SnoRNAs from the filamentous fungus Neurospora crassa: structural, functional and evolutionary insights

  • Na Liu1,

    Affiliated with

    • Zhen-Dong Xiao1,

      Affiliated with

      • Chun-Hong Yu1,

        Affiliated with

        • Peng Shao1,

          Affiliated with

          • Yin-Tong Liang1,

            Affiliated with

            • Dao-Gang Guan1,

              Affiliated with

              • Jian-Hua Yang1,

                Affiliated with

                • Chun-Long Chen2,

                  Affiliated with

                  • Liang-Hu Qu1Email author and

                    Affiliated with

                    • Hui Zhou1Email author

                      Affiliated with

                      BMC Genomics200910:515

                      DOI: 10.1186/1471-2164-10-515

                      Received: 03 July 2009

                      Accepted: 08 November 2009

                      Published: 08 November 2009

                      Abstract

                      Background

                      SnoRNAs represent an excellent model for studying the structural and functional evolution of small non-coding RNAs involved in the post-transcriptional modification machinery for rRNAs and snRNAs in eukaryotic cells. Identification of snoRNAs from Neurospora crassa, an important model organism playing key roles in the development of modern genetics, biochemistry and molecular biology will provide insights into the evolution of snoRNA genes in the fungus kingdom.

                      Results

                      Fifty five box C/D snoRNAs were identified and predicted to guide 71 2'-O-methylated sites including four sites on snRNAs and three sites on tRNAs. Additionally, twenty box H/ACA snoRNAs, which potentially guide 17 pseudouridylations on rRNAs, were also identified. Although not exhaustive, the study provides the first comprehensive list of two major families of snoRNAs from the filamentous fungus N. crassa. The independently transcribed strategy dominates in the expression of box H/ACA snoRNA genes, whereas most of the box C/D snoRNA genes are intron-encoded. This shows that different genomic organizations and expression modes have been adopted by the two major classes of snoRNA genes in N. crassa . Remarkably, five gene clusters represent an outstanding organization of box C/D snoRNA genes, which are well conserved among yeasts and multicellular fungi, implying their functional importance for the fungus cells. Interestingly, alternative splicing events were found in the expression of two polycistronic snoRNA gene hosts that resemble the UHG-like genes in mammals. Phylogenetic analysis further revealed that the extensive separation and recombination of two functional elements of snoRNA genes has occurred during fungus evolution.

                      Conclusion

                      This is the first genome-wide analysis of the filamentous fungus N. crassa snoRNAs that aids in understanding the differences between unicellular fungi and multicellular fungi. As compared with two yeasts, a more complex pattern of methylation guided by box C/D snoRNAs in multicellular fungus than in unicellular yeasts was revealed, indicating the high diversity of post-transcriptional modification guided by snoRNAs in the fungus kingdom.

                      Background

                      Eukaryotic rRNAs contain a large number of nucleotide modifications including 2'-O-methylation and pseudouridylation which are directed by box C/D snoRNAs and box H/ACA snoRNAs, respectively [1, 2]. These modifications are usually found in the conserved core regions of rRNAs, and they play important roles in ribosome function [3]. SnoRNAs are among the most numerous and functionally diverse non-coding RNAs currently known [4, 5], existing widely in eukaryotes including human [68], plants [911], yeasts [1215] and protists [1619], as well as in Archaea [20]. This indicates that they are ancient molecules that arose over 2-3 billion years ago [21]. In addition to guiding the posttranscriptional modifications of rRNAs in eukaryotes and Archaea, snoRNAs also interact with spliceosomal RNAs [22], tRNAs [23, 24], SL RNAs in trypanosomes [25], and at least one brain-specific mRNA in mammals [26]. Recently, snoRNA U50 was found to be a candidate tumor suppressor gene in prostate cancer [27]. The existence of substantial numbers of orphan snoRNAs indicates that snoRNAs also participate in diverse biological processes that remain to be identified [4].

