Characterization of three novel imprinted snoRNAs from mouse Irm gene

Characterization of three novel imprinted snoRNAs from mouse Irm gene

BBRC Biochemical and Biophysical Research Communications 340 (2006) 1217–1223 www.elsevier.com/locate/ybbrc Characterization of three novel imprinted...

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BBRC Biochemical and Biophysical Research Communications 340 (2006) 1217–1223 www.elsevier.com/locate/ybbrc

Characterization of three novel imprinted snoRNAs from mouse Irm gene Yu Xiao, Hui Zhou, Liang-Hu Qu

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Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Zhongshan University, Guangzhou 510275, People’s Republic of China Received 17 November 2005 Available online 4 January 2006

Abstract Most, if not all, of snoRNAs in mammals are intron-encoded, implying the expressional and functional relativeness between the snoRNA and their hosts. By computational analysis of an intron database extracted from 65 known mouse imprinted genes, three novel orphan box C/D snoRNAs were identified from Irm gene which is maternally expressed and related to human disorders. The snoRNAs were positively detected and found to express in all the mouse tissues except kidney. The imprinted snoRNAs exhibit stringent structures, but quite variable in locations at their host introns, suggesting their maturation probably through a splicing independent manner. We characterized Irm as a new kind of snoRNA host gene which has no protein-coding capacity and no 5 0 TOP structure in its mRNA. The newly identified snoRNAs appear mouse-specific, however, their function remains to be elucidated. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Box C/D snoRNA; Intron; Imprinted gene; Irm; 5 0 TOP

Around 98% of all transcriptional output in humans and mice is noncoding RNA. RNA-mediated gene regulation is prevalent in higher eukaryotes. Complex biological phenomena like RNAi, co-suppression, transgene silence, genomic imprinting, methylation, RNA editing, and possibly position-effect variegation and transvection involve RNAs [1]. In addition to rRNAs and tRNAs, intron-encoded RNAs now constitute the majority of species of transcripts in complex eukaryotes. One interesting subclass of these are small nucleolar RNAs (snoRNAs), the vast majority of which are processed from intronic RNAs derived from mRNA precursors of ribosomal protein, nucleolar protein, and elongation factor genes, as well as from other genes known as UHG whose exons no longer have any protein coding potential [2,3]. The nucleolus of the eukaryotic cell is densely packed with pre-ribosomal RNAs and discrete snoRNAs that are essential compo-

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Corresponding author. Fax: +86 20 84036551. E-mail address: [email protected] (L.-H. Qu).

0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.12.128

nents involved in the mechanism of pre-rRNA processing [2,4]. Maturation of pre-rRNA includes a subtle pattern of nucleotide modifications occurring mostly on the neonatal primary transcript. Nucleotide modifications in eukaryotic rRNAs are essentially of two types: methylations (the vast majority of which are added on the ribose) and pseudouridylations [5]. To date all snoRNAs (except the RNA component of RNase MRP) belong to two major classes, the C/D box and H/ACA box snoRNAs, which are named after their common sequence motifs and structural features [6]. Box C/D snoRNAs contain two short conserved sequence motifs, box C (5 0 -RUGAUGA-3 0 , where R is any purine) and box D (5 0 -CUGA-3 0 ), always located only a few nucleotides from the 5 0 , 3 0 ends, respectively, and usually brought into close adjacency by base pairing of the four or five terminal nucleotides [7]. In general, box C/D and box H/ACA snoRNAs are about 65–80 and 120–160 bp, respectively. Based on these features experimental Rnomics and computational screening of genomic sequences are powerful and universal methods to identify box C/D snoRNAs. These two methods remarkably

