gb-2007-8-10-r216 GBJ Research <p>The ribosomal protein genes and <it>Minute </it>loci of <it>Drosophila melanogaster</it></p> Marygold J Steven s.marygold@gen.cam.ac.uk Roote John j.roote@gen.cam.ac.uk Reuter Gunter reuter@genetik.uni-halle.de Lambertsson Andrew g.a.lambertsson@imbv.uio.no Ashburner Michael m.ashburner@gen.cam.ac.uk Millburn H Gillian g.millburn@gen.cam.ac.uk Harrison M Paul paul.harrison@mcgill.ca Yu Zhan zyu9@cs.mcgill.ca Kenmochi Naoya kenmochi@med.miyazaki-u.ac.jp Kaufman C Thomas kaufman@bio.indiana.edu Leevers J Sally sally.leevers@cancer.org.uk Cook R Kevin kcook@bio.indiana.edu

Growth Regulation Laboratory, Cancer Research UK London Research Institute, Lincoln's Inn Fields, London WC2A 3PX, UK

Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK

Institute of Genetics, Biologicum, Martin Luther University Halle-Wittenberg, Weinbergweg, Halle D-06108, Germany

Institute of Molecular Biosciences, University of Oslo, Blindern, Olso N-0316, Norway

Department of Biology, McGill University, Dr Penfield Ave, Montreal, Quebec H3A 1B1, Canada

Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan

Department of Biology, Indiana University, E. Third Street, Bloomington, IN 47405-7005, USA

 Genome Biology 1465-6906 2007 8 10 R216 http://genomebiology.com/2007/8/10/R216 17927810 10.1186/gb-2007-8-10-r216 17 6 2007 10 10 2007 10 10 2007 10 10 2007 2007 Marygold et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular characterization of Minute loci

A combined bioinformatic and genetic approach was used to conduct a systematic analysis of the relationship between ribosomal protein genes and Minute loci in Drosophila melanogaster, allowing the identification of 64 Minute loci corresponding to ribosomal genes.

Abstract

Background

Mutations in genes encoding ribosomal proteins (RPs) have been shown to cause an array of cellular and developmental defects in a variety of organisms. In Drosophila melanogaster, disruption of RP genes can result in the 'Minute' syndrome of dominant, haploinsufficient phenotypes, which include prolonged development, short and thin bristles, and poor fertility and viability. While more than 50 Minute loci have been defined genetically, only 15 have so far been characterized molecularly and shown to correspond to RP genes.

Results

We combined bioinformatic and genetic approaches to conduct a systematic analysis of the relationship between RP genes and Minute loci. First, we identified 88 genes encoding 79 different cytoplasmic RPs (CRPs) and 75 genes encoding distinct mitochondrial RPs (MRPs). Interestingly, nine CRP genes are present as duplicates and, while all appear to be functional, one member of each gene pair has relatively limited expression. Next, we defined 65 discrete Minute loci by genetic criteria. Of these, 64 correspond to, or very likely correspond to, CRP genes; the single non-CRP-encoding Minute gene encodes a translation initiation factor subunit. Significantly, MRP genes and more than 20 CRP genes do not correspond to Minute loci.

Conclusion

This work answers a longstanding question about the molecular nature of Minute loci and suggests that Minute phenotypes arise from suboptimal protein synthesis resulting from reduced levels of cytoribosomes. Furthermore, by identifying the majority of haplolethal and haplosterile loci at the molecular level, our data will directly benefit efforts to attain complete deletion coverage of the D. melanogaster genome.

 Bioinformatics Genetics Model organisms

Background

Ribosomes are sophisticated macromolecular machines that catalyze cellular protein synthesis in all cells of all organisms. They have an ancient evolutionary origin and are essential for cell growth, proliferation and viability. Though larger and more complex in higher organisms, both the structure and function of ribosomes have been conserved throughout evolution. Genetic approaches in Drosophila melanogaster have shown that disrupting ribosome function can result in an array of fascinating dominant phenotypes 12. Despite this, there has so far been no comprehensive inventory of genes encoding ribosome components in this organism, nor any systematic effort to determine their mutant phenotypes.

