Volume 12, Issue 5, Pages 1301-1307 (November 2003) Differential Phosphorylation and Subcellular Localization of La RNPs Associated with Precursor tRNAs and Translation-Related mRNAs  Robert V. Intine, Scott A. Tenenbaum, Amy L. Sakulich, Jack D. Keene, Richard J. Maraia  Molecular Cell  Volume 12, Issue 5, Pages 1301-1307 (November 2003) DOI: 10.1016/S1097-2765(03)00429-5

Figure 1 Non-Phospho-S366-Specific and Phospho-366-Specific Anti-La Antibodies Reveal that Phospho-La Is Much More Abundant Than Non-Phospho-La (A) Equal aliquots (2 μl) of a mock-treated and calf intestinal phosphatase (CIP)-treated HeLa cell extract were loaded in odd- and even-numbered lanes, respectively. Western blot detection was by Go (lanes 1 and 2), Ab1 (lanes 3 and 4), AbNP (lanes 5 and 6), and AbP (lanes 7 and 8). (B) Detection of hLa in HeLa cell extract (5 μl per lane) by isoelectric focusing (IEF) followed by Western blotting using Go (lanes 1 and 2), Ab1 (lanes 3 and 4), AbNP (lanes 5 and 6), and AbP (lanes 7 and 8) after CIP (+) or mock (−) treatment. Molecular Cell 2003 12, 1301-1307DOI: (10.1016/S1097-2765(03)00429-5)

Figure 2 Phospho-La Is Limited to the Nucleoplasm While Non-Phospho-La Is Nuclear and Cytoplasmic Immunofluorescence to visualize subcellular localization using Go (A), Ab1 (B), AbP (C), and AbNP (E). (D) and (F) show the DAPI stain superimposed on (C) and (E). Immunofluorescence intensity was calibrated to best reflect relative amounts of La in the nucleoplasm versus the cytoplasm. Molecular Cell 2003 12, 1301-1307DOI: (10.1016/S1097-2765(03)00429-5)

Figure 3 Precursor-tRNAs and Other Nascent pol III Transcripts Are Preferentially Associated with Phospho-La Rather Than Non-Phospho-La (A) Western blot of the La protein immunoprecipitated by nonimmune (NI, lane 1), Go (lane 2), AbP (lane 3), and AbNP (lane 4), using Go for detection of all lanes. (B) Northern blot analysis of immunoprecipitated RNAs to detect pre-tRNAMete, pre-tRNATyr, and pre-tRNAGly, using Abs indicated above the lanes. Note that 10% and 50% of the input (In) and immunoprecipitated material were loaded, respectively. (C) Northern blot analysis of RNAs remaining in the supernatants (10% loaded) of the IPs in (B). (D) hY5 RNA detection from the same IPs. For all panels in (B) and (D), a single blot was probed, stripped, and rehybridized sequentially. Another blot was probed sequentially for (C). Note that while lane 6 of (B) and (D) contained excess RNA relative to other lanes (see text), the supernatants in (C) contained equal relative amounts of RNA for all lanes. Molecular Cell 2003 12, 1301-1307DOI: (10.1016/S1097-2765(03)00429-5)

Figure 4 5′TOP-Containing mRNAs Are Preferentially Associated with Non-Phospho-La Rather Than Phospho-La (A) Ribosomal RNA rather than the normal spectrum of nascent RNA polymerase III transcripts are associated with npLa. Autoradiogram of RNAs prepared directly from extract of 32P-labeled HeLa cells (10% input, In, lane 2) or after immunoprecipitation (IP) with the antisera indicated above lanes 3–6 (NI, nonimmune). 32P-RNA remaining in the supernatants after IP are in lanes 7–10. Lanes 2 and 7–10 contain 10% of the amount of extract used for IP. Samples were electrophoresed in 10% polyacrylamide gel containing 8 M urea at ∼50°C. (B–G) RT-PCR analysis of RNAs coimmunoprecipitated with the La S366 isoforms. RNAs from input, supernatants, and IPs were purified in batch, and aliquots were analyzed for each transcript using the same relative amounts of RNA samples for each of the RT-PCRs. (B) S6 mRNA, (C) eEF1β mRNA, (D) L37 mRNA, (E) pre-tRNATyr, (F) hnRNP E2 mRNA, and (G) ferritin light chain (FeLC) mRNA. Lanes are as follows: Input (lane 1), IPs (odd-numbered lanes), and supernatants (even-numbered lanes) from nonimmune serum (Ni, lanes 2 and 3), AbP (lanes 4 and 5), and AbNP (lanes 6 and 7) as indicated. In all cases, heat inactivation of the reverse transcriptase enzyme prior to the RT step led to no product from the input (data not shown). Molecular Cell 2003 12, 1301-1307DOI: (10.1016/S1097-2765(03)00429-5)