Volume 6, Issue 2, Pages 339-348 (August 2000) Control of Transfer RNA Maturation by Phosphorylation of the Human La Antigen on Serine 366  Robert V.A. Intine, Amy L. Sakulich, Shashi B. Koduru, Ying Huang, Erik Pierstorff, John L. Goodier, Lon Phan, Richard J. Maraia  Molecular Cell  Volume 6, Issue 2, Pages 339-348 (August 2000) DOI: 10.1016/S1097-2765(00)00034-4

Figure 1 The Fission Yeast La Gene Encoding Sla1p Is Required for Accumulation of tRNASerUGAM Transcripts (A) A cloverleaf-like illustration of the intron-containing pre-tRNASerUGAM according to a representation in which anticodon nucleotides are base paired with intron nucleotides (Trotta and Abelson 1999). Nucleotides in the 5′ leader, 3′ trailer, and intron are in lower case. Three substitutions are shown in bold with underline (see text). (B–D) yAS99 (Sla1+) and yAS95 (Sla1−) were transformed with an integrating vector containing the tRNASerUGAM gene. After isolation in selective media, cells were grown in rich media (YES). Total RNAs from two Sla1+ and three Sla1− transformants are shown in this Northern blot. The same blot was hybridized and stripped sequentially with the probes indicated. Suppression of ade6-704, as revealed in a colorimetric plate assay (see below), is indicated below the lanes of (D). Identities of the species indicated to the left were determined by differential reactivity with oligonucleotide probes directed to the 5′ leader, 3′ trailer, and intron (not shown) (Van Horn et al. 1997). Molecular Cell 2000 6, 339-348DOI: (10.1016/S1097-2765(00)00034-4)

Figure 2 Sla1p Restores tRNASerUGAM-Mediated Suppression to Sla1− Cells (A) Representations of Sla1p constructs and a summary of their activities in yAS110 as demonstrated in the plate assays shown in (B). Shaded blocks represent conserved domains corresponding to RRM-1 and RRM-2 of human La (Kenan 1995) (Goodier et al. 1997; Fan et al. 1998). Note that the RRM-1 region of yeast La has been referred to as the La motif (Sobel and Wolin 1999). The nuclear localization signal (NLS) is referred to in the text. (B) Suppression assays in the absence and presence of thiamin. (C) Immunoblot analysis of the Sla1p constructs shown in (A) and (B) above in the absence of thiamin (except for lane 4; see below). Equal amounts of total protein, verified by SDS/PAGE and staining (not shown), were loaded. Lane 1, yAS99 (Sla1+); lanes 2–8, yAS95 (Sla1− ) transformed with pREP4-derived plasmids; lane 2, vector alone; lane 3, Sla1p 1–298; lane 4, Sla1p 1–298 (+ thiamin); lane 5, Sla1p 52–298; lane 6, Sla1p 171–298; lane 7, Sla1p 1–251; lane 8, Sla1p 1–284. Molecular Cell 2000 6, 339-348DOI: (10.1016/S1097-2765(00)00034-4)

Figure 3 A Distinct Activity of Human La Is Discernible in Fission Yeast (A) Suppression assay in yAS110 reveals differential activities of Sla1p and human (h)La. (B) Immunoblot of protein isolated from yAS110 transformed with pREP4-hLa 1–408 (lane 1) or pREP4 vector alone (lane 3); lane 2 contained HeLa extract as a control. (C) Northern blot analysis of cells transformed with vector alone (pREP4, lane 1), various Sla1 constructs (lanes 2–7), and hLa (lane 8) using a tRNASerUGAM-specific probe, MSer-UGA. Cells were grown in selective media. (D) The blot in (C) was reprobed for intron-containing tRNASerUGAM species. (E) The same blot was reprobed for intron-containing tRNALyscuu species. Suppression, as revealed in Figure 2, is summarized below the lanes. For (D) and (E), the nascent unprocessed precursors are indicated to the right. Molecular Cell 2000 6, 339-348DOI: (10.1016/S1097-2765(00)00034-4)