All ribosomes comprise a set of ribosomal proteins (RPs) surrounding a catalytic core of ribosomal RNA (rRNA). Bacteria possess a single type of ribosome composed of three rRNA molecules and typically 54 RPs. All eukaryotic cells, in contrast, contain at least two distinct types of ribosomes: cytoplasmic ribosomes (cytoribosomes) and mitochondrial ribosomes (mitoribosomes). Cytoribosomes are found on the endoplasmic reticulum and in the aqueous cytoplasm. They translate all mRNAs produced from nuclear genes and perform the vast majority of cellular protein synthesis. Each cytoribosome contains four different rRNAs and 78-80 cytoplasmic RPs (CRPs). Mitoribosomes consist of only two rRNA molecules and up to 80 mitochondrial RPs (MRPs). They are located in the mitochondrial matrix and synthesize proteins involved in oxidative phosphorylation encoded by those few genes retained in the mitochondrial genome. A third unique type of eukaryotic ribosome is found within the plastids (for example, chloroplasts) of plant and various algal cells. In all cases, distinct small and large ribosomal subunits exist that join together during the translation initiation process to form mature ribosomes capable of protein synthesis. (See references 3456 for general reviews of ribosomal structure and function.)

The protein components of ribosomes are interesting from several points of view. First, and most obviously, RPs play critical roles in ribosome assembly and function 7. Second, several RPs perform important extra-ribosomal functions, including roles in DNA repair, transcriptional regulation and apoptosis 68. Third, misexpression of human CRP and MRP genes has been implicated in a wide spectrum of human syndromes and diseases, including Diamond-Blackfan anaemia 9, Turner syndrome 10, hearing loss 11 and cancer 12. Fourth, mutations in the CRP genes of D. melanogaster are important tools for the study of growth, development and cell competition 2. Finally, many RPs are conserved from bacteria to humans, so their peptide and nucleotide sequences are useful for studying phylogenetic relationships 13.

The first eukaryotic CRPs characterized in detail were isolated from the rat cytoribosome 3. Individual proteins were separated by two-dimensional gel electrophoresis and named from their origin in the small (S) or large (L) subunit and their relative electrophoretic migration positions, for example, RPS9 or RPL28. Subsequent studies revealed that some protein spots contained non-ribosomal proteins or chemically modified versions of another CRP, and that some spots contained two co-migrating CRPs 35. Consequently, the nomenclature system used today contains numerical gaps as well as 'A' suffixes for those additional CRPs not resolved by the original electrophoresis (for example, RPL36A). Seventy-nine distinct mammalian CRPs are now acknowledged and their amino acid sequences and biochemical properties have been described 514. With the exception of RPLP1 and RPLP2, each of which forms homodimers in the cytoribosomal large subunit 15, all CRPs are present as single molecules in each cytoribosome 3.

Seventy-eight different mammalian MRPs have been described 6 and their individual amino acid sequences and biochemical properties have been determined 1617. Although the nomenclature of MRPs was originally based on electrophoretic properties, the current system reflects homology between mammalian MRPs and their bacterial orthologs 18. Thus, MRPS1 through MRPS21 are orthologous to Escherichia coli RPs S1-S21, while higher numbers have been assigned to the MRPs not found in bacteria. Gaps also exist in MRP numbering because a gap occurs in the bacterial enumeration or because there is no mammalian ortholog.

The RPs of D. melanogaster were first studied in the 1970s and early 1980s. Up to 78 individual CRPs were observed on two-dimensional gels 19202122232425262728293031 and about 30 were purified and analyzed biochemically 3233. A more recent characterization used mass spectrometry to identify 52 D. melanogaster CRPs 34, all of which are orthologous to known mammalian CRPs. The protein composition of Drosophila mitoribosomes has not been characterized biochemically to date.

CRPs and MRPs are encoded by the nuclear genome. Knowledge of the primary sequences of rat CRPs and bovine MRPs has led to the identification and mapping of the RP-encoding genes in many eukaryotic species 14. Indeed, systematic analyses of whole RP gene sets have been described for several organisms, including Saccharomyces cerevisiae 35, Arabidopsis thaliana 36 and humans 37383940. However, the complete set of D. melanogaster CRP and MRP genes has not been previously documented or characterized.

