Figure 2. Ro60 is a stable component of Y RNPs and is important for Y RNA integrity. (A) Mouse embryonic stem cell lysates were subjected to immunoprecipitation with antibodies against the trimethylguanosine (TMG) cap that is the 5′ end of many snRNAs (lanes 2–3), anti-Sm antibodies, which recognize the Sm proteins of the spliceosomal U snRNPs (lanes 4–5), anti-La (lanes 6–7) and anti-Ro60 antibodies (lanes 8–9). RNAs in immunoprecipitates (lanes 2,4,6,8), supernatants (lanes 3,5,7,9), and an equivalent amount of lysate (lane 1) were subjected to northern blotting to detect mY1 and mY3. As a control, the blot was reprobed to detect the spliceosomal U2 snRNA. Note that Y RNAs in the anti-La immunoprecipitate are slightly larger (lane 6) than the Y RNAs remaining in the supernatant (lane 7). (B) RNA extracted from wild-type and Ro60^−/− embryonic stem cells (lanes 1–2), brain (lanes 3–4), testis (lanes 5–6), and ovary (lanes 7–8) were subjected to northern blotting to detect mY1 and mY3. As a loading control, the blot was reprobed to detect 5S rRNA. (C) HEK293 cell lysates were subjected to immunoprecipitation with anti-Ro60 antibodies or nonimmune isotype control IgG. RNAs extracted from immunoprecipitates (lanes 4 and 5), supernatants (lane 2 and 3), and the starting lysate (lane 1) were subjected to northern blotting to detect hY RNAs. As a negative control, the blot was probed to detect the spliceosomal U6 snRNA. (DandE) To determine if Ro60 stabilizes hY RNAs, siRNAs against Ro60, or control non-target (NT) siRNAs were transfected into human keratinocytes. After 72 h, lysates were prepared and subjected to western blotting (D) to detect Ro60. Actin was used as a loading control. RNA extracted from the lysates was subjected to northern blotting (E) to detect hY RNAs. U6 snRNA was used as a loading control. Quantitation revealed that Ro60 was reduced by 83%, while hY1, hY3, hY4, and hY5 were reduced by 90%, 30%, 59%, and 60%, respectively.