Open Access

Identification and characterisation of non-coding small RNAs in the pathogenic filamentous fungus Trichophyton rubrum

Contributed equally
BMC Genomics201314:931

DOI: 10.1186/1471-2164-14-931

Received: 8 September 2013

Accepted: 20 December 2013

Published: 30 December 2013

Abstract

Background

Accumulating evidence demonstrates that non-coding RNAs (ncRNAs) are indispensable components of many organisms and play important roles in cellular events, regulation, and development.

Results

Here, we analysed the small non-coding RNA (ncRNA) transcriptome of Trichophyton rubrum by constructing and sequencing a cDNA library from conidia and mycelia. We identified 352 ncRNAs and their corresponding genomic loci. These ncRNA candidates included 198 entirely novel ncRNAs and 154 known ncRNAs classified as snRNAs, snoRNAs and other known ncRNAs. Further bioinformatic analysis detected 96 snoRNAs, including 56 snoRNAs that had been annotated in other organisms and 40 novel snoRNAs. All snoRNAs belonged to two major classes—C/D box snoRNAs and H/ACA snoRNAs—and their potential target sites in rRNAs and snRNAs were predicted. To analyse the evolutionary conservation of the ncRNAs in T. rubrum, we aligned all 352 ncRNAs to the genomes of six dermatophytes and to the NCBI non-redundant nucleotide database (NT). The results showed that most of the identified snRNAs were conserved in dermatophytes. Of the 352 ncRNAs, 102 also had genomic loci in other dermatophytes, and 27 were dermatophyte-specific.

Conclusions

Our systematic analysis may provide important clues to the function and evolution of ncRNAs in T. rubrum. These results also provide important information to complement the current annotation of the T. rubrum genome, which primarily comprises protein-coding genes.

Background

Numerous studies have demonstrated that non-coding RNAs (ncRNAs) are widely expressed in both prokaryotes and eukaryotes [14]. Furthermore, the number of ncRNAs substantially increases with the complexity of the organism, whereas the number of protein-coding genes remains relatively static. In bacteria, unicellular eukaryotes, and invertebrates, the coding sequences constitute approximately 95, 30, and 20% of the genomic DNA, respectively. In mammals, open-reading frames only account for approximately 1–2% of the genomes [59].

NcRNAs include highly abundant and functionally important RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA), as well as other small, stable RNAs, such as small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), RNase P and mitochondrial RNA processing (MRP) RNA, signal recognition particle (SRP) RNA, and telomerase RNA. These RNAs have been characterised and are involved in splicing, ribosome biogenesis, translation, and chromosome replication [10, 11]. Recent transcriptomic and bioinformatic studies have also identified an increasing number of new ncRNAs whose function has not been validated [1216]. Hence, the discovery and analysis of ncRNAs has become an important step in our understanding of genomic structure and will expand our knowledge of the function and the regulatory roles of ncRNAs in the cell cycle and development.

In recent years, ncRNAs have been identified using experimental methods and computational predictions in several fungi [3, 4, 1722]. A large number of non-coding RNA genes, including 33 box C/D snoRNA genes, have been predicted in the genome of Schizosaccharomyces pombe. Functional analyses of 20 Box H/ACA snoRNAs indicated that the snoRNAs evolved in coordination with rRNAs to preserve post-transcriptional modification sites among distant eukaryotes [3, 4, 20]. A comparative genomics analysis of seven different yeast species identified a substantial number of evolutionarily conserved, structured ncRNAs, suggesting their roles in post-transcriptional regulation [20]. NcRNAs that participate in the cleavage and processing of tRNAs were observed in Aspergillus fumigatus[21]. An extensive analysis of snoRNA genes from Neurospora crassa indicated a high diversity of post-transcriptional modification guided by snoRNAs in the fungus kingdom [22]. Thus far, the ncRNAs of dermatophytes have not been studied.

Trichophyton rubrum is the most common dermatophyte that can infect human keratinised tissue (skin, nails, and, rarely, hair) [2325]. T. rubrum has a 22.5-Mbp haploid nuclear genome consisting of five chromosomes that range in size from 3.0–5.8 Mbp and a 27-kbp circular mitochondrial genome [26]. The Broad Institute has sequenced the T. rubrum genome and predicted more than 8,700 protein-coding genes. However, apart from rRNAs and tRNAs, no other ncRNAs have been annotated and characterised within the T. rubrum genome [26]. In the present study, we constructed an ncRNA library (ranging from 70–500 nt) and identified ncRNAs in T. rubrum using an RNA-Seq method. A total of 352 ncRNA candidates were characterised, including 198 entirely novel ncRNAs and 154 known ncRNAs. We also analysed the sequence conservation, and genomic location of these ncRNAs in six other dermatophytes. Our results may guide further studies of the important roles of ncRNA in T. rubrum and provide important complementary information to the annotation of the T. rubrum genome.