                      SnoRNAs exhibit canonical sequence motifs and structural features. Box C/D snoRNAs carry the conserved box C (RUGAUGA, where R can be any purine) and D (CUGA) motifs near their 5' and 3' termini, respectively. Additionally, the variants of the C and D boxes, designated C' and D' box, are usually present internally [28]. Box H/ACA snoRNAs possess a hairpin-hinge-hairpin-tail secondary structure and two conserved sequence motifs, box H and box ACA. The hinge region contains the H box (ANANNA) and the tail consists of the ACA box located 3 nt before the 3' end [29, 30]. The snoRNAs exert their functions by base-pairing with their targets and recruit related proteins to the sites of modification [31]. Box C/D snoRNAs can form 10-21 basepairs (bp) with multiple cellular RNAs. The methylated nucleotide in the target RNA is usually positioned 5 nt upstream of the D or D' box of the snoRNAs, the so called "D/D'+5" rule [6]. In box H/ACA snoRNAs, two short antisense sequences in one or both of the two hairpin domains form 9-13 basepairs with rRNA sequences that flank the target uridine to be converted to pseudouridine. The pseudouridine is always located 14 to 16 nt upstream from the H box or the ACA box of the snoRNA [29, 30]. These structural and functional features of box C/D and H/ACA snoRNAs provide the parameters for identifying snoRNAs and their function.

                      The genomic organization of snoRNA genes displays great diversity in different organisms. In vertebrates, almost all snoRNA genes are located in the introns of host genes, with a few exceptions, such as U3 which are independently transcribed [4]. In plants and trypanosomatids, snoRNA genes are present in polycistronic clusters which encode both C/D and H/ACA snoRNAs [9, 17]. A particular genomic organization, the intronic gene cluster, was first found in rice and then in Drosophila melanogaster [32, 33]. Moreover, a unique genomic organization (dicistronic tRNA-snoRNA genes) has been characterized exclusively in plants [34]. The genomic organization of snoRNA genes differs largely in fungi. In the budding yeast Saccharomyces cerevisiae, apart from seven intronic snoRNA genes, the majority of snoRNA are encoded by independent genes as well as five polycistronic snoRNA gene clusters [12]. In contrast, most box C/D snoRNA genes from the fission yeast Schizosaccharomyces pombe are intron-encoded. This raises the question about the genomic organization and expression modes of snoRNA genes in the fungus kingdom. It is well known that multicellular fungi dominate the population of fungi. However, little is known about snoRNAs in multicellular fungi. It was thus of interest to determine the snoRNA genes from a multicellular fungi to shed light on these characteristics.

                      Neurospora crassa is a filamentous fungus sharing key components with animal cells in cellular physiology and genetics, contributing to the fundamental understanding of the genome defense system, DNA methylation, post-transcriptional gene silencing, cellular differentiation and development [35]. As a model eukaryote, the genome of N. crassa has been completely sequenced [36]. However, only four box C/D snoRNAs, snR39, snR52, snR60, snR61 (Rfam) were annotated in N. crassa . Recently, we identified three U3 snoRNA genes from N. crassa; each of them is independently transcribed and contains a small intron [37](Table 1). It is evident that the majority of the N. crassa snoRNAs remain to be identified. Meanwhile, a comparative genome analysis between yeast and multicellular fungi will provide insights into the evolution of snoRNA genes in the fungus kingdom. In this study, by combining computational and experimental methods, an extensive analysis of snoRNA genes from N. crassa was performed. Here, we present the first comprehensive list of two major families of snoRNAs from N. crassa, and further discuss the characteristics and evolutionary significance of the snoRNA genes.
                      Table 1

                      Box C/D snoRNAs identified in N. crassa

                             

                      Homologs

                       

                      Name a

                      Len b

                      Chr c

                      Exp d

                      Target site(s)

                      Match e

                      G p f

                      S. p

                      S. c

                      A. t

                      H. s

                      Location g

                      Nc CD1

                      125

                      III

                      C, N

                      26S-Am2242

                      10/0

                      D'

                      -

                      snR13

                      -

                      -

                      Intron

                      Nc CD2

                      99

                      V

                      C

                      26S-Um2379

                      13/0

                      D'

                      snR66

                      snR66

                      -

                      -

                      IR

                      Nc CD3

                      90

                      I

                      C, N

                      26S-Um2840

                      14/0

                      D'

                      -

                      -

                      snoR29

                        

                      Nc CD4

                      81

                      V

                      C, N

                      18S-Cm49

                      13/0

                      D'

                      -

                      -

                      -

                      -

                      Intron

                      Nc CD5

                      80

                      VI

                      C

                      26S-Am856

                      10/0

                      D

                      snR60-I

                      snR72

                      snoR72Y

                      -

                      Intron

                          

