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First published online 10 July 2007
doi: 10.1242/jcs.009357

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The mRNA-like noncoding RNA Gomafu constitutes a novel nuclear domain in a subset of neurons

¹ Nakagawa Initiative Research Unit, RIKEN, 2-1 Hirosawa, Wako 351-0198, Japan
² RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minamimachi, Chuo-ku, Kobe 650-0047, Japan
³ Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

View larger version (63K): [in this window] [in a new window]   Fig. 1. Identification of cell-type-specific genes expressed in a particular subset of retinal neurons. (A) Schematic drawing of the screening strategy. (1) E13.5 mouse retina was dissociated into single cells; (2) they were sorted into 96-well PCR tubes using FACS; (3) cDNAs were amplified by representative RT-PCR; (4) The cell types were retrospectively identified by the expression of Math5 (ganglion cells) and Crx (cone photoreceptor cells) and subtractive PCR was carried out using cDNA derived from single retinal ganglion (#3) and cone photoreceptor (#13) cells. (B,C) Expression pattern of cell-type-specific genes with unknown function in E14 retina obtained from putative retinal ganglion cells (B) or cone photoreceptor cells (C). Note that Th9 was assumed to be expressed in retinal ganglion cells in early migratory stages. GenBank accession numbers for #0.31, #1.46, #4.79 and #7.60 are AB294516, AB294517, AB294518, and AB294519, respectively. (D) Higher magnification images of in situ hybridization of E14 retina stained for #1.46 (left) and Math5 (right). Note that the transcript of #1.46 was exclusively localized to the nucleus. Bars, 20 µm (B,C); 5 µm (D).

View larger version (36K): [in this window] [in a new window]   Fig. 2. Gomafu RNA possesses mRNA-like characteristics. (A) Schematic illustration of the genomic structure of Gomafu. The region homologous to the EST clones AK053540 and AK028326 are shown in a bold line below. Arrowheads indicate positions of primer sets used to obtain overlapping cDNA. (B) Multiple tissue northern analysis of Gomafu RNA. Note that a single 9 kb band was detected only in the brain sample. (C) RNaseH northern analysis of Gomafu RNA. Note that no product greater than the predicted size was observed after RNaseH (RNH) digestion. Sizes of the full-length Gomafu RNA and RNaseH-digested fragments are indicated by filled and open arrowhead, respectively. The full-length transcript was detected in an overexposed blot, whose signal was relatively weak owing to insufficient transfer from the 1.5% agarose gel. (D) Splicing isoforms confirmed by RT-PCR. Frequency of each isoform among the cloned fragments is shown on the right. Arrowheads indicate positions of primer sets used to identify the splicing variants. GenBank accession numbers are listed on the right. (E) Northern blot analysis of poly(A)+ and total RNA derived from E14 brain. Gomafu RNA is enriched in the poly(A)+ fraction. (F) Northern blot analysis of stability of Gomafu RNA. Primary cultures of E15 brain cells were treated with -amanitin for indicated time. mRNA of Gomafu and -actin showed similar kinetics.

View larger version (101K): [in this window] [in a new window]   Fig. 3. Expression pattern of Gomafu during development analyzed by in situ hybridization. (A-C) Gomafu expression in the retina at E11.5 (A), P0 (B), and P7 (C). Note that the expression was restricted to the amacrine (am) and retinal ganglion cells (gc) at P7 (C). The inset in (A,B) shows higher magnification view. Arrowheads show the nuclear localization of the transcript. (D,E) Whole-mount in situ hybridization stained for Gomafu transcript at E8.5 (D) and E9.5 (E). Signals were observed in the entire neural tissues. Inset in E shows the signals obtained with the sense probe. (F-I) Gomafu expression in the brain at E14 (F), P0 (G), P7 (H), and adult (I). The horizontal line with intense staining in I is derived from artifactual folding of the section. (J-M,J'-M') Higher magnification images of the section shown in H. The signals were observed in the nucleus of mitral cells in the olfactory bulb (J,J'), pyramidal neurons of layer V in the cerebral cortex (K,K'), CA1 pyramidal neurons in the hippocampus (L,L'), and large neurons in the pontine nucleus in the hindbrain (M,M'). Nuclear counterstaining signals for DAPI are shown in J'-M' in light blue. (N,O) The marginal region of E14 retina was double labeled for Gomafu RNA (green) and a proliferating cell marker PCNA (magenta in N-N'') or BrdU (magenta in O-O''). Note that most of the Gomafu-expressing cells are post mitotic and labeled green in the merged images (N,O). Arrowheads indicate cells positive for both Gomafu RNA and the proliferation markers. Bars, 20 µm (A-C,J-O); 500 µm (D-I).

View larger version (129K): [in this window] [in a new window]   Fig. 4. Gomafu RNA forms a novel nuclear domain. (A-K) The subnuclear localization of Gomafu RNA was examined by fluorescent in situ hybridization and compared with various nuclear domain markers in E14 retinal cells (A-F) and in HeLa cells transfected with a vector expressing Gomafu cDNA (G-K). In D, cells were pre-treated with actinomycin D (ACTD). Note that Gomafu RNA signals (green) did not coincide with immunolabeled signals for SC35 (C,D,G), fibrillarin (Fbl, E,I), PML (F,J), PSP1 (H), and coilin (K), which are shown in magenta. (L-N) Double in situ hybridization of E14 retinal cells for Gomafu RNA (green) and other mRNA-like noncoding RNA Evf-1 (L), Xist (M) and Air (N), which were shown in magenta. All the images are single optical sections obtained using a laser-scanning confocal microscope. Bars, 2 µm.

View larger version (85K): [in this window] [in a new window]   Fig. 5. Intron-removed mature Gomafu RNA is localized to the nuclear matrix. (A-A'') Localization of Gomafu RNA in the nucleus of E14 retinal cell detected with probes against exon (green, A') and intron (magenta, A'') sequences. Note that intron probes detected two foci (arrows), whereas exon probes detected a number of dots diffusely distributed throughout the nucleus. (B-E) Distribution of Gomafu RNA (green) and Sytox-stained DNA (magenta) in the control (B,C) and nuclear-matrix-extracted (D,E) cells. E14 retinal cells (B,C) or Neuro2A cells transfected with a Gomafu cDNA expression vector (D,E) were subjected to fluorescent in situ hybridization. Note that Gomafu RNA remained intact after nuclear matrix preparation. (F) Northern blot analysis of Gomafu RNA in the biochemical fractions. Gomafu RNA was highly insoluble and was mostly fractionated into the nuclear matrix. Bars, 2 µm.

View larger version (93K): [in this window] [in a new window]   Fig. 6. Gomafu RNA escapes nuclear export. Gomafu RNA (green) and NLS-EGFP (magenta) were simultaneously detected in three independent heterokaryons consisting of three (A-A'''), four (B-B''') or five (C-C''') nuclei, respectively. In all the cases, NLS EGFP shuttled between the cytoplasm and nucleus and thus was observed in every nucleus of the heterokaryon (A',B',C'), whereas Gomafu RNA was detected only in the nucleus expressing this gene (A'',B'',C''; arrowheads). DAPI-stained nuclei of the heterokaryon are shown in A-C. The images were obtained using a conventional epifluorescent microscope. (D) Northern blot analysis of Gomafu RNA and mature product of UHG (U22 snoRNA host gene). Note that cytoplasmic UHG was stabilized upon inhibition of NMD by the cycloheximide treatment, whereas no change was observed in the amount of cytoplasmic (Cyto) Gomafu RNA. Bars, 5 µm.