8ior, himica ¢t Biophysica Acta. 1088 (1991) 151-154 © 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0167-4781/91/$03.50 ADONIS 0167478191000726

BBAEXP 90209 B B A R e p o r t - S h o r t S e q u e n c e - P a p e r

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Isolation of an active gene and of two pseudogenes for mouse U7 small nuclear R N A

A n d r e a s G r u b e r J, D o m i n i q u e Soldat i 2, M a y a Burri i . , a n d Danie l Schi imperl i i'-'

I Abteilung flit Entwicklungsbiologie. Zoologisches Institut der Umversitiit Bern. Bern and 2 lnstitut fiir Molekularbiologie H der Unwersitiit Ziirich, Ziirwh (Switzerland/

(Rec~vexl 13 November 1990)

Key words: Minor small nuclear ribonucleoprotein; Gene family: Histon¢ gene: RNA 3' processing: ( Mus muxculus)

Three U7 RNA-related sequences were isolated from mouse genomic DNA libraries. Only one of the sequences completely matches the published mouse U7 RNA sequence, whereas the other two apparently represent pseudogenes. The matching sequence represents a functional gene, as it is expressed after microinjection into Xenopus/aec/s oocytes. Sequence variations of the conserved c/s-acting 5 ' and 3' dements of U RNA genes may partly explain the low abundance of 127 RNA.

Gene expression in eukaryotes invelves complex RNA processing reactions which are. in part, catalysed by U snRNP particles [1]. The major snRNPs, U1, U2, U4/U6 and U5, participate in RNA splicing and are present in (2-10)- 10 s copies per cell. In contrast, the minor snRNPs are about 100-times less abundant. Two representatives of this group have been characterised to some extent: U7 snRNPs are involved in 3' processing of histone pre-mRNAs and U l l snRNPs are thought to play an important role in polyadenylation. Little is knowh about the genomic crganisation of minor U snRNA-encoding genes. A cluster containing five U7 genes from sea urchins has been isolated [2]. and at least three of these genes are functional [3]. We have recently isolated four human U7 pseudogenes [4]. A functional gene for mouse U7 RNA has recently been isolated (S.C. Phillips and P.C. Turner, 1990 meeting on RNA processing. Cold Spring Harbor Labo-atory, abstract p. 236), but has not yet been described in the literature. We have screened mouse genomic DNA libraries for a functional U7 gene and report on the isolation and characterisation of one such gene and of two U7 pseu- dogenes.

As probe for plaque screening of mouse genomic DNA libraries, we initially used four 16 nt long. 5'-

* Present address: lngenieurschule Burgdorf. Bur~doff. SwitzedargL Abbreviations: nL nucleotide(s): snRNA, small nuclear RNA: snRNP, small nuclear ribonucleoprotein; U, geae(s) encoding U snRNA.

Corrgsgxmdence: D. Schiimperli, Abteilung fur Entwickluagsbiologie, Zooiogisches lnstitut dot Universi~t Bern. Baltzerstrasse 4, 3012 Bern, Switzerland.

end-labelled oligonucleotides coveting the entire length of the complementary strand of mouse U7 RNA [4,5]. A BALB/c.A2G-Mx genomic library [6] (gift of H. Hug and M. Costas, University of Ziirich) and a BALB/c mouse liver genomic library (Clontech, Genofit, Geneva), both in ~ EMBL3, were screened. Hybridizing restriction fragments from three positive g recom- bi,~ants (MMU7.14, MMU7,15 and MMU7.23) were subcloned into plasmid vectors and sequenced by the didoaxy method [7] (Fig. 1A). MMU7.14 and MMU7.15 both originated from the BALB/c library and were found to be identical clones. None of the genes per- fectly match either of the two slightly differing pub- lished RNA sequences [5.8] (Fig. 1B). Furthermore, none of the typical U promoter and enhancer dements [9] are present, nor did we observe any expression of the genes after micro-injection into Xenopus laevis oocytes (data not shown). All these findings strongly suggest that the isolated sequences are pseudogenes, in contrast to four recently isolated human U7 pseudogenes [4], these rrouse genes are not followed by A-rich sequences typical for reverse transcribed pse,Jdogenes. The se- quence differences v,4th respect to U7 RNA are scattered over most of its length and even include single nucleo- tide insertions.

