Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization - PubMed (original) (raw)

Identification of brain-specific and imprinted small nucleolar RNA genes exhibiting an unusual genomic organization

J Cavaillé et al. Proc Natl Acad Sci U S A. 2000.

Abstract

We have identified three C/D-box small nucleolar RNAs (snoRNAs) and one H/ACA-box snoRNA in mouse and human. In mice, all four snoRNAs (MBII-13, MBII-52, MBII-85, and MBI-36) are exclusively expressed in the brain, unlike all other known snoRNAs. Two of the human RNA orthologues (HBII-52 and HBI-36) share this expression pattern, and the remainder, HBII-13 and HBII-85, are prevalently expressed in that tissue. In mice and humans, the brain-specific H/ACA box snoRNA (MBI-36 and HBI-36, respectively) is intron-encoded in the brain-specific serotonin 2C receptor gene. The three human C/D box snoRNAs map to chromosome 15q11-q13, within a region implicated in the Prader-Willi syndrome (PWS), which is a neurogenetic disease resulting from a deficiency of paternal gene expression. Unlike other C/D box snoRNAs, two snoRNAs, HBII-52 and HBII-85, are encoded in a tandemly repeated array of 47 or 24 units, respectively. In mouse the homologue of HBII-52 is processed from intronic portions of the tandem repeats. Interestingly, these snoRNAs were absent from the cortex of a patient with PWS and from a PWS mouse model, demonstrating their paternal imprinting status and pointing to their potential role in the etiology of PWS. Despite displaying hallmarks of the two families of ubiquitous snoRNAs that guide 2'-O-ribose methylation and pseudouridylation of rRNA, respectively, they lack any telltale rRNA complementarity. Instead, brain-specific C/D box snoRNA HBII-52 has an 18-nt phylogenetically conserved complementarity to a critical segment of serotonin 2C receptor mRNA, pointing to a potential role in the processing of this mRNA.

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Figures

Figure 1

Figure 1

Sequence alignments between the four human and mouse snoRNAs (A) and Northern blot analysis showing their tissue-specific expression (B-D). (A) Comparison between sequences of human and mouse snoRNAs with nucleotide substitutions indicated in red. Conserved box motifs, H and ACA for H/ACA box snoRNA and C, C′, D, and D′ for C/D box snoRNAs are underlined. For HBI-36/MBI-36 canonical location (8) of the two potential, bipartite antisense elements, AE1 and AE2, are shown by a thick overline (with dots corresponding to potential extensions of each element). Stem portions of the two-hairpin structure typical of H/ACA box snoRNA (8) are overlined by pairs of arrows in opposite orientation (hairpins 1 and 2 each contain a large internal loop). For C/D box snoRNAs, the 5-nt inverted sequence allowing formation of a typical 5′–3′ terminal stem–box C/D structure is shown by horizontal arrows. For mouse and human C/D box snoRNAs MBII-52/HBII-52 or MBII-85/HBII-85, respectively, the sequences shown are the consensus of the repeated genomic copies. For MBII-13 the 5′ terminal nucleotides of the snoRNA were not determined. In MBII-52/HBII-52, the conserved 18-nt antisense element complementary to an edited segment of the serotonin receptor 5-HT2C mRNA is also underlined (refs. and –; see Fig. 3.). (B) Northern blot analysis showing brain-specific expression of the four novel snoRNAs in mouse. (C) Northern blot analysis showing tissue-specific expression of the four novel snoRNAs in human. (D) Northern blot analysis showing expression of the rat homologues of MBI-36 and MBII-52 in different rat brain areas. On longer exposures, weak expression of MBI-36 snoRNA could also be observed in striatum, medulla, thalamus, olfactory bulb, and spinal cord. Probes for 5.8S rRNA and ubiquitous C/D box snoRNAs U21 and U40 provided internal controls. As frequently observed with other snoRNAs, some of the snoRNAs were revealed as doublets (or larger bands) reflecting the presence of some terminal heterogeneity. Sizes (in nucleotides) of the RNAs are indicated on the left.