Figure 4 RRM-3 and Other Motifs in the CTD of hLa Block tRNASerUGAM-Mediated Suppression (A) Extracts of S. pombe expressing mutant hLa proteins were examined by SDS/PAGE followed by staining (lower panel) and immunoblotting with antiserum specific for hLa (upper panel). (B) Results showing the activities of various constructs in the suppression assay. (C) The hLa proteins examined in this study are shown in schematized form with a summary of their suppression activities (indicated to the right). Positions of RRMs 1–3 are shown as are two potential nuclear localization signals (NLS). The SBM is represented by “+++”. Serine 366 is represented by an encircled S. An acidic region is represented by “− −.” Molecular Cell 2000 6, 339-348DOI: (10.1016/S1097-2765(00)00034-4)

Figure 5 RRM-3 and Other Motifs in the CTD of hLa Impede Processing of Pre-tRNASerUGAM and Endogenous Pre-tRNAs (A) Northern blot with a probe that detects precursor and mature tRNASerUGAM species. Suppression of strain yAS110 is summarized below the lanes. (B) The blot in (A) was reprobed for pre-tRNALyscuu intron-containing species (upper) and mature tRNALyscuu (lower). The species indicated to the right were determined by their differential reactivity with oligo probes directed to the 5′ leader, 3′ trailer, and intron (not shown). Molecular Cell 2000 6, 339-348DOI: (10.1016/S1097-2765(00)00034-4)

Figure 6 The SBM in the CTD of hLa Impedes a Rate-Limiting Step in tRNA Maturation (A) Predicted representations of the intron-containing pre-tRNASerUGAM (middle) and pre-tRNASerUGAM-G37:10 (right), according to a model in which anticodon nucleotides are base paired with intron nucleotides (Trotta and Abelson 1999). The parental species, pre-tRNASerUCA (left), is shown for comparison. Three tRNASerUGAM-specific nucleotides are shown as bold underlined text; intron sequences are shown in lower case except for the 37:10 position (arrowhead). The leaders and trailers are represented by solid lines. (B) Cells carrying tRNASerUGAM (strain yAS110, sectors 1–4) or tRNASerUGAM-G37:10 (strain ySK5, sectors 5–8) alleles were transformed with Sla1p, hLa 1–408, hLa Δ328–344, or pREP4, as indicated, and assayed for suppression. Molecular Cell 2000 6, 339-348DOI: (10.1016/S1097-2765(00)00034-4)

Figure 7 Phosphorylation of hLa on Serine 366 Promotes tRNA Expression (A) In vivo suppressor activities of pREP4 alone (sector 1), Sla1p (sector 2), hLa-S366 (sector 3), hLa-A366 (sector 4), and hLa-G366 (sector 5) in cells carrying the tRNASerUGAM-G37:10 allele. (B) Northern blot analyses of RNA from cells harboring the tRNASerUGAM allele (lanes 1–4) or the tRNASerUGAM-G37:10 allele (lanes 5–8) and transformed with various constructs. Lanes 1 and 5, pREP4; lanes 2 and 6, Sla1p; lanes 3 and 7, hLa-S366; lanes 4 and 8, hLa-G366. The upper panel was probed with an oligonucleotide (Mser-UGA) that detects all species of tRNASerUGAM and tRNASerUGAM-G37:10 transcripts. After radiodecay, the probe was stripped, and the blot was reprobed with an oligonucleotide specific for the 5′ leader (lower panel). (C) One-dimensional isoelectric focusing (IEF) of a purified isoform of unphosphorylated hLa after in vitro phosphorylation with [32P-γ]ATP and CKII (lane 1) or after mock treatment (lane 2) as described in the text. Lanes 1* and 2* reveal immunodetection of the hLa proteins present in lanes 1 and 2 (see text). (D) IEF immunoblot analysis of HeLa nuclear extract, and extract prepared from S. pombe cells expressing hLa-S366 and hLa-G366 after mock or CIP treatment as indicated above the lanes. Bands are labeled as discussed in the text. Lanes 7–10 represent a parallel analysis of the samples in lanes 3–6 to show that the artifact between lanes 5 and 6 is not reproducible. Molecular Cell 2000 6, 339-348DOI: (10.1016/S1097-2765(00)00034-4)