Two primate-specific small non-protein-coding RNAs in transgenic mice: neuronal expression, subcellular localization and binding partners - PubMed (original) (raw)

Two primate-specific small non-protein-coding RNAs in transgenic mice: neuronal expression, subcellular localization and binding partners

Tasneem Khanam et al. Nucleic Acids Res. 2007.

Abstract

In a rare occasion a single chromosomal locus was targeted twice by independent Alu-related retroposon insertions, and in both cases supported neuronal expression of the respective inserted genes encoding small non-protein coding RNAs (npcRNAs): BC200 RNA in anthropoid primates and G22 RNA in the Lorisoidea branch of prosimians. To avoid primate experimentation, we generated transgenic mice to study neuronal expression and protein binding partners for BC200 and G22 npcRNAs. The BC200 gene, with sufficient upstream flanking sequences, is expressed in transgenic mouse brain areas comparable to those in human brain, and G22 gene, with upstream flanks, has a similar expression pattern. However, when all upstream regions of the G22 gene were removed, expression was completely abolished, despite the presence of intact internal RNA polymerase III promoter elements. Transgenic BC200 RNA is transported into neuronal dendrites as it is in human brain. G22 RNA, almost twice as large as BC200 RNA, has a similar subcellular localization. Both transgenically expressed npcRNAs formed RNP complexes with poly(A) binding protein and the heterodimer SRP9/14, as does BC200 RNA in human. These observations strongly support the possibility that the independently exapted npcRNAs have similar functions, perhaps in translational regulation of dendritic protein biosynthesis in neurons of the respective primates.

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Figures

Figure 1

Northern blot analysis of RNA from BC200 and Gmo22 transgenic mice. Northern blot hybridization of total RNA isolated from various tissues of BC200 (−2271)—(A); BC200 (−515), BC200 (−250)—(B); Gmo22 (−383) and Gmo22 (+1)—(C) transgenic mice as indicated in the figures was performed. Hybridization was carried with 32P-labeled oligonucleotide probes: BC207–BC200 RNA-specific (A and B) and G22-3′-specific probe for G22 RNA (C). As a loading control, all blots were hybridized with 32P-labeled 7SL_3′ oligonucleotide—specific probe for 7SL RNA. Respective RNAs are indicated. (D) Schematic representation of various constructs having deletions at 5′ and 3′ flanking regions for BC200 and Gmo22 transgenic mice.

Figure 2

In situ hybridization of transgenic BC200 RNA. (A) In the hippocampus there is robust labeling in the cell bodies and dendrites of the hippocampus proper. (B) An enlargement of hippocampal area CA1 shows clear labeling in stratum radiatum containing the apical dendrites of the pyramidal cells. (C) In the cortex overlying the hippocampus, corresponding to primary somatosensory cortex, BC200 RNA is present in most cells in all the layers; however, dendritic localization is particularly evident in the large pyramidal cells of layer V. (D) Amygdala, BC200 RNA is unevenly distributed in the various nuclei of the amygdala. The basal lateral amygdala is more strongly labeled than is the central amygdala. (E) Dendrites in the basal lateral amygdala are clearly stained. Abbr. BLA, basal lateral amygdala; CA, central amygdala; DG, dentate gyrus; gc, granule cells; ml, molecular layer; pc, pyramidal cells; sl, stratum lucidum; so, stratum oriens; sr, stratum radiatum; WM, white matter; I–VI, cortical layers.