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increase the box C/D snoRNA members especially in yeast and plant. Recently computational method has been applied in screening box H/ACA snoRNAs [8]. Although in most of cases the C/D snoRNAs guide rRNA 2 0 -O-ribose methylation and the H/ACA snoRNA guide rRNA pseudouridylations, many orphan snoRNAs whose functions are still elusive have been identified in mammals [9], some of which are imprinted and expressed in a tissuespecific manner. In recent years, box C/D snoRNAs located at imprinted loci have been intensively studied in mouse and human [7,10–16]. Most imprinted snoRNAs are hosted in introns of imprinted genes like most of box C/D snoRNAs while others are harbored in the not-well-known transcripts. In the majority of cases, imprinted genes exist in clusters [17] with some exceptions of ‘‘microimprinted domains’’ [18,19]. Remarkably, two imprinted snoRNAs, MBII-52 and MBII85, are dozens of times repeated by tandem repetition. Interestingly, MBII-52 is involved in an RNA editing event in nucleolus [20]. These investigations show that snoRNA might have new function(s) other than splicing, methylation, and pseudouridylation. In order to better understand the complexity of genomic imprinting and roles of small RNAs in imprinting event, we focused our attention on the identification of novel imprinted snoRNAs and the features of their host gene. In the present study, we report three novel box C/D snoRNAs which nested in the introns of a mouse imprinted gene. The expression of the snoRNAs and the characteristic of host gene were further demonstrated and discussed. Materials and methods Computational analyses. There are about 65 known imprinted genes in mouse (sources: [21,22], NCBI). All introns of the well-defined genes were extracted by a Perl script. Introns of a few genes not fully annotated were extracted by hand according to the EST data from NCBI. Intergenic sequences between imprinted genes were also extracted. All these sequences were aggregated as a database with the consideration of mRNA alternative splicing. Software automatically searches this database for box C/D snoRNA-like sequences. The software is based on module recognition model created by Lowe and Eddy [23] with adaptations on our conditions. Furthermore, the expressions of outcome snoRNA candidates were confirmed by BLAST against mouse EST library at NCBI. Nucleic acid isolation. Total cellular RNA was isolated from different tissues of male mouse (Kunming strain from the Experimental Animal Center, ZhongShan University) by the method of Chomczynski and Sacchi [24], adapted to our conditions as follows. Mice were killed by severing spinal cord. After dissection tissues were immediately washed in chilled physiological saline to remove blood, packed with silver paper, quickly frozen in liquid nitrogen, and then stored at 70 °C. Frozen samples were then ground in mortar with frequent addition of nitrogen for an hour. A fraction enriched with small-sized RNAs was obtained according to a protocol described previously [25]. Briefly, 600 ml (1–2 mg) of total cellular RNAs was combined with 75 ml each of 50% PEG8000 and 5 M NaCl, mixed gently, incubated on ice for 1.5 h, and centrifuged at 15,000g for 10 min. After adding 1/10 vol of 3 M sodium acetate and 2 vols of 95% ethanol to the supernatant, small-sized RNA was spun down at 15,000g following incubation at 20 °C for 2 h, washed with 75% ethanol, dried briefly, and re-suspended in RNase-free water. RNA samples can be free of genomic DNA and hepatin, which sometimes makes samples sticky. Northern blot and reverse transcription analysis. A 60 lg aliquot of smallsized RNAs from different tissues of mouse was separated on a denaturing

8% polyacrylamide–8 mol/L urea gel at 300 V for 3 h and then was electrophoretically transferred onto a Hybond-N membrane (Amersham) by using a Trans-BlotSD semi-dry transfer (Bio-Rad). After electroblotting, the RNAs were fixed to the membrane by baking in an oven at 85 °C for 20 min and by UV cross-linking (UVC 515 multi-linker, Ultra-lum). The 5 0 ends of the DNA probes were labeled with [c-32P]ATP (Yahui) using T4 polynucleotide kinase (Takara) and submitted to purification according to standard laboratory protocols as described previously [26]. The membrane was pre-hybridized and then hybridized as previously described [27]. The Northern blots were quantified by using a phosphorimager apparatus (Typhoon 8600, Amersham Bioscience). Reverse transcription was carried out using AMV reverse transcriptase (Promega Catalog# M5101) according to its usage information but at 45 °C. cDNA cloning and sequencing. Reverse transcription was performed as described above to produce first strand of cDNA. cDNAs in the predicted size were gel-purified and tailed with poly(dG) at the 3 0 ends using terminal deoxynucleotidyl transferase (Takara), then amplified by PCR with primers used for Northern blot and BamHI(C)16, and finally cloned into pUC-18 plasmid vector as described previously [28]. The cDNA library was screened by PCR with the P47 and P48 universal primer pair. Only the recombinant plasmids carrying fragments of the right mass were chosen for sequencing which was performed with an automatic DNA sequencer (Applied Biosystems, 377) using the Big Dye Deoxy Terminator cycle-sequencing kit (Applied Biosystems). Novel RNA sequences were confirmed by BLASTN. DNA oligonucleotides. DNA oligonucleotides were synthesized by Sangon (Shanghai). BamHI(C)16, 5 0 -GGAATTCGGATCCCCCCCCCC CCCCCC-3 0 for cloning; P47, 5 0 -CGCCAGGGTTTTCCCAGTCACG AC-3 0 for screening cDNA library and sequencing; P48, 5 0 -AGCGGATA ACAATTTCACACAGGA-3 0 for screening cDNA library; The primers used for Northern blot and reverse transcription are as follows: snoRNAA, 5 0 -ACCTATAGTTGTGATGGATTGC-3 0 ; snoRNAB, 5 0 -TCAGACTC TCAGACGCATAGT-3 0 ; snoRNAC, 5 0 -TATCAGGGTTATGGATG AGATA-3 0 ; ImsnoRNA1, 5 0 -CCTCAGACTCTCAGACGCATAG-3 0 ; ImsnoRNA2, 5 0 -ACTCAGACTCCCAGATCTGTAG-3 0 ; ImsnoRNA 3, 5 0 -TCAGACTCCCAGACCTGTGA-3 0 ; snoRNAD, 5 0 -ACTCAGACT TCCGGACCTGTTC-3 0 ; snoRNAE, 5 0 -AGTCAGGGGATTTGA GCATTCT-3 0 and snoRNAF, 5 0 -CACTCAGAGCGACAGGGTTA-3 0 . The accession numbers for the novel snoRNAs reported in this paper are as follows: ImsnoRNA1 (DQ267100), ImsnoRNA2 (DQ267101), ImsnoRNA3 (DQ267102).

