Identification and characterization of the SMT3 cDNA and gene encoding ubiquitin-like protein from Drosophila melanogaster

Vol. 46, No. 4, November 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages 775-785

IDENTIFICATION AND CHARACTERIZATION OF THE SMT3 cDNA AND GENE ENCODING UBiQUITIN-LIKE PROTEIN FROM DROSOPHILA MELANOGASTER

1 , , and Hung-Wen Huang , Stephen C.-M. Tsoi 2 Y. Henry Sun 3

1,2,4 Steven S.-L. Li

1 Department of Biological Sciences, National Sun Yat-Sen

2 ,

University, Kaohsiung, Taiwan 80424, ROC, Dlvislon of

Intramural Research, National Institute of Environmental

Health Sciences, National Institutes of Health, Research

Triangle Park, North Carolina 27709, USA, and 3Institute of

Molecular Biology, Academic Sinica, Taipei, Taiwan 11529,

ROC.

Received June 18, 1998 ReceivedaRerrevision, August3,1998

A SMT3 cDNA encoding ubiquitin-like protein from

Drosophila melanogaster was isolated and sequenced.

Drosophila SMT3 genomic DNA was amplified by polymerase chain

reaction, and its nucleotide sequence was found to be

identical to that of the cDNA, indicating the absence of

intron in its protein coding region. The sequence of 90 amino

acids of Drosophila SMT3 exhibited 55%, 73%, 70% and 52%

identity to yeast SMT3, human SMT3A, SMT3B and SMT3C protein

The nucleotide sequence has been deposited in the GenBank

database under accession no. AF053083.

4To whom all correspondence should be addressed at NIEHS, NIH, RTP, NC27709, USA. Tel. 1-919-541-4253; FAX 1-919-541-7593; e-mail: LiSNIEHS.NIH.GOV

1039-9712/98/160775-11505.00/0 Copyright �9 1998 by Academic Press Australia.

775 All rights of reproduction in any farm reserved.

Vol. 46, No. 4, 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

sequences, respectively. Two Drosophila SMT3 transcripts of

2.5 kb and 1.5 kb were shown to be abundantly expressed in

embryo, larvae and adult tissues. The structural and

evolutionary relationships among ii SMT3 proteins from human,

mouse, Xenopus, Drosophila, nematode, Arabidopsis, rice,

Cicer, and yeast were also analyzed.

A family of ubiquitin-like proteins, including yeast

SMT3, human SMT3A, SHT3B and SMT3C, was recently discovered.

The yeast SMT3 gene was originally identified as a suppressor

of mutations in MIF2 gene, which encodes an essential protein

binding to the A+T-rich CDEII region of centromere DNA (i) .

Studies using temperature-sensitive mutants showed that the

loss of yeast Mif2p function results in chromosomes

missegregation, mitotic delay, and aberrant microtubule

morphologies (2). The Yeast Mif2 protein shares at least two

regions of similarity with mammalian centromere protein CENP-

C, an integral component of active kinetochores (3,4).

Human SMT3A gene was identified from genome project of

chromosome 21 (5). We have cloned human SMT3B (formerly

designated as HSMT3) CDNA (6). Human SMT3C protein was

independently isolated by several groups and denoted as

SU-MO-I (7), GMPI (8), PICI (9), UBLI (i0), sentrin (ii).

SUMO-I/GMPI was found to be covalently linked to the Ran

GTPase-activating protein RanGAPI, and attachment of SUMO-I

targets the otherwise cytosolic RanGAPI to the nuclear pore

complex. The modified form of RanGAPI also appeared to

associate with the mitotic spindle apparatus during mitosis

(7,8). PICI was shown to interact with the PML component of

nuclear multiprotein complex that is disrupted in acute

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Vol. 46, No. 4, 1998 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL

promyelocytic leukemia (9). UBLI was found to associate with

human RAD51/RAD52 proteins involved in DNA recombination and

DNA double-strand break repair (i0). Sentrin was shown to

interact with Fas/APO-I or TNF receptor i death domains, and

the overexpression of sentrin provided protection against

both anti-Fas/AP0-1 and TNF-induced cell death (ii) o

Here we report the cloning of a SMT3 cDNA, mRNA

expression, and characterization of genomic DNA from

Drosophila melanogater. We have also analyzed the structural

and evolutionary relationships of ii SMT3 protein sequences

from human, mouse, Xenopus, Drosophila, nematode,

Arabidopsis, rice, Cicer, and yeast.

MATERIALS AND METHODS

Isolation and characterization of SMT3 cDNA and gene.

Human SMT3C protein sequence was used as query to blast the

Drosophila EST database, and an EST (GenBank accession no.