Results

Identification of ncRNA candidates inT. rubrum

To obtain a global view of ncRNAs in T. rubrum, we extracted total RNA from the conidia and mycelia phases and generated a small RNA cDNA library with size-fractionated total RNA ranging in size from 70–500 bp. After sequencing on the 454/Roche sequencing platform, a total of 87,601 reads were obtained and mapped to the T. rubrum genome. Next, the reads that mapped to the same genomic loci were clustered, resulting in 4,432 unique contigs. After removing the coding RNA and matches to tRNAs and rRNAs, the remaining 352 clusters (corresponding to 56,550 reads) were considered ncRNA candidates. Of these candidates, 154 were predicted to align with Rfam sequences and the remaining 196 were novel ncRNA candidates (Figure 1; for detailed information, see Additional file 1: Table S1).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-14-931/MediaObjects/12864_2013_Article_5631_Fig1_HTML.jpg
Figure 1

Detection of ncRNA candidates in T. rubrum by sequencing a size-fractionated cDNA library. (A) The distribution of 87,601 reads from the constructed small cDNA library of T. rubrum in different RNA classes. (B) The numbers of ncRNAs from different regions in the T. rubrum genome. (C) The number of different classes of ncRNAs are displayed in brackets.

Characteristics of ncRNA candidates

Of the 352 identified ncRNA candidates, 234 mapped to loci within 1 kb of the closest coding gene, implying a possible functional relationship. Some of the ncRNA clusters located in the immediate vicinity of a protein-coding region might be processed from the 5′- or 3′-UTR of the corresponding mRNA. Among the 352 ncRNA clusters, 82 were intronic and 29 corresponded to non-annotated intergenic regions of the T. rubrum genome (Figure 1). To verify the expression and sizes of candidate ncRNAs, we selected the spliceosomal snRNAs U1, U2, U4, U5, and U6 and 15 randomly selected novel ncRNA candidates to use in northern hybridisation. The results are shown in Figure 2.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-14-931/MediaObjects/12864_2013_Article_5631_Fig2_HTML.jpg
Figure 2

Northern blotting analysis of T. rubrum ncRNA candidates. M. RiboRuler Low Range RNA Ladder (Fermentas), 1. snRNA U1, 2. snRNA U2, 3. snRNA U4, 4. snRNA U5, 5. snRNA U6, 6. Trnc_2843, 7. Trnc_3589, 8.Trnc_369, 9. Trnc_1414, 10. Trnc_293, 11. Trnc_305, 12. Trnc_1472, 13. Trnc_961, 14. Trnc_608, 15. Trnc_4262, 16. Trnc_1437, 17. Trnc_2618, 18. Trnc_3096, 19. Trnc_1686, 20. TRnc2844, and 21. 5.8S rRNA. The lengths and other information describing the ncRNAs from the northern blotting analysis are shown in Additional file 1: Table S1.

snRNA candidates

The spliceosome contains five essential small nuclear RNAs (snRNAs)—U1, U2, U4, U5, and U6—that are essential components for assembling the spliceosome and accomplishing the intricate task of intron removal from newly synthesised eukaryotic RNAs [17, 18, 27]. Here, we identified the genomic loci of snRNAs U1, U2, U5, and U6, each of which exhibited a unique genomic location. U5 and U6 were the most abundant snRNAs among our data, found in 15,583 and 9,034 reads, respectively. The expression of U2 and U4 was lower than the other snRNA candidates; we found only 163 reads of U2 and 146 reads of U4. These results are in agreement with those of the small ncRNA transcriptome analysis of another filamentous fungus, A. fumigatus[21, 28]. U4 was not initially identified in our data. To find the U4 genomic locus in T. rubrum, we downloaded the U4 sequences of A. fumigatus, A. oryzae, and A. niger from Rfam to use as query sequences to search for homologues in the T. rubrum genome using BLASTn. One genomic locus was identified. Corresponding reads assigned to the same locus had been sequenced and clustered in our data but had been eliminated because the percentage of ORF in the cluster was greater than 80%.

We aligned the T. rubrum snRNA U1, U2, U4, U5, and U6 candidates to the genomes of six T. rubrum-related dermatophytes to predict the homologues in these genomes by BLASTn. The homologues were compared using the multiple sequence alignment software ClustalW2, revealing that all snRNAs were highly conserved in these seven dermatophytes (Table 1). High variance was observed among the sequences and lengths of these snRNAs in T. rubrum and their homologues in other fungi; however, these snRNAs were conserved at the secondary structure level, with conserved regions in the hairpin loops (Additional file 2: Figure S2). These results correspond with previous reports on A. fumigatus[21].
Table 1

Conservation level of snRNAs in T. rubrum and related dermatophytes

   

Genome location

  

Name

Genes

Lena

Chromosome

Start

End

Position

Conserved in dermatophytes (% sequence identity)