                      26S-Um2383

                      12/0

                      D'

                      snR78

                      snR78

                      snoR37

                      U52

                       

                      Nc CD6

                      76

                      I

                      C

                      26S-Um2687

                      11/0

                      D'

                      snR51-I

                      snR51

                      -

                      U41

                      Exon

                      Nc CD7

                      84

                      VI

                      C

                      26S-Gm2250

                      12/0

                      D'

                      snR75

                      snR75

                      U15

                      U15

                      Intron

                      Nc CD8

                      85

                      I

                      C

                      26S-Gm2751

                      13/0

                      D'

                      snR48

                      snR48

                      -

                      U60

                      IR

                      Nc CD9

                      81

                      I

                      C

                      18S-Gm1122

                      14/0

                      D

                      snR41-II

                      snR41

                      -

                      -

                      Intron

                      Nc CD10

                      104

                      I

                      C, N

                      26S-Um1039

                      12/0

                      D

                      -

                      -

                      -

                      -

                      Intron

                          

                      26S-Am3264

                      13/0

                      D'

                      -

                      -

                      -

                      -

                       

                      Nc CD11

                      79

                      I

                      C

                      18S-Am793

                      13/0

                      D'

                      snR53

                      snR53

                      snoR53Y

                      -

                      Intron

                          

                      U6-Am47

                      11/0

                      D'

                      snR53

                      -

                      -

                      mgU6-47

                       
                          

                      26S-Am356

                      15/0

                      D

                      -

                      -

                      -

                      -

                       

                      Nc CD12

                      85

                      VI

                      C

                      26S-Cm2159

                      10/0

                      D'

                      -

                      snR76

                      Ath119b

                      HBII-180

                      Intron

                      Nc CD13

                      84

                      VI

                      C

                      18S-Am538

                      12/0

                      D

                      snR41-I

                      snR41

                      snR41Y

                      U62A/B

                      Intron

                      Nc CD14

                      75

                      IV

                      C

                      26S-Am2288

                      10/0

                      D'

                      -

                      -

                      U79

                      U79

                      Intron

                      Nc CD15

                      73

                      I

                      C, N

                      18S-Am159

                      11/0

                      D'

                      -

                      -

                      -

                      -

                      Intron

                          

                      tRNAThr-Um114

                      12/1

                      D

                      -

                      -

                      -

                      -

                       

                      Nc CD16

                      122

                      II

                      C

                      26S-Gm2357

                      13/0

                      D'

                      snR81

                      snR190

                      -

                      HBII-99

                      Intron

                          

                      26S-Gm1907

                       

                      D'

                      -

                      -

                      -

                      U50

                       

                      Nc CD17

                      73

                      I

                      C

                      18S-Am154

                      13/0

                      D

                      -

                      -

                      -

                      U45A/C

                      Intron

                          

                      26S-Gm2875

                      12/0

                      D'

                      -

                      -

                      snoR34

                      HBII-210

                       

                      Nc CD18

                      88

                      V

                      C, N

                      18S-Cm1004

                      11/0

                      D

                      snR79

                      snR79

                      -

                      -

                      Intron

                      Nc CD19

                      75

                      III

                      C

                      18S-Gm1423

                      13/0

                      D'

                      snR56

                      snR56

                      snoR19

                      U25

                      Intron

                      Nc CD20

                      75

                      VI

                      C, N

                      26S-Um2372

                      13/0

                      D

                      snR88

                      -

                      snoR58

                      -

                      Intron

                      Nc CD21

                      69

                      VI

                      C

                      18S-Am28

                      13/0

                      D'

                      snR74

                      snR74

                      U27

                      U27

                      Intron

                      Nc CD22

                      79

                      I

                      C

                      26S-Um1866

                      14/0

                      D'

                      snR62

                      snR62

                      U34

                      U34

                      Intron

                          

                      18S-Um893

                      11/0

                      D

                      -

                      -

                      -

                      -

                       

                      Nc CD23

                      72

                      V

                      C, N

                      26S-Gm2773

                      11/0

                      D'

                      snR38

                      snR38

                      snoR38Y

                      snR38

                      Intron

                      Nc CD24

                      75

                      VI

                      C

                      18S-Um575

                      13/0

                      D'

                      snR77

                      snR77

                      snoR77Y

                      HBII-135

                      Intron

                          