AA264131) was found to contain homologous sequence. The two

polymerase-chain-reaction (PCR) primers based on the EST

sequence were: forward 5'-CATGTCTGACGAAAAGAAGGGAGG-3' and

reverse 5'-GTGGCGCTCATAAGATTACTTAF-3'. The mixed cDNAs

prepared from Drosophila adult cDNA library (Stratagene, La

Jolla, CA) were used as templates for PCR with the Expand

High Fidelity (Boehringer Mannheim) containing a mixture of

thermostable Taq and Pwo DNA polymerase. PCR conditions were:

1 cycle of 2 min. denaturation, 5 cycles (denaturation, 30

sec at 94~ annealing, 30 sec at 62~ and elongation, 2

min. at 68~ and 25 cycles (denaturation, 30 sec at 94~

annealing and elongation, 2 min. at 68~ The PCR-amplified

777


fragment was sequenced and labeled using PCR-Digoxigenin

(DIG) labeling kit (Boehringer Mannheim). This labeled DNA

probe was then used to screen the cDNA library, and a

positive cDNA clone was isolated. Two additional PCR primers

indicated in Fig. 1 were used to amplify SMT3 DNA using

templates of genomic DNA (BIOS laboratories, New Haven, CT,

USA) and cloned SMT3 cDNA. The sizes of PCR-amplified

fragments from SMT3 cDNA and genomic DNA were compared on

1.0% agarose gel. The purified DNA fragments amplified from

these two templates were labeled with the Dye Terminator kit

(Perkin-Elmer, Foster City, CA, USA), and both strands of the

amplified DNAs were completely sequenced using automatic DNA

sequencer (Applied Biosystem, USA, model 377).

Northern blot analysis. The poly(A§ from embryo,

larvae and adult were obtained from Clontech (Palo Alto, CA).

Approximately 2 ~g of each poly(A+)-RNAs were ran on a 1.2%

formaldehyde agarose gel for 3 hours at i00 volts and

transferred upward to a Hybond-N § membrane (Amersham Life

Science, Cleveland, OH) by capillary action for an hour. The

membrane was fixed by UV crosslinking and then hybridized

with DIG-labeled probe (25 ng/ml) in DIG Easy hybridization

solution. Prehybridization and hybridization conditions were

strictly followed according to the Genius System User's guide

(version 3.0) except the temperature was 45~ The specific

SMT3 transcripts were detected by DIG luminescent detection

kit.

Analyses of structural and evolutionary relationships

among SMT3 proteins. The complete amino-acid sequence of

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Drosophila SMT3

CACGCCCGGCATTCGACGCTCCGCAAAAGAAAAAAAAACTTTTTTGACCACTTA GCAGC TTCAACAAGCAAC CAAAAAATCAACATGTCTGACGAAAAGAAGGGAGGT

M S D E K K G G 8

GAGACCGAGCACATCAACCTGAAGGTCCTCGGCCAGGACAACGCCGTCGTCCAG E T E H I N L K V L G Q D N A V V Q 26 TTCAAGATCAAGAAGCACACACCCTTGAGGAAGCTGATGAACGCCTACTGCGAC F K I K K H T P L R K L M N A Y C D 44 CGTGCCGGACTCTC CATGCAGGTGGTGCGCTTCCGTTTCGACGGACAGCCCATC R A G L S M Q V V R F R F D G Q P I 62 AACGAGAACGACAC TCCGACCTCGCTGGAGATGGAGGAGGGCGACACCATCGAG N E N D T P T S L E M E E G D T I E 80 GTTTACCAGCAGCAGAC TGGTGGCGCTCCATAAGATTACTTAGTTAAGTTAGTT V Y Q Q Q T G G A P * 90 AC TC CTC TTACAAC TACACAC TTAAAACAAAAAAGAAAAAAAATACAAGAAAAA C CACAAAAGCAAAAACACAACAACAACAACATGAAGAATCCAACAAACCAGGC C CTAAGAATCGATTGAATATGCTTTTAGTACAACTGTAGATTCTAAATGCGTCTG TGTGCGTTAATAACAAAAACATTTGCAGACAAGAAAATGGT

Fig. i. The nucleotide and deduced amino acid sequences of

Drosophila SMT3 cDNA.

The stop codon TAA is indicated by an asterisk. Two PCR primers used are underlined.

Drosophila melanogaster SMT3 protein was deduced from the

cDNA and gene sequences determined in this investigation

(Genbank accession no. AF053083). The names of the organisms

and the Genbank accession numbers of other reported SMT3

proteins are as follows: Human, Homo sapiens (SMT3A, X99584;

SMT3B, L76416; SMT3C, X99586); mouse, Mus musculus (SMT3C,

AF033353), African frog, Xenopus laevis (SMT3C, Z97073);

nematode, Caenorhabditis elegans (U94830); Thale cress,

Arabidopsis thaliana (X99609); Rice, Oryza sativa (X99608);

chickpea, Cicer arietinum (AJ001901) and yeast, Saccharomyces

cerevisiae (U27233). The amino-acid sequences were aligned

using the multiple alignment program in GENEWORK computer

package based on the method of Feng and Doolittle (12). The

structural and evolutionary relationships were analyzed using

UPGMA method from the same computer program (13).