Results Identification and expression analysis of three novel snoRNAs from a mouse imprinted gene A large number of snoRNA-like sequences were screened from imprinted intronic and intergenic sequences, which show some partiality for certain genes. In this study, we focused our attention on eight intron-encoded snoRNA candidates that share an imprinted host named Irm (AF498294), a novel maternally expressed gene located at mouse chromosome 12qF1 (Fig. 1). Interestingly, two brain-specific snoRNAs, MBII-343 and MBII-426, have been reported for this locus [10]. In addition, two other snoRNAs and a candidate sequence were located at thousands of bp upstream of Irm gene. With Northern blot and reverse transcription, only three, named ImsnoRNA1, ImsnoRNA2, and ImsnoRNA3, among the eight candidates were positively detected and found to express in a tissue-restricted pattern (Fig. 2A). Although the two known snoRNAs from Irm gene were previously reported as brain-specific, our data showed that the three novel snoRNAs express in all the tissues except kidney.

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Fig. 1. Schematic characterization of Irm gene. Part of genomic contig NT_039553 is represented by a blue line. Linked green boxes are exons of Irm. Three novel snoRNAs and snoRNA-like sequences are indicated by red bars and broken red bars (A–F), respectively. Black bars represent snoRNAs identified previously at Irm locus. TATA-box and poly(A) signals are also indicated. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)

Fig. 2. Positive detection and expression analyses of the three novel snoRNAs. (A) Northern analyses of the novel snoRNAs in nine different tissues of male adult mouse as specified at the top of each lane. (B) Reverse transcription analyses of the novel snoRNAs with total cellular RNA extracted from adult male mouse brain.

Reverse transcription was used to map the 5 0 end of the novel snoRNAs (Fig. 2B), and the products of the reverse transcription were cloned and sequenced. The cDNA sequences are identical to the prediction from their genes. It is showed by sequence alignment that the novel snoRNAs exhibited some similarity to MBII-343 (Fig. 3), suggesting a group of orphan snoRNAs that possess no sequence complementarity to rRNAs or snRNAs in a mouse imprinted gene.

The mouse imprinted snoRNAs are structurally stringent but variable in locations at their host introns All of the three novel snoRNAs possess the typical structure of the box C/D snoRNA, that is, (i) a typical box C and box D with existence of box C 0 and box D 0 in most of the cases, (ii) the necessity of 5 0 , 3 0 terminal stem, and (iii) a single nucleotide between 5 0 terminal stem and

Fig. 3. Sequence alignment of snoRNAs harbored inside Irm gene. Dashes denote gaps and bold dots denote sequence identities. Terminal stem sequences are underlined.

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Fig. 4. Sequence features of mouse imprinting snoRNAs. Underlines indicate nucleotides involved 5 0 , 3 0 terminal stem. C, D 0 , C 0 , and D boxes are aligned, respectively. Distances of the snoRNAs from the 3 0 splicing sites of their host introns are determined by EST sequences. Only MBII-49 has no boxD 0 . n.d., not determined.

boxC (Fig. 4). Intriguingly, the five silent snoRNA candidates have one or more deviations from standard snoRNA motif. We examined all the mouse imprinted box C/D snoRNAs and found that they all possess above stringent structures. The necessity of 5 0 , 3 0 terminal stem hints that the imprinted snoRNAs are mostly processed in a splicing independent manner [29]. It has been shown that most of guide snoRNAs in mammals have an intronic position close to 3 0 splice site [29], however, the location of the

imprinted snoRNAs in their host introns is quite variable (Fig. 4). Irm is a new kind of snoRNA host gene In all cases, the snoRNA-containing introns occur within two kinds of transcripts. One is for coding certain proteins and the other is non-protein-coding known as UHGs like UGH, U17HG, gas5, and so on, the function

Table 1 Two types of snoRNA host gene Host gene

Sequence from 5 to +10a

Host of orphan snoRNAs (without 5 0 TOP) Irm ATGACGAGGTTTCGG

SnoRNA type

Target of snoRNAb

GenBank Accession No.