Accession

Trnc_3904

U1

196

supercont2.8

159538

159733

5′UTR

M. gypseum (98%), M. canis (98%), A. benhamiae (100%)

KC353306

Trnc_774

U2

201

supercont2.1

3545014

3545214

3′UTR

T. tonsurans (98%), T. equinum (98%), M. gypseum (97%), T. verrucosum (99%), M. canis (96%), A. benhamiae (99%)

KC353051

Trnc_1437

U4

264

supercont2.17

13253

15593

Intergenic

T. tonsurans (100%), T. equinum (100%), M. gypseum (99%), A. benhamiae (100%)

KC353100

Trnc_681

U5

211

supercont2.1

3061687

3061897

5′UTR

T. tonsurans (92%), T. equinum (92%), M. gypseum (95%), T. verrucosum (93%), M. canis (91%), A. benhamiae (100%)

KC353044

Trnc_1782

U6

104

supercont2.2

1801544

1801647

3′UTR

T. tonsurans (100%), M. gypseum (100%), M. canis (99%), A. benhamiae (100%)

KC353131

Lena: the cDNA length of the snRNA. Conserved in dermatophytes (% sequence identity): the sequence identity of homologous snRNAs in other dermatophytes compared to T. rubrum; Accession is the accession number in GenBank.

snoRNAs

In eukaryotic cells, two major classes of small nucleolar ncRNA (snoRNA) have been identified: C/D box snoRNAs, which are involved in the 20-O-methylation of ribosomal, spliceosomal, and transfer RNAs (the latter in Archaea only), and H/ACA snoRNAs, which guide pseudouridylation in these RNA species [29, 30].

To predict the two classes of snoRNAs and their putative targets in our data, we used the Snoscan and SnoGPS programs, defining the potential target sequences as the 5.8S, 18S, and 25S rRNAs of T. rubrum and all snRNAs identified in our data [17, 18]. We identified 96 snoRNAs, including 58C/D box snoRNAs (46 had homologues in other organisms) and 38H/ACA snoRNAs (nine had homologues in other organisms). We identified 37C/D box snoRNAs as putative targets, most of which were predicted to guide methylation of 18S and 25S rRNAs. We also identified five C/D box snoRNAs (TRnc_801, TRnc_3573, TRnc_4113, TRnc_1272, and TRnc_1271) that were predicted to guide the methylation of snRNAs U1, U2, and U5. Of the 37C/D box snoRNAs, 22 had different modification sites in target rRNA or snRNA sequences. No rRNA or snRNA targets were identified in the remaining 21C/D box snoRNAs (Table 2). Additionally, the 30 identified H/ACA box snoRNAs were identified as guiding the pseudouridylation of 45 sites in rRNAs (Table 3. Detail information about potential base-paring between H/ACA box snoRNAs and rRNA shown in Additional file 3: Figure S3), whereas no pseudouridine sites were predicted on any snRNAs.
Table 2

C/D box snoRNA candidates identified in T. rubrum

  

Genome position

Homologues

  

Name

Lena

Chromosome

Start

End

Location

Accession1

Genes

Putative target(s)

Accession2

TRnc_1010

87

supercont2.10

749220

749306

3′UTR

RF00477

snosnR66

 

KC353070

TRnc_1157

95

supercont2.11

539262

539356

Intron

RF00093

SNORD18, U18

25S: Am651, Gm654; 18S: Am1159

KC353075

TRnc_1271

242

supercont2.12

280437

280196

Intron

RF01152

sR1

25S: Am2268, Am3277,Cm964,Cm961;U5: Cm103; 18S: Am1540

KC353083

TRnc_1272

265

supercont2.12

280712

280448

Intron

RF01152

sR1

25S: Cm964, Cm961;18S: Um604; U5: Cm103

KC353084

TRnc_1299

109

supercont2.13

24837

24729

Intron

RF00593

snoU83B

 

KC353086

TRnc_1359

97

supercont2.14

159345

159441

Intron

RF00475

snosnR69

25S: Cm3322

KC353090

TRnc_1366

215

supercont2.14

179253

179467

3′UTR

RF01152

sR1

 

KC353091

TRnc_1449

234

supercont2.17

97081

97314

5′UTR

RF01191

SNORD121A

18S: Cm673, Gm234

KC353101

TRnc_1560

77

supercont2.2

546818

546894

3′UTR

RF01139

sR2

 

KC353110

TRnc_1603

358

supercont2.2

766347

766704

3′UTR

RF00345

snoR1

 

KC353115

TRnc_1709

154

supercont2.2

1400380

1400533

5′UTR

RF01193

snoR20a

 

KC353124

TRnc_1825

309

supercont2.2

1958330

1958022

3′UTR

  

25S: Um2301; Um769

KC353137

TRnc_1841

143

supercont2.2

2090171

2090313

3′UTR

RF01144

sR17

 

KC353138

TRnc_2011

127

supercont2.3

74633

74759

3′UTR

RF00441

snoZ242

 

KC353147

TRnc_2018

306

supercont2.3

117035

116730

3′UTR

  

18S: Um628

KC353149

TRnc_2027

96

supercont2.3

166668

166763

Intron

RF01281

snoR35

 

KC353150

TRnc_2179

431

supercont2.3

961995

961565

Intergenic

  

25S: Um413

KC353160

TRnc_2265

87

supercont2.3

1276133

1276219

Intron

RF01197

snR39

25S: Gm808

KC353164

TRnc_2283

233

supercont2.3

1301587

1301819

5′UTR

  