The failure to isolate a completely matching U7 gene suggested that the probe and hybridisation conditions used were not suflrlciently stringent. We therefore desig- ned a phage "1"7 RNA polym~.~¢ riboprobe comple- mentary to nt 3047 of moc~ U7 snRNA. Omission of the last 15 nt should prevent the probe from folding back on itself, due to the hairpin-loop present in the/37 sequence. This probe, when synthesised in vitro using

152

A MU7.14/15 (X 54747)

1TTACTTAGAG GGCTbACACT 61CTGTCTGAA~ AGTGAA~ACA

IZ1TCQGAAAACC .C_~_GAATTAC 181. AAGACCAAGA ATTTiTTATA

MMU7.23 (X 54746)

I CCCCCTTAAC ACTAATTGAG 61ACTGAAGCTC CTTTCTCTGT

121TACAATTGGC TATGTTTAAA 181 TCATAAAATG CTGATTCCTG 241 AAATAAACGC AGAGTCTTTG 301 GGCCACA~T ~iTTAG~AT 361 AAiTTTGTCT AGCATGTTTT 421 CCCCTCTGCC CTGTGTGATA 481GGGTCAAACA AGTTAAAAAG 541 GAAGGCTGTC AAGATCCACA

GCTGTTTTTA GAACGTTTTC TACCAGCTAA CCAGAGAAGG 60 GCTCTTTTAG AATTTTT~TT T_!.TCTGGCTTT CTGATTTTGA 120 TTTTAGATTT T1TTTCTAAC AGGAT~TTCG ACTTGGTCGG 180 TAGCAG 206

AAAATGCCTT ACAGCTGGAT CTCATGGAGG CATTTCCTCA 60 GATGACTCCA GCTGTGTCAA GTTGACACAA AACTAGTCAG 120 AAAAATGTAC TTGCTCTCTC TAAGAATCAA GGAGCTAAGG 180 GGATAATCAA ATGGAGAATT GGGGTACATA ATTGATTTGT 240 GTCTACAACG GATTTACCCA GAGAGAACTG CTTGGATTGT 300 Tll"TCTArar.AGGATTTCTGA CTTTGCTCAG MAA£CCAAG 360 CTGGCCTAGG ATAGATTTCA TTTAGGTGTC TCMACCCCAT 420 TAGAACTAGA TAAAGGCTTG AAGATTTGGT AAGTAAAAAT 480 GCAGTGACAT GTATATTAAA GGCTTTAAAT GAGTAATTAA S40 ATCTTTTGCA AGATTTTTAG AGTTTATATG AAA S93

B to 2.o 30 4o 5Q 6.0 U7 RNA AAGUG • U UACAGC UCUUUUAGAAUUUGUCUAGeCAGGUUUU~:UGACUUCGGUC~AAAACCCCU

BP Sm Pal i ndrOme 14/15 G AAC T TTT T C T T A AA 73 TT GCC T A T C A AA

Fig. i , Mouse U7 pseudogenes. (A) Nuclentide sequence. Specific restriction fragments from three positive ~, recomhinants (MMU7.14. MMU7.15 and MMUT.23) were subcloned into plasmid vectors and subjected to dideoxy sequencing [7]. MMUT.14 and MMUT.15 were found to be identical clones, The U7-related sequences are underlined and shown in bold letters. GenBank accession codes are given in parentheses. (B) Sequence alignments. The published U7 sequence [5] with some of its prominent features is shown on top. BP, region involved in base pairing with histone pre-mRNA; Sm, region involved in interaction with common snRNP proteins (Sin antigens). For the two pseudogenes, only the variant nneleotides

are shown. Small black squares indmcate insertions in one or both of the pseudogenes with respect to U7 RNA.

[a-32p]UTP and hybridised to a Southern blot contain- ing EcoRl-, Pstl- and Hindlll-digested mouse genomic DNA, gave rise to only three hybridising bands (data not shown). This probe was then used to screen a C57BL/6 mouse brain genomic library, again in EMI~L3 (constructed by W. Wille, University of K(51n).