                      U5-Am62

                      15/0

                      D

                      -

                      -

                      -

                      -

                       

                      Nc CD25

                      81

                      I

                      C, N

                      26S-Am846

                      13/0

                      D'

                      -

                      -

                      -

                      -

                      Intron

                      Nc CD26

                      77

                      I

                      C

                      U2-Gm183

                      11/0

                      D'

                      -

                      -

                      -

                      -

                      Intron

                      Nc CD27

                      85

                      VI

                      C

                      26S-Am2904

                      15/0

                      D'

                      snR71

                      snR71

                      U29

                      U29

                      Intron

                          

                      26S-Cm2906

                      15/0

                      D'

                      snR69

                      snR69

                      snoR69Y

                      -

                       

                      Nc CD28

                      78

                      II

                      C

                      26S-Am1845

                      12/0

                      D'

                      -

                      -

                      snoR33

                      U95

                      Intron

                          

                      26S-Am1859

                      14/0

                      D

                      -

                      -

                      -

                      -

                       

                      Nc CD29

                      101

                      II

                      C

                      U2-Am31

                      16/0

                      D

                      -

                      -

                      -

                      SCARNA9

                      Intron

                          

                      5.8S-Am42

                      14/0

                      D'

                      -

                      -

                      snoR9

                      -

                       

                      Nc CD30

                      84

                      I

                      C, N

                      18S-Cm584

                      12/0

                      D'

                      -

                      -

                      -

                      -

                      IR

                      Nc CD31

                      101

                      VI

                      C, N

                      26S-Cm2917

                      13/0

                      D'

                      snR73

                      snR73

                      U35

                      U35

                      Intron

                      Nc CD32

                      82

                      VII

                      C

                      18S-Am161

                      11/0

                      D

                      -

                      -

                      snoR18

                      U44

                      Intron

                          

                      18S-Um167

                      11/0

                      D'

                      -

                      -

                      snoR122

                      U45A/B

                       

                      Nc CD33

                      102

                      III

                      C

                      26S-Am2218

                      13/0

                      D

                      snR63

                      snR63

                      U46

                      U46

                      Intron

                      Nc CD34

                      69

                      IV

                      C

                      18S-Um1265

                      12/0

                      D'

                      snR55

                      snR55

                      snoR34

                      U33

                      Intron

                      Nc CD36

                      90

                      I

                      C

                      26S-Cm2299

                      10/0

                      D'

                      snR64

                      snR64

                      snoR44

                      U74

                      Intron

                      Nc CD37

                      97

                      II

                      C

                      Cleavage

                        

                      U14

                      U14

                      U14

                      U14

                      Intron

                          

                      18S-Cm411

                      15/0

                      D

                      U14

                      U14

                      U14

                      U14

                       

                      Nc CD38*

                      80

                      IV

                      C

                      26S-Am1114

                      12/0

                      D'

                      snR61

                      snR61

                      U38

                      U38

                      Intron

                          

                      26S-Am2858

                      10/0

                      D'

                      -

                      -

                      -

                      -

                       

                      Nc CD39

                      73

                      I

                      C

                      26S-Am897

                      13/0

                      D'

                      snR83

                      -

                      -

                      -

                      Intron

                          

                      26S-Am375

                      11/0

                      D

                      -

                      -

                      -

                      -

                       

                      Nc CD40

                      107

                      IV

                      C, N

                      26S-Am2062

                      11/0

                      D'

                      -

                      -

                      -

                      -

                      Intron

                      Nc CD41

                      72

                      II

                      C

                      18S-Gm1423

                      12/0

                      D'

                      snR56

                      snR56

                      snoR19

                      U25

                      Intron

                          

                      26S-Cm1489

                      12/0

                      D

                      -

                      -

                      U49

                      mgh28S-2409

                       

                      Nc CD42*

                      92

                      II

                      N, R

                      18S-Am417

                      12/0

                      D

                      snR52

                      snR52

                      -

                      U83

                      Intron

                      Nc CD43

                      96

                      VI

                      N, R

                      18S-Gm559

                      11/0

                      D'

                      snR80

                      -

                      -

                      -

                      Intron

                      Nc CD44

                      85

                      IV

                      N, R

                      18S-Um1227

                      15/0

                      D'

                      snR82

                      -

                      snoR14

                      HBII-55

                      Intron

                          

                      26S-Cm776

                      11/0

                      D

                      -

                      -

                      -

                      -

                       