18S: Am1105; 25S: Am499, Am1453

KC353165

TRnc_2405

317

supercont2.3

1975149

1975465

3′UTR

  

25S: Gm1738

KC353175

TRnc_2419

204

supercont2.3

2045771

2045974

5′UTR

RF01125

sR4

18S: Am350, Gm698, Cm701;25S: Gm215, Cm3127

KC353177

TRnc_2421

182

supercont2.3

2046135

2046316

5′UTR

RF00016

SNORD14, U14

18S: Um50, Cm379;25S: Cm2352

KC353178

TRnc_2498

172

supercont2.3

2451919

2452090

5′UTR

RF00527

  

KC353188

TRnc_2545

119

supercont2.3

2657688

2657806

3′UTR

RF01188

snR56

18S: Gm1389,Am385

KC353195

TRnc_2569

192

supercont2.3

2759920

2759729

Intron

RF01297

sR40

 

KC353197

TRnc_2594

143

supercont2.3

2859175

2859033

Intron

RF01305

sR51

 

KC353199

TRnc_2691

158

supercont2.4

233433

233276

Intergenic

  

5.8S: Gm87

KC353216

TRnc_2782

128

supercont2.4

669565

669438

5′UTR

RF00630

P26

18S: Cm534; 25S: Cm1583, Cm1196, Cm3233

KC353223

TRnc_2936

246

supercont2.4

1403883

1403638

Intron

RF00312

snoZ206

25S: Gm1378

KC353235

TRnc_3227

139

supercont2.5

625518

625380

Intron

RF00594

SNORD86, U86

KC353256

TRnc_3297

138

supercont2.5

896392

896529

3′UTR

RF00610

SNORD110

KC353262

TRnc_338

135

supercont2.1

1643180

1643314

Intron

RF01223

snR13

25S: Am2267

KC353022

TRnc_3425

202

supercont2.6

22581

22782

3′UTR

  

25S: Gm911

KC353267

TRnc_3426

98

supercont2.6

23000

23097

3′UTR

   

KC353268

TRnc_3438

173

supercont2.6

91295

91467

5′UTR

RF01291

snoU97, SNORD97

KC353269

TRnc_3573

95

supercont2.6

964586

964680

Intron

RF00530

snoMe28S-Cm2645

25S: Cm2324, Um2867; U2: Um43

KC353276

TRnc_3654

191

supercont2.7

14823

15013

3′UTR

RF01140

sR20

18S: Gm832

KC353284

TRnc_3667

191

supercont2.7

59063

59253

3′UTR

RF00529

snoMe28S-Am2589

KC353285

TRnc_3778

101

supercont2.7

777627

777727

5′UTR

RF00471

snosnR48, snr46

18S: Am721; 25S: Gm2780; Am2243

KC353293

TRnc_3833

109

supercont2.7

1124537

1124429

3′UTR

RF01273

sR34

 

KC353299

TRnc_3855

288

supercont2.7

1281447

1281734

Intron

RF01127

sR42

 

KC353305

TRnc_3911

80

supercont2.8

194694

194773

Intron

RF00213

snoR38

25S: Gm2799

KC353308

TRnc_4113

681

supercont2.8

1152047

1152727

Intron

RF01274

sR45

25S: Cm1856,Cm1673; 18S: Am833; U2: Am155

KC353324

TRnc_415

103

supercont2.1

1918108

1918210

Intron

RF01121

Sr38

 

KC353027

TRnc_4250

192

supercont2.9

658585

658394

3′UTR

  

18S: Cm373

KC353339

TRnc_4259

104

supercont2.9

693331

693434

5′UTR

RF00276

SNORD52, U52

25S: Um2408

KC353340

TRnc_4260

95

supercont2.9

695194

695288

Intergenic

RF01178

snoR77Y,snR77

18S: Um565, Am564

KC353341

TRnc_4261

138

supercont2.9

695445

695582

Intergenic

RF01209

snR76

18S: Cm1674;25S: Cm2184, Am2266, Cm3294, Cm1758

KC353342

TRnc_4262

273

supercont2.9

695588

695860

Intergenic

RF01185

snR75, U15

25S: Gm2275

KC353343

TRnc_4263

157

supercont2.9

695917

696073

Intergenic

RF00086

SNORD27, U27, snR74

25S: Cm1179

KC353344

TRnc_4264

88

supercont2.9

696179

696266

5′UTR

RF01207

snR73,U35

25S: Cm3333

KC353345

TRnc_4267

100

supercont2.9

703004

703103

3′UTR

  

18S: Um525, Gm527

KC353346

TRnc_4316

97

supercont2.9

861468

861372

5′UTR

RF01223

snR13

 

KC353347

TRnc_4336

162

supercont2.9

996654

996493

Intron

  

18S: Gm1089

KC353348

TRnc_608

234

supercont2.1

2701229

2701462

3′UTR

RF01202

sn2991

5.8S: Cm137

KC353041

TRnc_640

129

supercont2.1

2869815

2869687

3′UTR

RF00300

snoZ221

 