779


RESULTS AND DISCUSSION

Characterization of SMT3 cDNA and gene. A Drosophila SMT3

cDNA clone was isolated and its sequence of 581 nucleotides

determined (GeneBank accession no. AF053083). This SMT3 cDNA

contains a protein-coding sequence of 270 nucleotides, 5' (85

nucleotides) and 3' (226 nucleotides) untranslated regions

(Fig. i) . The sizes of PCR-amplified fragments, including

protein-coding region, from SMT3 cDNA and genomic DNA were

found to be the same on 1.0% agarose gel (Fig. 2), and their

sequences of 463 nucleotides were determined to be identical.

These results indicated the absence of intron in the protein-

coding region of Drosophila SMT3 gene. It is of interest that

the protein-coding sequence of nematode SMT3 gene is

interrupted by two small introns of 56 and 50 bp at coden

nos. 22-23 and 56, respectively (14).

Northern blot Analysis. The results of Northern blot

analysis indicated that two SMT3 transcripts of 2.5 kb and

1.5 kb were expressed in embryo, larvae and adult tissues,

although the transcript of 1.5 kb is most abundantly present

in larvae sample (Fig. 3). These size differences may be due

to either different polyadenylation sites used or alternative

splicing of the same SMT3 gene. It is also possible that they

represent different transcripts of two similar SMT3 genes.

Further, one might wonder why the small SMT3 protein of 90

amino acids requires such large transcripts.

Amino acid sequence comparison among SMT3 proteins. The

sequence of 90 amino acids deduced from the Drosophila SMT3

780

Vol. 46, No. 4, 1998 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL

1 2 3 Kb b p

4 6 3 ~

I 1.0

0.5

Fig. 2. Size comparison of PCR:amplified fragments using

Drosophila genomic DNA and SMT3 cDNA as templates.

Lane i, genomic DNA as template; lane 2, SMT3 cDNA as

template; lane 3, size marker of 1 kb DNA ladder (BRL Life

Technologies).

I 2 3 Kb

2.5

1.5

Fig. 3. Northern blot analysis of Drosophila SMT3

transcripts.

Lane i, larvae; lane 2, adult; lane 3, embryo.

781

Vol. 46, No. 4, 1998 BIOCHEMISTRYand MOLECULAR BIOLOGY INTERNATIONAL

~ A

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cDNA and gene sequences determined in this investigation was

aligned with the SMT3 protein sequences from human, mouse,

Xenopus, Drosophila, nematode, Arabidopsis, rice, Cicer and

yeast available in the databases (Fig. 4). The deduced

sequence of 90 amino acids from the Drosophila SMT3 cDNA and

gene exhibited 55%, 73%, 70% and 52% identity to the yeast

SMT3, human SMT3A, SMT3B and SMT3C protein sequences,

respectively. Human and mouse SMT3C proteins have identical

sequences. There are 21 identical residues out of Iii

positions compared among these ii SMT3 sequences. The

Human SMT3A

Human SMT3B

Drosophila SMT3

Human SMT3C

Mouse SMT3C

- - Xenopus SMT3C

Nematode SMT3

Arabidopsis SMT3

Rice SMT3

Cicer SMT3

Yeast SMT3

Fig. 5. Structural and evolutionary relationships among ii

SMT3 proteins.

The graphical display of structural and evolutionary

relationships among the ii SMT3 was obtained using UPGMA

methods (13).

783


significant amino acid identity and similarity of these SMT3

proteins among different species indicate their functional

importance. The SMT3C proteins from human, mouse and Xenopus

were reported to exhibit diverse functions (7-11, 15-16).

However, the exact function(s) of human SMT3A and SMT3B,

Drosophila, nematode and plant SMT3 proteins remain to be

elucidated experimentally. The structural relationships among

these ii SMT3 proteins from human, mouse, Xenopus,

Drosophila, nematode, Arabidopsis, rice, Cicer and yeast were

analyzed and the evolutionary tree constructed using UPGMA

method (13). Results are presented in Fig. 5. Drosophila SMT3

is clustered with human SMT3A and SMT3B, while nematode SMT3

is clustered with human, mouse and Xenopus SMT3C. Three plant

SMT3 are clustered into a separate group, and the yeast SMT3

is the out group.

ACKNOWLEDGMENTS

We thanks Drs. Frank Johnson and Po-Chuen Chan for

reading the manuscript. This investigation was supported in

part by grants NSC85-2732-B-II0-002 and NSC86-2313-B-II0-002

from National Science Council of Taiwan, ROC.

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Identification and characterization of the SMT3 cDNA and gene encoding ubiquitin-like protein from Drosophila melanogaster

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