Several D. melanogaster RP genes were initially identified by virtue of their dominant 'Minute' mutant phenotypes 2, which include prolonged development, low fertility and viability, altered body size and abnormally short, thin bristles on the adult body. All of these phenotypes may be explained by a cell-autonomous defect in protein biosynthesis: the production of each bristle, for example, requires a very high rate of protein synthesis in a single cell during a short developmental period. Merriam and colleagues reported the first unequivocal molecular link between a Minute locus and a CRP gene in 1985 41. Since then, 14 additional Minute loci have been definitively linked with distinct CRP genes 2424344454647484950515253. However, there are at least 35 genetically validated Minute loci that have not yet been associated with a specific gene and there may be additional Minute genes to be discovered. Several investigators have hypothesized that all Minute loci encode protein components of ribosomes (reviewed in 2). Whether this is truly the case and whether both CRP and MRP genes are associated with Minute phenotypes are open and intriguing questions.

Many Minute loci were originally identified from the phenotypes of flies heterozygous for a chromosomal deletion 5455 and all Minute point mutations studied in depth have been found to be loss-of-function alleles 2. This indicates that Minute phenotypes can be attributed to genetic haploinsufficiency; that is, a single gene copy is not sufficient for normal development. (Note that X-linked mutations that cause Minute phenotypes in heterozygous females are lethal in hemizygous males.) The most popular explanation for the haploinsufficiency of Minute loci is that they correspond to RP genes and that RPs are required in equimolar amounts: halving the copy number of a single RP gene limits the availability of the encoded RP, thereby reducing the number of functional ribosomes that are assembled in the cell and impairing protein synthesis 2. While this idea is consistent with the available data, there may be other explanations.

The reduced fertility and viability associated with many Minute loci makes the recovery of deletions uncovering them rather difficult - the mutant strains are too weak to maintain as stable heterozygous stocks. In fact, some Minute loci are known only from the phenotypes of transient aneuploids 5456. This means that several chromosomal regions containing a Minute locus are not uncovered by current deletion collections 57. This is frustrating for researchers because deletions are basic tools for mutational analysis and are widely used for mapping new mutations and identifying genetic modifiers. Efforts to maximize deletion coverage of the D. melanogaster genome would benefit from a systematic assessment of the relationship between RP genes and Minute loci. It would allow the isolation of deletions that flank haploinsufficient RP genes as closely as possible, or the design of transgenic constructs or chromosomal duplications to rescue the haploinsufficiency of deletions uncovering Minute genes.

Here, we report the systematic identification, naming and characterization of all the CRP and MRP genes of D. melanogaster. We have used this information, together with phenotypic data obtained from examining mutation and deficiency strains, to assess the correspondence between RP genes and Minute loci. We find that 66 of the 88 CRP genes identified are, or are very likely to be, haploinsufficient and associated with a Minute phenotype, whereas MRP genes and the remaining 22 CRP genes are not. Significantly, we show that all but one of the known Minute loci in the genome correspond to CRP genes - the single exception encodes a subunit of an essential translation initiation factor. Together, these results identify the majority of haploinsufficient loci in the D. melanogaster genome that significantly affect viability, fertility and/or external morphology, and also provide a mechanistic framework for understanding the Minute syndrome and the phenotypic effects of aneuploidy.

Results

Identification of D. melanogaster ribosomal protein genes

In order to conduct an exhaustive survey of Drosophila CRP and MRP genes, we first performed a series of BLAST searches using human RP sequences as queries, because both CRPs and MRPs have been well-characterized in humans 56. Tables 1 and 2 list the genes we identified together with their cytological locations. Where necessary, D. melanogaster genes were named or renamed according to the standard metazoan RP gene nomenclature proposed by Wool and colleagues 55859 and approved by the HUGO Gene Nomenclature Committee 18, whilst still conforming to FlyBase 60 conventions - that is, CRP genes are given an 'Rp' prefix and MRP genes have an 'mRp' prefix. The seven exceptions to this standard RP nomenclature are mostly genes originally named to reflect a mutant phenotype, for example, the string of pearls (sop) gene encodes RpS2 61 and bonsai encodes mRpS15 6263. In these cases, the original gene symbol has been preserved, with the apposite RP symbol given as a synonym.