KC353043

TRnc_801

488

supercont2.1

3681448

3681935

3′UTR

RF00012

U3

18S: Um418; 25S: Cm1363, Cm1633, Cm1983, Cm3165; U1: Cm45

KC353053

TRnc_821

210

supercont2.1

3768831

3768622

Intergenic

  

18S: Cm1301,25S: Cm880

KC353055

TRnc_985

153

supercont2.10

686423

686575

Intron

RF00494

snoU2_19

 

KC353066

Name: the C/D box snoRNAs were numbered according to the order of identification. Lena: the cDNA length of the snoRNA. Homologues: homologues in Rfam or other organisms. Accession1 is the accession number in Rfam; Accession2 is the accession number in GenBank; Genes are homologous gene names in other organisms [1922]. Putative target(s): the predicted modified nucleotides within rRNAs or snRNAs using the Snoscan package.

Table 3

H/ACA box snoRNA candidates identified in T. rubrum

  

Genome location

Homologues

  

Name

Lena

Chromosome

Start

End

Position

Accession1

Genes

Putative target

Accession2

Trnc_1355

371

supercont2.14

142837

142467

5′UTR

  

18S-Ψ1434

KC353088

Trnc_1370

133

supercont2.14

187697

187565

5′UTR

RF01134

sR30

 

KC353092

Trnc_203

308

supercont2.1

996485

996178

5′UTR

  

18S-Ψ803

KC353013

Trnc_2045

228

supercont2.3

296293

296520

5′UTR

  

25S-Ψ2867,18S-Ψ489

KC353151

Trnc_2579

349

supercont2.3

2792710

2793058

5′UTR

  

18S-Ψ611

KC353198

Trnc_2999

290

supercont2.4

1720998

1721287

5′UTR

  

25S-Ψ2135

KC353240

Trnc_3005

214

supercont2.4

1748930

1749143

5′UTR

  

25S-Ψ1081

KC353241

Trnc_3218

332

supercont2.5

584674

585005

5′UTR

  

18S-Ψ573,25S-Ψ681,25S-Ψ2635

KC353255

Trnc_3509

433

supercont2.6

608530

608098

5′UTR

  

25S-Ψ2545,25S-Ψ1671

KC353274

Trnc_5

289

supercont2.1

19982

20270

5′UTR

  

25S-Ψ2329

KC352999

Trnc_910

468

supercont2.10

343107

343574

5′UTR

  

18S-Ψ12

KC353060

Trnc_1407

234

supercont2.16

54707

54474

3′UTR

  

25S-Ψ1155

KC353095

Trnc_1472

188

supercont2.2

69663

69850

3′UTR

RF01258

snR10

 

KC353105

Trnc_1776

326

supercont2.2

1789188

1788863

3′UTR

RF01231

snoR74

18S-Ψ1593,18S-Ψ412

KC353129

Trnc_1893

344

supercont2.2

2393882

2393539

3′UTR

  

25S-Ψ312

KC353142

Trnc_2452

323

supercont2.3

2170039

2169717

3′UTR

  

25S-Ψ2650

KC353184

Trnc_2596

324

supercont2.3

2882125

2881802

3′UTR

  

18S-Ψ1336

KC353200

Trnc_2843

225

supercont2.4

976176

976400

3′UTR

RF01251

snR3

25S-Ψ2120,25S-Ψ2251

KC353227

Trnc_3023

182

supercont2.4

1839416

1839597

3′UTR

  

25S-Ψ759,25S-Ψ1558,25S-Ψ520

KC353242

Trnc_3387

226

supercont2.5

1472165

1472390

3′UTR

  

18S-Ψ565,25S-Ψ2404

KC353265

Trnc_3741

180

supercont2.7

491853

492032

3′UTR

RF01247

snR32

 

KC353292

Trnc_4007

239

supercont2.8

722404

722166

3′UTR

  

18S-Ψ1344

KC353317

Trnc_64

306

supercont2.1

267027

267332

3′UTR

  

25S-Ψ2714

KC353002

Trnc_817

188

supercont2.1

3719705

3719892

3′UTR

  

18S-Ψ267,18S-Ψ1697

KC353054

Trnc_920

310

supercont2.10

389299

389608

3′UTR

  

25S-Ψ116,18S-Ψ1213

KC353061

Trnc_1698

360

supercont2.2

1345609

1345968

Intron

  

18S-Ψ1026

KC353122

Trnc_2075

96

supercont2.3

425677

425772

Intron

RF00405

SNORA44

 

KC353153

Trnc_2172

126

supercont2.3

922150

922025

Intron

RF00406

SNORA42

 

KC353159

Trnc_2443

106

supercont2.3

2090244

2090349

Intron

RF00428

SNORA38

 

KC353182

Trnc_2531

75

supercont2.3

2617075

2617001

Intron

RF00415

SNORA30

 

KC353194

Trnc_2606

280

supercont2.36

2106

2385

Intergenic

  

25S-Ψ1054

KC353202

Trnc_2618

322

supercont2.36

8062

8383

Intergenic

  