Five strongly hybridising recombinants were isolated and found by restriction mapping to be overlapping clones from the same genomic location. A 3.3 kb Sacl fragment hybridising with the U7 probe was snbeloned into pSP64. This DNA was then used for dideoxy sequencing [7] (Fig. 2A). The sequence of the gene

MMUT.B21 (X 54748) -481CCATGGAGCC CAAGCTGGCC TTGAACTTCT CAGCTTTTGT GCTGGTATCA CAGAAATGTG -422 -421 GCGCTATGCC CGGTCGgcgC TGATGTTCCA GAGTCTTTGC AGTGTGGGGA ACTTGTAAGG -362 -361 ATGAGACTAC AAGACATCGG GCCACATCGC CTGCCACTAC TTAAGTCCGA TTCACTTCGG -302 -301CTTTAGCTCC AAGCCTTTAA TCTCGCGAAG CTCTTTTTTT TTTTTTAACA ACATAGGAGC -242 -241 TGTGATTGGC TGTTTTCAG¢ CAATCAGCAC TGACTCATTT GCATAGCCTT TACAAGCGGT -182 -181CACAAACTCA AGAAACGAGC GGTTTTAATA GTCTTTTAGA ATATTGTTTA TCGAACCGAA -122 -121TAAGGAACTG TGCTTTGTGA TICACATATC AGTGGAGGGG TGTGGAAATG GCACCTTGAT -62 41CTCACCCTCA TCGAAAGTGG AGTTGATGTC CTTCCCTGGC TCGCTACAGA CGCACTTCCG -Z -1 CAAgTGTTAC AGCTCTTTTA GAATTTGTCT ~AGGTTTT CTGACTTCGG TCGGAAJ~CC 59 60 CCTCCCAATT TCACTGGTCT ACkATGAAAG CAAAACAGTT CTCTTCCCCG CICCCCGGTG 119

120 TGTGAGAGGG GCTTTGATCC TTCTCTGGTT TCCTAGGAAA CC~CGTATGTG CTAGAGCCAC 179 180 GCTCTGAGAC TTCC 193

B ENm~UCER TATGC~AT -197/-205 Consensus: YATGYARAT

TATA Box CTCACC£Tf~AI~GAAAGTG -61/-39 Cc~sensus: STSACCGTGWSTRAARRTG

3' SX6NAL GTCTACAAIGAaAG~ 76/89(÷14I*27) Cc s e n s u s : GTYYNNAAARRYAGA

Fig 2. (A) Nucleotide sequence of a f~ ~ctional mouse U7 gene. Five overlapping clones were isolated. A 3.3 kb Sacl restriction fragment from clone MMUT.B21 was subcloned into ptasmid pSP04 and subjected to dideoxy sequencing [7]. Numbering starts with the first nnelentidc of the U7 coding sequent . The sequence encoding, I J7 RNA is underlined and shown in bold letters. Lower case letters represent nuclentide ambiguities due high G + C content/secondary stmcturt The GenBank accession code is given in parentheses. (B) Putative regulatory elements. Putative regulatory elements are compared with the consemas sequences of the conserved U gone enhancer, TATA Box, and 3' signal [9 I. Bold face, underlined letters

indic~¢e nneleotides that differ from the consensus sequences.

Control B21 total r.__-nntrol B21 oo~tes oocyt~ mo~eRNA oocytes oocym

M 5 5 2 .2 .02 1 5 10 1 5 10

9o ql)

76 ~ ; tRNA

67 ~ ~ U7 RNA ~ u7 (~)

153

primer (16)

A B Fig. 3. Expression of the mouse U7 gene after microinjection into Xenopus laet.~.v oocytes. Oocytes were injected [12] with 4 ng of either clone MMU7.B21 or a subclone from mouse hlstone clone 53 [13] (control oocytes). (A) Gel electrophoresis of labelled total RNA equivalent to five oocytes after injcction in the presence of [a-32p]GTP. The positions of (endogenous} tRNAs and of the U7 RNA products are indicated. M, size marker (Hpall digest of pBR322, sizes indicated in nuclcotides. (B) Primer extension analysis with 4igonucleotide cB complementary to nt 17-33 of mouse U7 RNA [5]. Either 2, 0.2 or 0.02 pg of total RNA from K21 mouse mastocytoma cells or !. 5 or I0 oocyte equivalents of total RNA from injected oocytes were used. The positions of the primer and of the U7-speclfic extension product (sizes in nucleotides in parentheses)

are indicated.

exactly corresponds to that of mouse U7 RNA as previously determined by Soldati and Schfimperli [5[