<p>Table 1</p>

The CRP genes of D. melanogaster

D. melanogaster gene

Human CRP

Symbol*

CG number

Location†

BLAST E value‡

RPSA

sta/RpSA

CG14792

X: 2B1

3e-94

RPS2

sop/RpS2

CG5920

2L: 30E1

e-118

RPS3

RpS3

CG6779

3R: 94E13

e-106

RPS3A

RpS3A

CG2168

4: 101F1

e-100

RPS4

RpS4

CG11276

3L: 69F6

e-121

RPS5

RpS5a

CG8922

X: 15E5-7

1e-98

RpS5b

CG7014

3R: 88D6

3e-96

RPS6

RpS6

CG10944

X: 7C2

e-103

CG11386

CG11386

X: 7C2

2e-15

CG33222

CG33222

X: 7C2

2e-15

RPS7

RpS7

CG1883

3R: 99E2

1e-74

RPS8

RpS8

CG7808

3R: 99C4

1e-82

RPS9

RpS9

CG3395

3L: 67B11

8e-92

RPS10

RpS10a

CG12275

3R: 98A14

4e-43

RpS10b

CG14206

X: 18D3

8e-52

RPS11

RpS11

CG8857

2R: 48E8-9

1e-58

RPS12

RpS12

CG11271

3L: 69F5

2e-43

RPS13

RpS13

CG13389

2L: 29B2

2e-74

RPS14

RpS14a

CG1524

X: 7C6-7

3e-71

RpS14b

CG1527

X: 7C8

3e-71

RPS15

RpS15

CG8332

2R: 53C8

6e-62

RPS15A

RpS15Aa

CG2033

X: 11E11-12

2e-65

RpS15Ab

CG12324

2R: 47C1

6e-65

RPS16

RpS16

CG4046

2R: 58F1

2e-69

RPS17

RpS17

CG3922

3L: 67B5

5e-52

RPS18

RpS18

CG8900

2R: 56F11

2e-69

RPS19

RpS19a

CG4464

X: 14F4

5e-48

RpS19b

CG5338

3R: 95C13

1e-43

RPS20

RpS20

CG15693

3R: 93A1

7e-50

RPS21

oho23B/RpS21

CG2986

2L: 23B6

3e-30

RPS23

RpS23

CG8415

2R: 50E4

8e-70

RPS24

RpS24

CG3751

2R: 58F3

7e-55

RPS25

RpS25

CG6684

3R: 86D8

1e-38

RPS26

RpS26

CG10305

2L: 36F4

3e-47

RPS27

RpS27

CG10423

3R: 96C8

2e-39

RPS27A

RpS27A

CG5271

2L: 31E1

8e-80

RPS28

RpS28a

CG15527

3R: 99D2

1e-21

RpS28b

CG2998

X: 8E7

2e-23

RpS28-like

CG34182

2L: 30B3

1e-07

RPS29

RpS29

CG8495

3R: 85E8

5e-23

RPS30

RpS30

CG15697

3R: 93A2

6e-32

RPLP0

RpLP0

CG7490

3L: 79B2

e-122

RpLP0-like

CG1381

2R: 46E5-6

3e-10

RPLP1

RpLP1

CG4087

2L: 21C2

3e-34

RPLP2

RpLP2

CG4918

2R: 53C9

5e-32

RPL3

RpL3

CG4863

3R: 86D8

0.0

RPL4

RpL4

CG5502

3R: 98B6

e-141

RPL5

RpL5

CG17489

2L: h35/40B

e-120

RPL6

RpL6

CG11522

3R: 100C7

8e-58

RPL7

RpL7

CG4897

2L: 31B1

2e-75

RpL7-like

CG5317

2L: 33C1

3e-32

RPL7A

RpL7A

CG3314

X: 6B1

e-102

RPL8

RpL8

CG1263

3L: 62E7

e-119

RPL9

RpL9

CG6141

2L: 32C1

1e-66

RPL10

Qm/RpL10

CG17521

3L: h47/80A

7e-98

RPL10A

RpL10Aa

CG3843

3R: 88D10

3e-65

RpL10Ab

CG7283

3L: 68E1

3e-95

RPL11

RpL11

CG7726

2R: 56D7

6e-83

RPL12

RpL12

CG3195

2R: 60B7