25S-Ψ1062

KC353205

Trnc_2621

406

supercont2.36

8934

9339

Intergenic

  

25S-Ψ1689

KC353206

Trnc_2636

203

supercont2.36

19276

19478

Intergenic

  

18S-Ψ217,25S-Ψ1890

KC353210

Trnc_2898

393

supercont2.4

1199167

1198775

Intron

  

25S-Ψ1718,25S-Ψ36

KC353231

Trnc_3585

281

supercont2.6

1065274

1064994

Intron

  

18S-Ψ867,25S-Ψ111

KC353278

Trnc_4006

251

supercont2.8

710950

711200

Intron

RF01263

snR191

18S-Ψ935,25S-Ψ1239,25-Ψ2245

KC353316

Name: the H/ACA box snoRNAs were numbered according to the order of identification. Lena: the cDNA length of the snoRNA. Homologues: homologues in Rfam or other organisms. Accession1 is the accession number in Rfam; Accession2 is the accession number in GenBank; Genes are homologous gene names in other organisms [1922]. Putative target(s): the predicted modified nucleotides within rRNAs using SnoGPS package.

Other types of ncRNA inT. rubrum

We also identified 51 other ncRNA genomic loci, such as pri-miRNAs or pre-miRNAs, RNAse MRP, and telomerase RNA. miRNAs related transcriptional loci were the most widely distributed ncRNAs in the T. rubrum genome; for example, the mir-598 miRNA family had 13 transcriptional regions and mir-533 had eight. In our data, these miRNA homologies of ncRNAs, which varied from 70–270 bp, were much longer than the lengths of mature miRNAs (18–25 bp), they may be pri- or pre-miRNAs candidates.

Evolutionary conservation of the ncRNAs inT. rubrum

To analyse the evolutionary conservation of ncRNAs in T. rubrum, we used BLASTn to align the sequences of all 352 ncRNAs to the genomes of six related dermatophytes: T. equinum, T. tonsurans, T. verrucosum, A. benhamiae, M. gypseum, and M. canis. The loci of 102 of these sncRNAs were also identified in all six genomes (Additional file 4: Table S4). We found that the sequences of these sncRNAs were highly conserved, with sequence identities above 85%. Of the 352 ncRNAs, ten had no hits in other genomes and might be specifically expressed in T. rubrum (Table 4). To further analyse the conserved ncRNAs in dermatophytes, we employed BLASTn to align all of the sncRNAs with the NCBI non-redundant nucleotide database (NT) after excluding Arthrodermataceae. These BLASTn results were processed by MEGAN4, which placed each ncRNA sequence in a node in the NCBI taxonomy [31].
Table 4

The ncRNA candidates specifically expressed in T. rubrum

    

Genome location

 

Name

Class

Reads

Lena

Supercontig

Start

End

Position

Accession

Trnc_20

 

1

94

supercont2.1

48466

48559

3′UTR

KC353103

Trnc_1456

 

1

94

supercont2.18

53193

53100

3′UTR

KC353000

Trnc_2606

snoRNA;H/ACA-box

2

280

supercont2.36

2106

2385

Intergenic

KC353202

Trnc_2609

 

4

255

supercont2.36

4048

4302

Intergenic

KC353203

Trnc_2621

snoRNA;H/ACA-box

97

406

supercont2.36

8934

9339

Intergenic

KC353206

Trnc_2633

 

297

597

supercont2.36

17132

17728

Intergenic

KC353209

Trnc_2636

snoRNA;H/ACA-box

1

203

supercont2.36

19276

19478

Intergenic

KC353210

Trnc_2640

 

2

71

supercont2.36

21309

21379

Intergenic

KC353211

Trnc_2649

 

2

79

supercont2.36

23976

24054

Intergenic

KC353212

Trnc_3096

 

1

201

supercont2.4

2153644

2153444

3′UTR

KC353244

Lena: the cDNA length of the ncRNAs; Accession is the accession number in GenBank. This table shows the lengths and genomic loci of ten ncRNAs that might be specifically expressed in T. rubrum. These ncRNAs have no hits assigned to the NCBI NT database using BLASTn.

As shown in Figure 3, a total of 179 ncRNA sequences were classified under cellular organisms, with 166 clustered to the Eukaryota node (approximately 47.2% of the total 352 ncRNAs). Of these ncRNAs, 97 were assigned to Fungi, indicating that these ncRNAs were conserved in fungi; all snRNAs were assigned to this node. Of the ncRNAs under the Fungi taxonomic level, 16 and 44 were assigned to Onygenales and Trichocomaceae, respectively, supporting the close relationship between the dermatophytes and the fungi in these families. Seventy-three ncRNAs were assigned to phyla distantly related to fungi, including three assigned to the root, seven to cellular organisms, 27 to the Eukaryota node, 30 under Bilateria, and six under Bacteria. These results suggest that some ancient ncRNAs are preserved in T. rubrum.
https://static-content.springer.com/image/art%3A10.1186%2F1471-2164-14-931/MediaObjects/12864_2013_Article_5631_Fig3_HTML.jpg
Figure 3

MEGAN phylogenetic analysis of T. rubrum ncRNA candidates. A MEGAN tree with the taxonomic affiliation of 352 ncRNAs that were identified by BLASTN of all sequences in NT after excluding Arthrodermataceae according to NCBI taxonomy. Each circle of the MEGAN tree represents a taxon in the NCBI taxonomy database and is labelled by its name and the number of snRNAs that were assigned to the taxon and not to a subtaxon. The size of the circles represents the number of ncRNAs.

