthetic DNA fragments into the XbaI and XhoI sites of pW(Xb/Xh). Nucleotides which are 2′-O-methylated (m) or pseudouridylated (Ψ) in rat U6, U3, and U2 snRNAs and human 5.8S rRNA are indicated. (B) Subcellular localization of the W-U6-1 ribosomal minigene transcript. Upon transfection of the pW-U6-1 minigene into mouse cells, RNA samples (200 ng) extracted either from total cells (T) or from cytoplasmic (Cy), nuclear (Nu), nucleoplasmic (Np), and nucleolar (No) fractions were analyzed by RNase A and T1 protection by use of antisense RNA probes specific to the W-U6-1 transcript (upper panel), U3 snoRNA (middle panel), or U4 snRNA (lower panel). Lane C represents a control mapping with E. coli tRNA. Lane M, size markers in nucleotides.