7e-75

RPL13

RpL13

CG4651

2L: 30F3

1e-68

RPL13A

RpL13A

CG1475

3R: 83B6-7

3e-68

RPL14

RpL14

CG6253

3L: 66D8

2e-30

RPL15

RpL15

CG17420

3L: h50-52/80F

5e-90

RPL17

RpL17

CG3203

X: 6C10

5e-72

RPL18

RpL18

CG8615

3L: 65E9

3e-71

RPL18A

RpL18A

CG6510

2R: 54C3

3e-68

RPL19

RpL19

CG2746

2R: 60E11

4e-83

RPL21

RpL21

CG12775

2L: 40A-B

4e-66

RPL22

RpL22

CG7434

X: 1C4

4e-40

RpL22-like

CG9871

2R: 59D3

2e-24

RPL23

RpL23

CG3661

2R: 59B3

1e-68

RPL23A

RpL23A

CG7977

3L: 62A10

7e-52

RPL24

RpL24

CG9282

2L: 34B10

7e-55

RpL24-like

CG6764

3R: 86E5

8e-14

RPL26

RpL26

CG6846

3L: 75E4

1e-59

RPL27

RpL27

CG4759

3R: 96E9-10

1e-43

RPL27A

RpL27A

CG15442

2L: 24F3

5e-60

RPL28

RpL28

CG12740

3L: 63B14

3e-31

RPL29

RpL29

CG10071

2R: 57D8

8e-14

RPL30

RpL30

CG10652

2L: 37B9

3e-46

RPL31

RpL31

CG1821

2R: 45F5

5e-47

RPL32

RpL32

CG7939

3R: 99D3

6e-58

RPL34

RpL34a

CG6090

3R: 96F10

2e-29

RpL34b

CG9354

3R: 85D15

1e-29

RPL35

RpL35

CG4111

X: 5A11

2e-38

RPL35A

RpL35A

CG2099

3R: 83A4

1e-35

RPL36

RpL36

CG7622

X: 1B12

3e-33

RPL36A

RpL36A

CG7424

2L: 28D3

3e-43

RPL37

RpL37a

CG9091

X: 13B1

2e-39

RpL37b

CG9873

2R: 59C4

1e-31

RPL37A

RpL37A

CG5827

2L: 25C4

2e-38

RPL38

RpL38

CG18001

2R: h46/41C-E

2e-25

RPL39

RpL39

CG3997

2R: 60B7

3e-18

RPL40

RpL40

CG2960

2L: 24E1

2e-69

RPL41

RpL41

CG30425

2R: 60E5

8e-08

*Additional gene synonyms exist in most cases [60]. Bold font indicates CRP-like genes, putative pseudogenic fragments (CG11386 and CG33222) or the member of a duplicate gene pair that is expressed in a small number of tissues and/or at relatively low levels. †Computed cytological position is given for euchromatic genes (Genome Release 5 [60]). The cytological and h-band locations for heterochromatic genes are based on data in reference [151] or estimated from images of in situ hybridizations of BACs to polytene chromosomes (RpL5 and RpL21) [152]. The h-band location of RpL15 was provided by B Honda (personal communication). ‡Expect (E) value obtained from a BLASTp search of the D. melanogaster annotated proteome (Genome Release 5.1) with human RefSeq CRP sequences. (E values corresponding to RpL15 and RpS28-like were obtained from a BLAST search using Release 5.3.) Where multiple protein isoforms exist, the highest scoring hit is given.