                      Nc CD45

                      102

                      I

                      N, R

                      26S-Um3021

                      11/0

                      D

                      -

                      -

                      -

                      -

                      IR

                      Nc CD46A

                      89

                      VII

                      N, R

                      26S-Am635

                      11/0

                      D'

                      U18

                      U18

                      U18

                      U18

                      Intron

                      Nc CD46B

                      89

                      I

                      N, R

                      26S-Am635

                      12/0

                      D'

                      U18

                      U18

                      U18

                      U18

                      Intron

                      Nc CD47*

                      75

                      III

                      N, R

                      26S-Gm785

                      16/0

                      D

                      snR39b

                      snR39b

                      snR39BY

                      snR39b

                      Intron

                      Nc CD48*

                      91

                      V

                      N, R

                      26S-Am797

                      14/0

                      D'

                      snR60-I

                      snR60

                      U80

                      U80/U77

                      Intron

                          

                      26S-Gm888

                      17/0

                      D

                      snR60-II

                      snR60

                      U80

                      U80

                       

                      Nc CD49

                      91

                      I

                      N, R

                      26S-Um2682

                      12/0

                      D

                      -

                      snR67

                      -

                      -

                      Intron

                          

                      18S-Am971

                      13/0

                      D'

                      snR54

                      snR54

                      U59

                      U59A/B

                       

                      Nc CD50

                      98

                      I

                      N, R

                      26S-Cm1418

                      15/0

                      D'

                      U24

                      U24

                      U24

                      U24

                      IR

                      Nc CD51

                      87

                      VII

                      N, R

                      26S-Am1430

                      13/0

                      D'

                      U24b

                      U24

                      U24

                      U76

                      Intron

                      Nc CD52

                      177

                      VII

                      C, N

                      tRNA-Am43

                      11/0

                      D'

                      -

                      -

                      -

                      -

                      IR

                          

                      tRNALeu-Am90

                      12/0

                      D'

                      -

                      -

                      -

                      -

                       

                      Nc CD53

                      212

                      IV

                      C, N

                      Orphan

                        

                      -

                      -

                      -

                      -

                      IR

                      Nc CD54

                      125

                      V

                      C, N

                      26S-Um667

                      10/0

                      D

                      -

                      -

                      -

                      -

                      IR

                      Nc CD55

                      137

                      IV

                      C, N

                      Orphan

                        

                      -

                      -

                      -

                      -

                      Intron

                      Nc U3A

                      262

                      I

                      C, N

                      Cleavage

                        

                      -

                      -

                      -

                      -

                      RE

                      Nc U3A-2

                      184

                      I

                      C, N

                         

                      -

                      -

                      -

                      -

                      RE

                      Nc U3A-3

                      75

                      I

                      C, N

                         

                      -

                      -

                      -

                      -

                      RE

                      Nc U3B

                      270

                      I

                      C, N

                      Cleavage

                        

                      -

                      -

                      -

                      -

                      RE

                      Nc U3B-2

                      191

                      I

                      C, N

                         

                      -

                      -

                      -

                      -

                      RE

                      Nc U3C

                      275

                      II

                      -

                      Cleavage

                        

                      -

                      -

                      -

                      -

                      RE

                      a The box C/D snoRNAs were numbered according to the order of identification. b Len, cDNA length of the snoRNA. c Chr, chromosomal location of snoRNA gene. d Exp, expression situation. C, N, R, snoRNA was identified by cDNA library, northern blotting analysis, and reverse transcription analysis, respectively. e target match, (Watson-crick pairs+G*U)/mismatch. f Gp, guide position. g IR, Intergenic region; RE, Repeat element. The genes marked with asterisks indicate that the genes were annotated in the Neurospora crassa database but were not detected by experimental methods. The data for S. p snoRNAs were cited from Luo (2004) [38] and Bi et al. (2007) [39]. A. t snoRNAs and modifications are from the plant database http://​bioinf.​scri.​sari.​ac.​uk/​cgi-bin/​plant_​snorna/​conservation. S. c snoRNAs and modifications are from the yeast snoRNA database at UMass-Amberst http://​people.​biochem.​umass.​edu/​fournierlab/​snornadb/​main.​php. Abbreviation: S. p, S. pombe; S. c, S. cerevisia; A. t, A. thaliana; H. s, H. sapiens.

