Aberrant Nuclear Trafficking of La Protein Leads to Disordered Processing of Associated Precursor tRNAs  Robert V Intine, Miroslav Dundr, Tom Misteli, Richard J Maraia  Molecular Cell  Volume 9, Issue 5, Pages 1113-1123 (May 2002) DOI: 10.1016/S1097-2765(02)00533-6

Figure 1 Human La Residues 316–332 Are Required for tRNA-Mediated Suppression and Normal pre-tRNA Processing (A) In vivo suppressor activities of pRep4 vector (sector 1), hLa1–408 (sector 2), spLa1–298 (sector 3), hLa26–408 (sector 4), hLaΔ328–344 (sector 5), hLa1–315 (sector 6), and hLaΔ316–332 (sector 7) in ySK5 cells. (B) Immunoblot of hLa proteins expressed in S. pombe as indicated above the lanes. (C) Northern blot of RNAs from ySK5 cells expressing various La constructs as indicated above the lanes, probed with an oligonucleotide specific for intron-containing pre-tRNASerUGA species. (D) The blot in (C) was stripped and reprobed with an oligonucleotide specific for spliced tRNASerUGA species. Arrowhead points to aberrant spliced tRNA species. Note that two blemishes (air bubbles) in lane 5 can serve to orient the positions of bands in (C) and (D). Suppression activity is indicated below the lanes. Molecular Cell 2002 9, 1113-1123DOI: (10.1016/S1097-2765(02)00533-6)

Figure 2 The Distribution of Processing Intermediates of Endogenous pre-tRNA Is Disordered by hLa Proteins Lacking Residues 316–332 (A–D) Northern blot analyses of precursor and mature tRNALysCUU species in fission yeast transformed with various constructs as indicated above the lanes. Probes were specific for: (A) intron-containing pre-tRNALysCUU species, (B) 5′ leader-containing pre-tRNALysCUU species, (C) 3′ trailer-containing pre-tRNALysCUU species, and (D) spliced tRNALysCUU species. Schematic representations of the normal nascent pre-tRNA, intermediates, and spliced species are indicated to the left with probes represented by thick black bars; the position and schematic representation of an aberrant spliced species is indicated to the right. Molecular Cell 2002 9, 1113-1123DOI: (10.1016/S1097-2765(02)00533-6)

Figure 3 NRE-Deficient hLaΔ316–332 Fails to Concentrate in Fission Yeast Nuclei because It Is Exported in an LMB-Sensitive Manner S. pombe cells were mock treated (A–F) or treated with Leptomycin B (LMB, [G–L]). Cells transformed with empty pRep4 vector (A–B and G–H), hLa1–408 (C–D and I–J), or hLaΔ316–332 (E–F and K–L) were stained with anti-hLa antibody (A, C, E, G, I, and K) and also with DAPI (B, D, F, H, J, and L) as indicated. The relatively less stained circular regions indicated by arrows in (E) are revealed as nuclei by DAPI in (F). Molecular Cell 2002 9, 1113-1123DOI: (10.1016/S1097-2765(02)00533-6)

Figure 4 Nuclear Localization of GFP-hLa in Primate Cells Involves Nuclear Retention (A–H) Mammalian expression vectors containing GFP-hLa fusions were transfected into CMT3 cells. Cells were visualized after staining with DAPI (A–D) and green fluorescence (E–H). Transformation was with GFP-hLa1–408 (A and E), GFP-hLa1–363 (B and F), GFP-hLa1–315 (C and G), and hLaΔ316–332 (D and H). (I) The GFP-La fusion proteins and endogenous La protein were visualized by immunoblotting using anti-La (Go) antibody. (J–O) Localization of GFP-hLa1–408 (J–L) and GFP-hLaΔ316–332 (M–O) in the absence (J and M) and presence (K and N) of LMB. (L) and (O) show DAPI staining of (K) and (N). Molecular Cell 2002 9, 1113-1123DOI: (10.1016/S1097-2765(02)00533-6)

Figure 5 Aberrant Spliced tRNA Species Are Stably Associated with hLaΔ316–332 Native extracts of fission yeast cells expressing hLa proteins were subjected to RNA purification (lanes 1–3) or immunoprecipitated with anti-hLa (lanes 5–7) or nonimmune sera (lanes 10–12), and then RNA was purified and examined by Northern blotting. The various pre-tRNALysCUU intermediates or spliced species are diagrammed to the left; thick gray rectangles represent exons and gray lines represent introns; open rectangles represent aberrant spliced species; the probes are shown as thick black bars, and their position on the pre-tRNALysCUU-related species is indicated as for Figures 2A–2D. (A), intron probe; (B), trailer probe; (C), leader probe; and (D), spliced probe. Lanes 4, 8, 9, and 13 contain tRNALysCUU-related standards to serve as markers and hybridization controls (see text). Molecular Cell 2002 9, 1113-1123DOI: (10.1016/S1097-2765(02)00533-6)

Figure 6 Conserved NRE Function in Distantly Related La Proteins and in a Homologous System (A) The sequences of La CTDs spanning the regions corresponding to the NRE through the WAM-like region of hLa were aligned by ClustalW (MacVector) and adjusted manually (see text). Columns whose amino acids share ≥55% identity or similarity were boxed and shaded. Amino acids numbering is indicated to the left. (B) Suppression plate assay of wild-type and mutant spLa proteins in ySK5 as indicated. (C) Immunoblot of the most relevant strains in (B), probed with anti-spLa serum. (D) Northern blots showing aberrant pre-tRNALysCUU; species (arrows) detected with the 5′ leader probe (upper panel) and the probe specific for spliced tRNALysCUU (lower panel). Note that lane 4 shows RNA from hLaΔ316–332 cells for comparison with spLaΔ234–255 in lane 3. The position of the nascent pre-tRNALysCUU which is stabilized by spLa1–298 (lane 2) and spLa1–284 (lane 6) is indicated. spLa1–251 is an inactive suppressor that does not stabilize nascent pre-tRNALysCUU (lane 5) and therefore produces a hybridization pattern indistinguishable from the vector control, pRep4 (lane 1). Quantitative monitoring after stripping of the upper blot ensured that the aberrant species in the lower blot was detected by the splice-specific probe, and the same results were obtained when the probe order was reversed (data not shown). Molecular Cell 2002 9, 1113-1123DOI: (10.1016/S1097-2765(02)00533-6)

Figure 7 Localization of Wild-Type and Mutant spLa Proteins in Fission Yeast and Transfected Mammalian Cells (A–T) Fission yeast cells transformed with the constructs indicated to the left were processed for anti-spLa immunofluorescence (A–I and K–S) and DAPI staining (B–J and L–T) in the absence (A–J) and presence (K–T) of LMB. (U-Z) Subcellular localization of GFP-spLa fusion proteins expressed in mammalian (CMT3) cells and visualized for DAPI (U, W, and Y) and green fluorescence (V, X, and Z), as indicated. Molecular Cell 2002 9, 1113-1123DOI: (10.1016/S1097-2765(02)00533-6)