Figure 3

In situ hybridization of transgenic BC200 and G22 RNA. (A) Dendritic localization of Gmo22 RNA, substantial labeling was observed in the CA1, CA2 and CA3 regions of the hippocampus. At higher magnification of cells in the CA1 region, labeling is evident up to the distal ends of the dendrites. In the dentate gyrus (DG), labeling in the dendritic field was more intense than in the CA1-CA3 dendritic fields. The control, with S probe did not show any labeling. (B) There is no significant labeling observed for G22 RNA in Gmo +1 transgenic animals with either the AS probe or the contol (S) probe. The higher magnification further confirms that there is no specific labeling. (C) Dendritic localization of BC200 RNA, intense signal was observed in the CA1, CA2 and CA3 areas of the hippocampus and in some of the cortical layers (Figure 2). The stratum lucidum does not show any labeling. The higher magnification reveals strong labeling in the cell bodies as well as the dendritic field of CA1. The dentate gyrus shows very low signal if at all in comparison to other areas of the brain. The control (S) probe does not show any labeling. (D) In situ hybridization experiments with (AS) probes for both BC200 RNA and G22 RNA on the sections of wild-type (WT) mice, as a control, did not show any significant labeling as evident at both lower and higher magnifications. (E) In situ hybridization showing the localization of tubulin mRNA. There is intense labeling in all the regions of hippocampus, CA1, CA2 and CA3, and also the cells in the dentate gyrus. In the cortex, the cells in almost all the layers are stained intensely. The signal is distinctly confined to the somatic region in all the cells that are labeled. The higher magnification further shows that the dendritic field in the hippocampus is devoid of any labeling.

Figure 4

BC200 RNA expression in mouse embryos. (A) E15 embryos, sagittal middle sections, S control. (B) E15 embryos, sagittal central sections through the spinal cord; AS probe. (C) E18 embryos, sagittal lateral sections through the retina; AS probe. The BC200 signal is seen in brain and retina. (D) E18 embryos, sagittal central sections through the spinal cord; AS probe. The BC200 signal is seen in brain, spinal cord and retina.

Figure 5

Comparison of human and transgenic mouse BC200 RNPs. S2 extracts from both human and transgenic mouse brains were separated on a native gel and the resulting blot was hybridized with 32P-labeled oligonucleotide (BC207) complimentary to the unique region of the human BC200 RNA gene. The first lane contains in vitro transcribed BC200 RNA.

Figure 6

Immunoprecipitation of BC200 and G22 RNPs from transgenic mouse brain. (A and B) Samples of the S2 fractions from BC200 (−2271) and Gmo22 (−383) transgenic mouse brains (same amount in each case) were immunoprecipitated with antibodies against PABP and SRP9 proteins. RNA was extracted from the precipitated complexes and separated on 7 M urea/8% polyacrylamide gels, and the resulting transfer blots were hybridized as described (see Materials and Methods). As a control in each case, RNA from complexes immunoprecipitated from the crude extract using pre-immune serum (PABP and SRP9) was loaded. In vitro transcribed BC200 RNA and G22 RNA were loaded as an additional control (first lane in each case).

Figure 7

Interaction of human PABP with BC200 RNA and G22 RNA detected by gel retardation analysis. (A and B) Interaction of human PABP with G22 RNA and BC200 RNA, respectively. 32P-labeled RNAs were incubated with human recombinant PABP protein as follows: free RNA, no protein was added; lanes 1–12: 1, 2, 3, 4, 6, 8, 12, 16, 25, 32, 50 and 62 nM PABP was added; in (B) lanes 13 and 14 represent increasing concentrations of PABP up to 100 and 125 nM, respectively. RNP complex formation between (C) 32P-labeled G22 RNA and PABP; (D) 32P-labeled BC200 RNA and PABP, in the presence of unlabeled G22 RNA and BC200 RNA, respectively for (C and D). (E) Competition assay between 32P-labeled G22 RNA and PABP, in the presence of unlabeled BC200 RNA. (F) Competition assay between 32P-labeled BC200 RNA and PABP, in the presence of unlabeled G22 RNA. Lanes indicated as follows: free RNA, no protein or competitor RNA was added; contr. RNP, initial RNP complex formed in the presence of 25 nM of PABP, no competitor RNA was added; (C–E) lanes 1–7: 2, 10, 20, 40, 80, 150 and 200 nM competitor RNA was added to the initial RNP complex; lanes 8 for (C and D), represent the same conditions as lanes 7, except that no PABP protein was added in reactions. (F) Lanes 1–11: 2, 4, 8, 17, 35, 70, 140, 200, 280, 400 and 560 nM G22 RNA was added to the initial RNP complex.

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Two primate-specific small non-protein-coding RNAs in transgenic mice: neuronal expression, subcellular localization and binding partners - PubMed (original) (raw)