Reference or source

n.d.

AF498294

n.d.

AF274590 NM_009682 AK133920 NM_175152 BC009928 Z31557 AK034512

This work; J.P. Hagan, direct submission This work; V.M. Houtzager direct submission This work; [34] This work; [35] This work; [35] This work; [36] This work; [37] This work; [35]

X67267

[32]

BC002242 AK010989 AK014003 AK032757

This This This This

AK002579 AW141322 AK010286 BC003809

This work; [35] This work; N.H. Lee, et al. direct submission This work; [35] This work; [36]

AK028204 AK088716 NM_009429

This work; [35] This work; [35] This work; [38]

Hippi

GTGCGGGCGGCGAAG

MBII-426,MBII-343, ImsnoRNA1, 2, 3 (C/D) MBII-163 (C/D)

Ap3s2 Atp2b1c Thap3c Dyskerin(DKC1)d Cctz-1 for Tcp-1 protein Serotonin receptor 5-HT2 c

TGGTACCGAGAGAAG TCGCGGTGCAGCGTG CCGCCGAGCCGCCAT AAATCGCATTGCGCA TTCGAGAAGACCCCG AGAGGGAGGTCTGAG

MBI-106 (C/D) MBII-244 (C/D) MBI-15 (H/ACA) MBI-87 (H/ACA) MBI-79 (H/ACA) MBI-36 (H/ACA)

n.d. n.d. n.d. n.d. n.d. n.d.

Host of rRNA or snRNA modification snoRNAs (with 5 0 TOP) Gas5 CTCGGCCTTTCGGAG U74-81, U44, U47 (C/D) Sorting nexin 5 TCACGAGATCTCGCG MBI-43 (C/D) rpL13 TCTAGGCCTTTCCGC MBII-202 (C/D) MBII-240 (C/D) rpL37 TCCCTGTCTTCCGGT rpS12 GTGCCGCTTTTCCCC MBII-429 (C/D) MBI-42 (H/ACA) rpL23 AACTTCCTTTCTGAC MBI-3 (H/ACA) TTTCGCCCGTGTCCC MBI-20 (H/ACA) rpP2d

28S 18S 28S 18S 28S 18S 28S 28S 28S

rpL27a Tcp-1c

CTCCTCCCTTTTCCCT GTGGTCCCCGCCGTG

rpL32-3A rpS16 TPT1

ATTAGGCTTCTTCCT GTTTAGCCTTTTCCG TCACGCTTTTCCGCC

a b c d

MBI-28 (H/ACA) MBI-39 (H/ACA) MBI-125 (H/ACA) MBI-141 (H/ACA) MBI-142 (H/ACA) MBI-161 (H/ACA)

rRNA rRNA rRNA rRNA rRNA rRNA rRNA rRNA rRNA

28S rRNA 18S rRNA U2 snRNA 18S rRNA 28S rRNA 28S rRNA

Pyrimidines are underlined. Transcription start sites (+1) are in boldface. rRNA and snRNA targets are predicted in [9] except snoRNAs from gas5. mRNAs are checked with genomic data by genome BLAST. Thus some mRNAs have been modified by several bases. All the host genes are from mouse except Dyskerin and rpP2 that are from human and rat, respectively.

work; work; work; work;

[36] [35] [35] [35]