Apart from the classified ncRNAs, the remaining 170 ncRNA candidates had no significant similarity to any nucleotide sequence in NT, including 154 unassigned ncRNAs and 16 ncRNAs with no hits. Of these unclassified ncRNAs, 27 existed in and were conserved in all six dermatophytes, indicating that these 27 ncRNAs were dermatophyte-specific ncRNAs (Table 5).
Table 5

The ncRNA candidates specifically expressed in dermatophytes

   

Genome location

 

Name

Lena

Reads

Chromosome

Start

End

Position

Accession

Trnc_817

188

323

supercont2.1

3719705

3719892

3′UTR

KC353054

Trnc_733

174

1

supercont2.1

3371115

3371288

3′UTR

KC353049

Trnc_2676

156

2

supercont2.4

110438

110593

3′UTR

KC353213

Trnc_3999

178

5

supercont2.8

672734

672557

3′UTR

KC353314

Trnc_1167

177

1

supercont2.11

544895

545071

3′UTR

KC353076

Trnc_2448

161

1

supercont2.3

2123075

2122915

5′UTR

KC353183

Trnc_4219

104

1

supercont2.9

449429

449532

5′UTR

KC353335

Trnc_956

241

2

supercont2.10

559285

559525

5′UTR

KC353063

Trnc_305

97

579

supercont2.1

1515685

1515781

Intron

KC353018

Trnc_500

203

1

supercont2.1

2298649

2298447

Intron

KC353035

Trnc_1792

251

1

supercont2.2

1856556

1856806

Intron

KC353132

Lena: the cDNA length of the ncRNAs; Accession is the accession number in GenBank. This table shows the lengths and genomic loci of ten sncRNAs that might be specifically expressed in dermatophytes. These ncRNAs were conserved in all six dermatophytes but have no homologues in NT.

Discussion

RNA is emerging as a central player in cellular regulation, with active roles in multiple regulatory layers, including transcription, RNA maturation, RNA modification, and translational regulation [32]. Recent studies have revealed an unexpected complexity of regulatory RNAs, even in bacteria [2, 33]. In the present study, we first used an RNA-Seq method to analyse the ncRNAs in the genome of the dermatophyte fungus T. rubrum. We identified 352 sncRNA candidates, including snRNAs, snoRNAs, miRNAs, and other types of ncRNAs; 196 novel ncRNAs were predicted. We further confirmed the genomic loci of these ncRNAs in T. rubrum. This work provides an important complement to the current annotation of the T. rubrum genome, which is currently comprised primarily of protein-coding genes.

Five types of snRNAs (U1, U2, U4, U5, and U6) were identified, and their secondary structures were predicted by RNAfold [27]. We found these snRNAs to be highly conserved among dermatophytes. We also detected 96 snoRNAs, including 55 that were annotated in other organisms and 41 that were novel snoRNAs. Using the Snoscan and snoGPS programs, we bioinformatically identified their potential target sites on rRNAs and snRNAs. miRNAs have been previously reported in some fungi, such as S. pombe, but have not been found in A. fumigatus[21, 34]. In our data, we detected 68 genomic loci corresponding to 12 miRNA families; the lengths of these ncRNAs varied from 80–270 bp, suggesting that they were pri-miRNAs or pre-miRNAs [35]. To analyse the evolutionary conservation of ncRNAs, we aligned the 352 snRNAs to six other dermatophyte genomes and the NT database; we found 27 dermatophyte-specific ncRNAs and 11 T. rubrum-specific ncRNAs.

Conclusions

In this study, sequences for ncRNAs were obtained in T.rubrum and characterized by sequence comparison to know ncRNAs in other organisms, some of which were presumably functionally characterized in other work. This will prove to be a valuable resource but real understanding of regulatory mechanisms will come from followon work from this strong beginning.

Methods

Strain and culture conditions

The T. rubrum strain BMU01672 was grown on potato glucose agar (Difco) at 28°C for ten days to produce conidia. The conidia were isolated as previously reported, introduced into YPD medium (2% dextrose, 2% Bacto-Peptone, and 1% yeast extract), and incubated at 28°C with constant shaking at 200 rpm (Innova 4230 Refrigerated Incubator Shaker; New Brunswick Scientific, Edison NJ) [36]. After culture, the mycelia were harvested and ground to a powder in liquid nitrogen for RNA extraction.