The isolated U7 gene contains an inverted version of the conserved U enhancer element [9], centered at nt - 2 0 0 (Fig. 2B). Moreover, there are somewhat mod- ified versions of the snF.NP-specific TATA box-like element and 3' signal. It therefore seemed likely that we had isolated an active gene. This was tested by micro- injection of one of the original ~ recombinants (B21) into Xenopus laevis oocytes. After injection with [a- 32p]GTP and overnight incabation, a group of labelled transcripts slightly shorter than tRNA could be de- tected which were absent ..n control oocytes injected with a clone from a mouse histone gene cluster (Fig. 3A). We tested if these trarscripts originated from the mouse U7 gene by a primer extension assay. R N A prepared from oocytes injected with the same DNAs, but in the absence of labelled nucleotide, were in- cubated w!th reverse transcriptase and oligonucleotide cB complementary to nt 17-33 of mouse U7 R N A [5]. The corresponding region of X. laevis U7 R N A differs in sequence from mouse U7 R N A so that only very faint extension products are formed with Xenopus R N A samples (Wittop Koning- T., unpublished observation, see control oocytes in Fig. 3B). in contrast, R N A from even a single oocyte injected with ), B21 (approx. 8- l0 T gene copies) yields a very strong extension product corresponding to more than 2 pg of total mouse R N A (approx. 10 5 cells). We conclude from this result that the isolated U7 gene is relatively efficiently expressed and therefore represents a functional gene. The fact that multiple bands are observed in Fig. 3A suggests some

heterogeneity at the 3' end. Precursor RNAs containing short 3' extensions have previously been observed for mammalian U! genes injected into Xenopus oocytes 1101.

An important question is why U7 R N A is so much less abundant than the major snRNAs. One possible reason could be a lower gene copy number. The number of functional UI genes has been estimated to be about 30 in mammalian genomes [9]. Another possibility could be that the signals for transcription initiation, termina- tion a n d / o r R N A processing are less efficient than for the major snRNPs. The functional gene shows several sequence variations relative to the conserved TATA box and 3' elements of U genes (Fig. 2B) which could be responsible for a reduced rate of U7 R N A production. Finally, the Sm binding site varies somewhat from the eukaryotic consensus R A U U U K U ( U ) G R which could have implications for the efficiency with which the produced U7 R N A is incorporated into stable snRNP particles. For yeast U5 RNA, it has been shown that weakening of the Sm binding site leads to reduced U5 RNA levels [11]. With a functional gene at hand, these possibilities will now become testable.

We thank E. Kurt for exceflent technical assistance. This work was supported by the States of Zfirich and Bern and by grants 3036.87 and 3100-27753.89 of the Swiss National Science Foundation.

Rderences

! Birnstiel. M.L. (1988) Structure and function of major and minor small nuclear ribonucleoprotem panicles. Springer. BeTfin.

154

2 De I_orenzi. M.. Rohrer. U. and Bimstiel. M.L. (1986) Proc. Natl. Acad. Sci. USA 83. 3243-3247.

3 Southgate. C. and Busslinger, M. (1989) EMBO J. 8. 539-549. 4 Soldali. D. and Schiimperli, D. 0990) Gene 95. in press. 5 Soldati. D. and Scbiimperli. D. (1988) Mol. Cell. Biol. 8. 1518-

1524, 6 Hug, H.. Costas. M.. Staeheli, P., Aebi, M. and Weissmann. C.

(1988) Mol. Cell. Biol. 8. 3065-3079. 7 Sanger. F.. Nicklen. S. and Coulson, A.R. (1977) Ptoc. Natl. Acad.

Sci. USA 74, 5463-5467. 8 Cotten. M.. Gick. O.. Vasserot. A., Schaffner. G and Birnstiel.

M.L. (1988) EMBO J. 7. 801-808.

9 Dahlberg. a.E. and Lund, E. 0988) in Structure and function ot major and minor small nuclear ribonucleoprotein particles (Bim- stiel. M.L, ed.), pp. 39-70, Springer. Berlin.

10 Neuman de Vegvar. H.E, and Dahlberg, J.E. (1990) Mol. Cell. Biol. 10. 3365-3375.

11 Jones. M.H. and Guthrie, C. (1990) EMBO J, 9, 2555-2561. 12 Kressmann. A., Clarkson. S.G., Pirrotta. V. and Birnstiel, M.L.

(1978) Proc. Natl. Acad. Sci. USA 75. 1176-1180. 13 Gruber, A.. Streit, A., Reist. M.. lknninger. P., I~hni. R. and

Schiimperli. D. (1990) Gene 95, in press.