Figure 2

Figure 2

Genomic organization of the repeated MBII-52 snoRNA genes in mouse (A), processing of the MBII-52 snoRNA from one repeat unit (B), and immunoprecipitation of MBII-13, MBII-52, and MBII-85 snoRNAs with an anti-trimethyl cap antibody (C). A) Schematic organization of the tandemly repeated units containing mouse snoRNA MBII-52-genes and the construct used for expressing the MBII-52 snoRNA locus in mouse cells. A_Bgl_II–Xho_I DNA fragment of the mouse 1.9-kb repeat unit was inserted into the eukaryotic vector pRCEN downstream from a cytomegalovirus (CMV) promoter (not drawn to scale). In the sequence of the insert, the Ipw bipartite exon G is indicated in red, the intron is in lowercase type, and the MBII-52 snoRNA (blue) is in uppercase type (the underlining denotes the location of the primer used for the reverse transcription, see_B). (B) Transient expression of MBII-52 snoRNA in transfected mouse L929 cells. The snoRNA was assayed by primer extension performed on total RNA from mouse L929 cells transfected with the construct depicted in A (lane 2) or an unrelated DNA (lane 3). Splicing of exons G1 onto G2 has been experimentally checked in this system (data not shown). Lane 1 shows control detection of MBII-52 performed on total brain RNA (from rat). The U2 snRNA content of each sample was assayed in parallel through reverse transcription with another appropriate primer to provide a control for gel loading. Lane M contains DNA marker (size in nucleotides). (C) Lack of immunoprecipitation of MBII-13, MBII-52, and MBII-85 snoRNAs with an anti-trimethyl cap antibody. Lanes: 1, input (total RNA from mouse brain); 2, RNA fraction precipitated by the R1131 antibody. The three snoRNAs were assayed by Northern blot hybridization with specific oligonucleotide probes, and positive controls were 5′ trimethyl-capped U2 snRNA and U3 snoRNA probed with appropriate oligonucleotides. Markers sizes in nucleotides are indicated to the left.

Figure 3

Figure 3

Chromosomal location of the human HBII-13,HBII-52, and HBII-85 snoRNA genes (A) and their lack of expression, assayed by Northern blot analysis, in a PWS patient (B) or in a PWS mouse model (C). (A Upper) Overview of the proximal chromosomal 15q region containing genes for PWS and Angelman syndrome (not drawn to scale). Blue boxes, paternally expressed genes (italic type)/transcribed sequences; red boxes, maternally expressed genes; open boxes, biallelically expressed genes or genes with unknown imprint status. (Lower) Fine mapping of the three snoRNA genes with regard to known genes or expressed sequences distal to SNRPN. Each vertical line below the map represents one snoRNA gene. Horizontal lines indicate the PAC and bacterial artificial chromosome clones. (B) Lack of expression of HBII-13, HBII-52, and HBII-85 snoRNAs in the brain of a PWS patient. RNA samples were taken from the cortex of a human control sample (wt brain), a PWS patient (PWS brain), and an Angelman syndrome patient (AS brain). (C) Lack of expression of MBII-13, MBII-52, and MBII-85 snoRNAs in a PWS mouse model (25). Total brain RNA samples of a wild-type mouse (wt brain), a paternal imprinting center (IC) deletion strain (IC+/ko), and a phenotypically normal mouse with a Snrpn deletion (Snrpn+/ko) were analyzed.

Figure 4

Figure 4

Structure of the serotonin receptor 5-HT2C_gene containing the intron-encoded HBI-36 gene (Upper Left) and potential base-paired interaction between the serotonin receptor 5-HT2C mRNA and C/D box snoRNA HBII-52 (Lower). (Upper Left) The six exons of the 5-HT2C gene including the alternative splice site in exon V are indicated with the location of the HBI-36 snoRNA gene within the second intron shown by a red bar (most introns of the 5-HT2C gene are extremely large; not drawn to scale). (Upper Right) Location of 47 copies of the HBII-52 snoRNA genes with respect to the PAR-4 gene in the PWS locus on chromosome 15 (see Fig. 3_A) is indicated on the right. (Lower) Potential base pairing of HBII-52 snoRNA with the editing sites/alternative splice site of exon V. The corresponding guide duplex should direct ribose methylation to the nucleotide paired to the fifth nucleotide upstream from box D (–7), i.e., the adenosine at position C. The adenosine at position C is one out of four sites of adenosine-to-inosine editing within the serotonin receptor mRNA (ref. ; the four sites of edition in 5-HT2C receptor mRNA are denoted by solid arrows and labeled A–D). The alternative splicing site present in the serotonin receptor 5-HT2C mRNA (36) is indicated by a red arrow.

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