RNA extraction and cDNA library construction

Total RNA was extracted from conidia and mycelia using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Same amount of total RNA from conidia and mycelia was mixed and pooled on a denaturing 8% polyacrylamide gel [7 M urea and 1× TBE buffer (90 mM Tris, 64.6 mM boric acid, 2.5 mM EDTA, pH 8.3)]. We collected gel bands containing RNAs of 70–500 bp, excluding the 5.8S rRNA band. RNAs were passively eluted and then ethanol-precipitated. RNA size and concentration were quantified with the Agilent 2100 Bioanalyser and the Agilent RNA 6000 Pico Kit according to the manufacturer’s protocols. The fractionated RNA was dephosphorylated with FastAP (Fermentas) and ligated to the 3′-adaptor oligonucleotide (UUUUGACCACGGTACCCAG, RNA is underlined) by T4 RNA ligase (Promega). Subsequently, the RNA was reverse transcribed using oligo 3RT (CTGGGTACCGTGGTCAAA) and converted into double-stranded cDNA with a SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen). The ds-cDNA was purified using the MinElute Reaction Cleanup Kit (Qiagen) according to the manufacturer’s protocol.

454/Roche sequencing and data bioinformatic analysis

For 454/Roche sequencing, approximately 5 μg of the size-fractionated cDNA sample (70–500 bp) was blunted. The pieces were then ligated with short adaptors prior to amplification and sequencing. The sequencing run was performed using the method of Margulies et al.[37].

After 454 sequencing, the 5′ and 3′ adaptors were removed from the reads. Genome data for T. rubrum and six related dermatophytes (Trichophyton equinum, Trichophyton tonsurans, Trichophyton verrucosum, Arthroderma benhamiae, Microsporum gypseum, and Microsporum canis) were downloaded from the Broad Institute web site (http://www.broadinstitute.org/annotation/genome/dermatophyte_comparative/MultiDownloads.html).

The high-quality reads were mapped to the genome using BLAST (version 2.2.22) (Eval < 1e − 5). Then, reads that were 80% mapped to the genome were clustered according to their genomic position and assembled into contigs according to the genomic sequence at the corresponding loci. The ORFs in the contigs were predicted using getorf in the EMBOSS program (version 6.3.1). Contigs with less than 80% ORF were aligned to TrED EST sequences and the NCBI non-redundant protein sequence database (NR) [38, 39]. The clusters with no hits in the TrED EST sequences and NR were used for the following steps: (1) alignment to non-coding RNA sequences with rRNA sequences downloaded from Rfam and GenBank [40], (2) identification of tRNAs with tRNAscan-SE (version 1.1) [41], and (3) alignment of clusters to Rfam sequences using HMMER (version 3.0) [42] and INFERNAL (version 1.0.2). The criteria for identification of known ncRNAs were as follows: (1) percentage of ORF less than 80%, (2) no hits in NR, (3) not mRNA, and (4) with homologues in Rfam [Eval (HMMER and INFERNAL) < 0.01]. For new ncRNA identification, the criteria were as follows: (1) percentage of ORF less than 80%, (2) no hits in NR, (3) not mRNA, (4) not rRNA, (5) not tRNA, and (6) no hits in Rfam (Eval > 0.01).

Analysis of snRNAs folding and predication of snoRNAs putative targets

T. rubrum snRNAs are compared with the homologs in other fungi using the multiple sequence alignment software ClustalW2. The secondary structures of aligned sequences are predicted by RNAalifold [28]. The putative targets of snoRNAs were predicted by Snoscan and SnoGPS programs [17, 18]. The potential target sequences as the 5.8S, 18S, and 25S rRNAs of T. rubrum were downloaded from GenBank under the accession number JX431933.

To predict the two classes of snoRNAs and their putative targets in our data, we used the Snoscan and SnoGPS programs, defining the potential target sequences as the 5.8S, 18S, and 25S rRNAs of T. rubrum and all snRNAs identified in our data [17, 18].

Northern blot analysis

For the northern blot analysis, 10 μg of total RNA was separated by electrophoresis on an 8% polyacrylamide gel containing 7 M urea and then electrotransferred onto a nylon membrane (Hybond-N+; Amersham) using a semi-dry blotting apparatus (BioRad). A total of 24–30 mer DNA oligonucleotides antisense to snRNAs and 15 randomly selected ncRNA candidates were end-labelled with (γ32P)-ATP and hybridised at 45°C for 16 hr. After stringency washes, the blots were exposed to phosphor storage screens, which were then scanned with a Typhoon 9200 imager (GE Healthcare).

Nucleotide sequence accession numbers

The 352 ncRNAs sequences of T. rubrum were submitted to GenBank under the following accession numbers: KC352999 – KC353350.

Notes

Declarations

Acknowledgements

This work was supported by the National Nature Science Foundation of China (Grant No. 30870104), the National High Technology Research and Development Program of China (Grant No. 2012AA020303), the National Science and Technology Major Project of China (Grant No. 2013ZX10004-601), and an intramural grant from the Institute of Pathogen Biology, Chinese Academy of Medical Sciences (Grant No. 2006IPB008).

Authors’ Affiliations

(1)
MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College
(2)
Bioinformatics Laboratory, Institute of Biophysics, Chinese Academy of Sciences

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This article is published under license to 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. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.