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of whose exons (if any) is unknown. Irm gene, like UHGs, has no long enough ORF for coding protein and no favorable Kozak consensus sequence (TTTTGAATGA, ATG is the start codon) that is needed for effective translation initiation [30]. All the UHGs from mammals are characteristic of the 5 0 TOP structure [31–33], however, Irm gene does not belong to the 5 0 TOP gene family which is believed to be a common feature of snoRNA host gene no matter whether it encodes a protein or not with the exception of mouse hsc70 gene [32,33]. We have examined other hosts of snoRNAs that were identified previously (Table 1) [9]. Almost all hosts of the snoRNAs that execute their function on rRNAs, both box C/D and box H/ACA, have 5 0 TOP structure. However, the hosts of orphan snoRNAs whose functions are still obscure do not possess a 5 0 TOP structure. Interestingly, the host genes with no 5 0 TOP structure are all proteincoding except Irm. The absence of 5 0 TOP structure in Irm suggests a regulatory pathway different from the 5 0 TOP gene family. That means snoRNAs, as the products of 5 0 TOP-less transcripts, are not related with ribosome synthesis. It has been shown that the antisense RNAs commonly regulate imprinted genes. From EST database, many EST records were found to be complementary to the 3 0 end of Irm mRNA. These antisense RNAs might be involved in the expression of Irm gene and of course snoRNAs hosted within it. It is also worth noting that introns of Irm gene are remarkably larger than those of UHGs whose introns are very short. Therefore, Irm is a non-coding RNA gene, but does not belong to the known UHG family. Discussion To a certain extent the five intron-encoded snoRNAs from Irm are similar in sequence although it is not as strong as the cases of MBII-52 and MBII-85 (Fig. 3) [7,13]. In the latter cases, the snoRNAs are dozens of times repeated with little diversity between copies. The relatively low similarity between them implies a local duplication event earlier than that occurred in repeating MBII-52 and MBII-85. Why mouse cells have these dozens of copies of snoRNAs expressed at a single time is not clear. This manner offers a possible sharp increase in dosage instantly. Evolutionarily slight variation between copies might give possibility to the formation of molecules of new function. It is natural to suppose that the five snoRNAs from Irm gene might have evolved from the same origin. And even those silent snoRNA-like sequences have randomly evolved from the same origin and thus lost their activity. At the same time the other two transcripts, Rian and MBII-343 mRNAs, are located at Irm locus. Previously they were believed to be non-protein-coding just like host transcripts of MBII-52 and MBII-85 [10,16]. Our analysis of EST sequences demonstrated that MBII-343 mRNA is a splicing fragment of Irm gene and Rian might be

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another species of transcript derived from a different splicing manner. In most cases, if not all, the functions of snoRNAs are based on recognition of RNA targets by specific base pairing as other ncRNAs do [39]. Through NCBI BLAST no transcript is found to be complementary to the potential function region, i.e., 10–20 bp upstream of box D and box D 0 of three novel snoRNAs. Their functions remain conjectural. In contrast to previous results, we have shown that the expressions of the three novel snoRNAs are tissuerestricted. Their host might express in the same manner. This suggestion is supported by the analysis of Irm Unigene. Unigene has been applied to identify tissue-enriched/specific gene expression in mouse [40]. Irm’s unigene (Irm and Rian belong to the same unigene cluster), Mm.293263, shows that 75 sequences represent this locus including mRNAs, ESTs, and gene predictions supported by transcribed sequences. Among these transcripts 28 sequences are from whole brain or parts of brain and others from fetal liver, whole embryo body, pancreas, stem cell, retina, placenta, synovial fibroblasts, and a pooled cDNA library (limb, maxilla, and mandible). This suggests that Irm gene may not exclusively but highly express in brain. It implies that the targets of these snoRNA, if they have any function, are present in most of mouse tissues but plentiful in brain and testis. The 5 0 terminal oligopyrimidine tract (5 0 TOP) is a capproximal structural element (composed of an uninterrupted stretch of 4–14 pyrimidines) that confers very stringent translation regulation. In conditions of nutrient deprivation, lack of growth factors, contact inhibition, or after initiation of a differentiation program, the translation of 5 0 TOP-containing mRNAs is potently repressed [41,42]. It suggests that 5 0 TOP tracts participate in the regulation of transcription of snoRNA-containing genes, balancing the synthesis of translation-associated components with machinery that generates the ribosome [32]. Gas5 and UHGs are coding for snoRNAs involved in rRNA modification with no protein product. With consideration of ribosome synthesis these non-coding genes also need to be regulated by 5 0 TOP. It seems that the hosts of orphan snoRNAs, no matter whether coding for protein or not, do not need to be regulated by 5 0 TOP. Although being a non-coding RNA gene, Irm was recently suggested to be involved in a complex multigenic basis for linked human disorders [43]. It is interesting to know whether the snoRNAs encoded by Irm’s introns also play roles in the process related to Irm gene. Acknowledgments We thank Xiao-Hong Chen for her technical assistance. This research is supported by the National Natural Science Foundation of China (key project 30230200) and the funds from the Ministry of Education of China (No. 20030558034) and the National Basic Research Program (No. 2005CB724600).

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