 <p>Table 2</p>

The MRP genes of D. melanogaster

D. melanogaster gene

Human MRP

Symbol*

CG number

Location†

BLAST E value‡

MRPS2

mRpS2

CG2937

2L: 25B1

1e-69

MRPS5

mRpS5

CG40049

3L: h47/80A-B

4e-63

MRPS6

mRpS6

CG15016

3L: 64B2

2e-20

MRPS7

mRpS7

CG5108

2L: 31D11

6e-38

MRPS9

mRpS9

CG2957

3R: 84E4

8e-81

MRPS10

mRpS10

CG4247

3R: 88E3

1e-33

MRPS11

mRpS11

CG5184

3R: 89E11

8e-26

MRPS12

tko/mRpS12

CG7925

X: 3A3

1e-33

MRPS14

mRpS14

CG32531

X: 18C7

1e-35

MRPS15

bonsai/mRpS15

CG4207

2R: 58F3

1e-15

MRPS16

mRpS16

CG8338

2R: 50E1

2e-24

MRPS17

mRpS17

CG4326

2R: 60C1

2e-14

MRPS18A

mRpS18A

CG31450

3R: 85A3

8e-13

MRPS18B

mRpS18B

CG10757

2L: 38B6

4e-33

MRPS18C

mRpS18C

CG9688

3R: 99F4

7e-23

MRPS21

mRpS21

CG32854

3R: 87E8

3e-22

MRPS22

mRpS22

CG12261

3R: 98B6

3e-38

MRPS23

mRpS23

CG31842

2L: 34D6

2e-20

MRPS24

mRpS24

CG13608

3R: 95E6

2e-31

MRPS25

mRpS25

CG14413

X: 12F1

1e-49

MRPS26

mRpS26

CG7354

3L: 75B9

3e-11

MRPS27

NA

NA

NA

No hit

MRPS28

mRpS28

CG5497

2R: 55E2

1e-27

DAP3/MRPS29

mRpS29

CG3633

2R: 58E1

7e-72

MRPS30

mRpS30

CG8470

X: 13E18

3e-21

MRPS31

mRpS31

CG5904

3L: 72C2

5e-35

MRPS33

mRpS33

CG10406

3R: 89B16

3e-30

MRPS34

mRpS34

CG13037

3L: 72E1-2

8e-06

MRPS35

mRpS35

CG2101

3L: 62F4

2e-72

MRPS36

NA

NA

NA

No hit

MRPL1

mRpL1

CG7494

3R: 84F9-10

8e-25

MRPL2

mRpL2

CG7636

3L: 68A7

2e-56

MRPL3

mRpL3

CG8288

X: 13E14

5e-52

MRPL4

mRpL4

CG5818

2L: 35F1

6e-70

MRPL9

mRpL9

CG31478

3R: 88F1

6e-17

MRPL10

mRpL10

CG11488

2L: 21B4

8e-20

MRPL11

mRpL11

CG3351

3R: 88C3

4e-29

MRPL12

mRpL12

CG5012

3L: 66E5

2e-13

MRPL13

mRpL13

CG10603

2L: 37B1

2e-47

MRPL14

mRpL14

CG14048

X: 3A1

3e-31

MRPL15

mRpL15

CG5219

3L: 77C3

4e-74

MRPL16

mRpL16

CG3109

X: 2B14

3e-58

MRPL17

mRpL17

CG13880

3L: 61B3

8e-21

MRPL18

mRpL18

CG12373

2R: 49C2

3e-22

MRPL19

mRpL19

CG8039

3R: 85A5

1e-56

MRPL20

mRpL20

CG11258

3L: 69F5

8e-21

MRPL21

mRpL21

CG9730

3L: 76A3

2e-19

MRPL22

mRpL22

CG4742

X: 15A7-8

4e-41

MRPL23

mRpL23

CG1320

3L: 62D7

4e-28

MRPL24

mRpL24

CG8849

2L: 25B4

2e-45

MRPL27

mRpL27

CG33002

2L: 24F3

2e-10

MRPL28

mRpL28

CG3782

2L: 25B5

5e-27

MRPL30

mRpL30

CG7038

X: 4C11

3e-15