Xenopus U8 snoRNA Binding Protein Is a Conserved Nuclear Decapping Enzyme  Trina Ghosh, Brian Peterson, Nenad Tomasevic, Brenda A Peculis  Molecular Cell  Volume 13, Issue 6, Pages 817-828 (March 2004) DOI: 10.1016/S1097-2765(04)00127-3

Figure 1 cDNA for X29 Protein The complete nucleotide sequence for X29 is shown with a translation below. The initiating methionine is indicated in bold. Underlined peptides were obtained by Edman sequencing or MS/MS. The NUDIX consensus sequence is boxed, with the adjacent glutamic acid residues essential for catalysis indicated. The KK in the NUDIX domain indicates the X29 EE→KK mutation (see text). Degenerate oligonucleotides corresponding to peptides labeled #7 and #8 (dashed underline) yielded the 350 bp fragment by RT-PCR. X29 Genbank Accession Number, AY423379. Molecular Cell 2004 13, 817-828DOI: (10.1016/S1097-2765(04)00127-3)

Figure 2 Sequences and Structures in U8 RNA Required for X29 Binding (A) Gel mobility shift competition assays were performed using full-length m7G cap U8 RNA (lane 1, 6, 11, and 16). Addition of X29 results in complex formation (lanes 2, 7, 12, and 17). Four different cold competitor RNAs were added at 30, 60, or 100 molar fold excess, as indicated. A schematic of full-length U8 RNA and the three truncated constructs used for competitions, U8 5′40 (lanes 7–10), U8 5′40CD (lanes 12–15), and U8 3′100 (lanes 17–20), are shown above the relevant data panel. Mobility of free RNA and the X29-U8 complex is indicated. (B) Labeled U8 RNA synthesized in the presence of cap analogs, as indicated, was used in gel mobility shift assays performed in the presence of 20 mM EDTA. Molecular Cell 2004 13, 817-828DOI: (10.1016/S1097-2765(04)00127-3)

Figure 3 Western Blot Analysis Shows X29 Protein Is Nuclear in Xenopus Oocytes Western blot analysis was performed using overexpressed His-tagged X29 protein (lanes 1 and 5), crude extract from Xenopus ovary (lanes 2 and 6), five oocyte cytoplasmic equivalents (lanes 3 and 7), or five oocyte nuclear equivalents (lanes 4 and 8) incubated with antibodies directed against X29 or LSm 8 (a nuclear protein). Mobility of molecular mass markers is indicated on the left. Arrow indicates LSm 8. Overexpressed X29 migrates slower (at 32 kDa) than the endogenous X29 protein (at 29 kDa, indicated by “*”) due to the presence of epitope tags. Molecular Cell 2004 13, 817-828DOI: (10.1016/S1097-2765(04)00127-3)

Figure 4 Immunofluorescence in Xenopus Tissue Culture Cells Shows X29 Is Primarily Nucleolar Xenopus tissue culture cells were grown and fixed on glass slides, then incubated with antibodies against X29 or fibrillarin, as indicated. The center panels are a merged image of the antibody plus DAPI staining of the DNA to define the nucleus. In (B), fibrillarin staining is visible in the three cells in interphase; fibrillarin relocates during mitosis (Ochs et al., 1985). Molecular Cell 2004 13, 817-828DOI: (10.1016/S1097-2765(04)00127-3)

Figure 5 X29 Protein Contains Intrinsic Decapping Activity that Releases m7GDP from U8 RNA Demonstrating High Substrate Specificity (A) Decapping reactions were performed with m7G-capped U8 RNA (lane 1) with control proteins, BSA (750 ng) (lanes 2 and 7), purified nuclear LSm complexes (lanes 3 and 8), or with X29 protein (lanes 4 and 9), X29 EE→KK mutant protein (lanes 5 and 10), or human H29K protein (lanes 6 and 11) with Mg+2. (B) An aliquot of the reaction products in (A) was treated with NDPK (lanes 7–9), as indicated. (C) SDS gel (14% Tris-glycine) containing 1 μg each of overexpressed X29 protein, X29 EE→KK mutation, and human H29K protein, stained with Coomassie blue. Size markers are shown on the left. (D) m7G cap-labeled RNAs, as indicated, were incubated with Mg+2, without (−) or with (+) two levels (1X or 5X) of X29 protein. RNAs used: U8 snoRNA, U3 snoRNA, U13 snoRNA, 5S rRNA, and NO38 (mRNA with no polyA tail). (E) Decapping reactions performed with m7G U8 RNA, with (+) or without (−) X29, incubation for 30 min with either Mg+2 or Mn+2. (F) Decapping of 5S RNA by X29 in Mn+2. X29 was added as indicated (1X and 5X protein) to m7G 5S or GpppG 5S. At 2 or 30 min of incubation time points were taken. (H) Time course of decapping of GpppG U8 RNA incubated with X29 in the presence of 1 mM Mn+2. Addition of NDPK to the reaction confirmed this was GDP as it was converted to GTP, migrating barely above the origin. In all TLC panels, standards were simultaneously developed on the same plate and their mobilities are indicated. (G) Time course of decapping GpppG U8 RNA incubated with X29 in the presence of 1 mM Mg+2. Molecular Cell 2004 13, 817-828DOI: (10.1016/S1097-2765(04)00127-3)

Figure 6 X29 Cleaves Capped RNAs and Leaves a 5′ Monophosphate (A) U8 or U3 RNAs were transcribed in the presence of GpppG (G), m7GpppG (m7G), or m227G pppG (TMG) cap analogs as indicated. RNAs were incubated in a decapping reaction then ligated to a radiolabeled tag RNA of 30 nt in length via a bridge oligo (schematic at top, see text and Experimental Procedures). Ligation products were resolved on a denaturing 8% acrylamide gel. Autoradiography displayed only the ligated products. Capped RNAs are not a substrate for ligation since a 5′ monophosphate is required. (B) Xenopus oocytes were microinjected with 5 pMol-labeled U8 RNA synthesized with a GpppG or no cap (pppG). At the time interval indicated (in minutes), total RNA was isolated from the oocytes and resolved on a denaturing acrylamide gel. Autoradiography demonstrates the instability of uncapped U8 RNA in vivo. Molecular Cell 2004 13, 817-828DOI: (10.1016/S1097-2765(04)00127-3)

Figure 7 Overexpression of X29 In Vivo Affects Pre-rRNA Processing Cells were transiently transfected with X29 plasmid and harvested at the time points indicated (hours after transfection) and total RNA or protein was isolated. (A) Western blot analysis of proteins resolved on a 14% SDS gel, detected with anti-X29 antibody. (B) Northern blot analysis of RNA resolved on a denaturing 8% acrylamide gel. The membrane was sequentially hybridized with probes specific for U8 RNA and ITS2. 12S is a 3′ extended precursor to 5.8S rRNA containing approximately 150 nt of ITS2. The increase in RNA levels at 72 hr may be due to loss of plasmid in the transient transfection; protein levels decrease slightly at the 72 hr time point. Bands on Northerns were quantitated on a Fuji Phosphorimager, normalized to hybridization signal of 5S rRNA. U8 levels drop from 100% at T = 0 to 93%, 71%, and 87% over the three time points shown; 12S levels drop from 100% at T = 0 to 87%, 73%, and 79%. (C) Quantification from independent repetitions (n = 3) of the data shown in (B). In these transient transfections, in situ hybridization indicated about 30% of the cells express exogenous X29. Molecular Cell 2004 13, 817-828DOI: (10.1016/S1097-2765(04)00127-3)