Fig. 1

Distinct Subcellular Localization of Minor- and Major-Class snRNAs

(A) In situ hybridization of paraffin-embedded tissue sections from adult zebrafish with LNA probes for U2, U12, and U6atac snRNAs. Control hybridizations with mismatch (mm) probes for U12 and U6atac snRNA are shown on the right. Hybridization of digoxygenin (DIG)-labeled LNA probes was detected by an anti-DIG antibody coupled to alkaline phosphatase and NBT/BCIP staining. Arrows point at nuclear (U2) or perinuclear and cytoplasmic staining (U12, U6atac).

(B) In situ hybridization of whole-mount zebrafish embryos (24 hpf) using DIG-labeled LNA probes for U2 and U6atac snRNAs, or a 5 mismatch (mm) probe for U6atac snRNA. Arrowheads indicate the center of nuclei. Arrows point to cytoplasmic staining (U6atac). Scale bar indicates 25 μm.

(C) Fluorescence in situ hybridization of NIH 3T3 mouse fibroblasts using LNA probes for the snRNAs indicated, visualized by confocal fluorescence microscopy. Nuclear/DNA counterstaining was performed with DRAQ5. Scale bar indicates 20 μm.

(D) Detection of U2 and U6atac snRNA upon nuclear-cytoplasmic fractionation of NIH 3T3 cells. Nuclear (n) and cytoplasmic (c) fractions were obtained by differential lysis of plasma and nuclear membranes by nonionic detergents for the times indicated. Total RNA from the fractions was reverse transcribed and analyzed by RT-PCR. -RT corresponds to RT-PCR reaction lacking reverse transcriptase. Amplification reactions were not in plateau phase as verified by different numbers of PCR cycles.