                      Results

                      Identification of 55 box C/D and 20 box H/ACA snoRNAs from N. crassa

                      We initially carried out the genome-wide analysis of snoRNAs from N. crassa by employing the snoscan [12] and snoGPS programs [13]. From this database search, 89 box C/D and 131 box H/ACA snoRNA candidates were predicted (see Methods). To validate the snoRNA candidates and identify more novel snoRNAs from N. crassa, the box C/D and box H/ACA snoRNA-specific library of N. crassa were respectively constructed from mixed-stage mycelium and spores using anchored primers (18, and see Methods). To exclude the highly abundant clones and enrich the novel RNA species in our analysis, the radiolabelled oligonucleotides were used to screen the cDNA libraries (~1800 clones in the box C/D and ~ 4000 clones in box H/ACA snoRNA libraries). Subsequently, a total of 338 and 278 clones from box C/D and box H/ACA snoRNA libraries were sequenced, respectively. Taken together, 65 snoRNAs including 45 box C/D (Table 1) and 20 box H/ACA snoRNAs (Table 2) were identified. Twenty eight box C/D snoRNAs from the cDNA library were covered by the snoscan results. However, only three H/ACA snoRNAs overlapped with snoGPS results. Because the data from the computational search of H/ACA snoRNAs may include excessive false-positive candidates, they were not included for further analyses in this study.
                      Table 2

                      Box H/ACA snoRNA genes in N. crassa

                            

                      Homologs

                       

                      Name a

                      Len b

                      Chr c

                      Exp d

                      Target site (s)

                      G p e

                      S.p

                      S.c

                      A.t

                      H.s

                      Location f

                      Nc ACA1

                      136

                      III

                      N, R

                      18S-Ψ105

                      H

                      -

                      snR44

                      -

                      ACA36

                      Intron

                          

                      26S-Ψ1037

                      ACA

                      -

                      snR44

                      -

                      -

                       

                      Nc ACA2

                      159

                      III

                      N, R

                      26S-Ψ1868

                      H

                      -

                      -

                      -

                      -

                      IR

                          

                      26S-Ψ2313

                      ACA

                      -

                      snR82

                      -

                      -

                       

                      Nc ACA3

                      217

                      V

                      C, N

                      26S-Ψ401

                      H

                      -

                      -

                      -

                      -

                      IR

                          

                      26S-Ψ2095

                      ACA

                      -

                      snR3

                      -

                      ACA6

                       

                      Nc ACA4

                      206

                      II

                      C, N

                      18S-Ψ463

                      H

                      -

                      snR189

                      -

                      -

                      IR

                      Nc ACA5

                      187

                      I

                      C, N

                      18S-Ψ996

                      ACA

                      -

                      snR31

                      snoR5

                      ACA8

                      IR

                      Nc ACA6

                      165

                      VII

                      C, N

                      26S-Ψ940

                      H

                      -

                      snR8

                      -

                      ACA56

                      IR

                          

                      26S-Ψ1105

                      ACA

                      snR5

                      snR5

                      -

                      -

                       

                      Nc ACA7

                      167

                      V

                      C, N

                      Orphan

                      ACA

                      -

                      -

                      -

                      -

                      IR

                      Nc ACA8

                      192

                      II

                      C, N

                      18S-Ψ1509

                      ACA

                      -

                      -

                      -

                      -

                      IR

                      Nc ACA9

                      201

                      V

                      C, N

                      Orphan

                       

                      -

                      -

                      -

                      -

                      Intron

                      Nc ACA10

                      309

                      II

                      C, N

                      Orphan

                       

                      -

                      -

                      -

                      -

                      3'UTR

                      Nc ACA11

                      176

                      I

                      C, N

                      Orphan

                       

                      -

                      -

                      -

                      -

                      IR

                      Nc ACA12

                      296

                      II

                      C, N

                      Orphan

                       

                      -

                      -

                      -

                      -

                      IR

                      Nc ACA13

                      208

                      I

                      C, N

                      Orphan

                       

                      -

                      -

                      -

                      -

                      IR

                      Nc ACA14

                      234

                      II

                      C, N

                      26S-Ψ984

                      ACA

                      snR5

                      snR5

                      snoR81

                      ACA52

                      IR

                      Nc ACA15

                      233

                      II

                      C, N

                      26S-Ψ2902

                      H

                      -

                      snR37

                      -

                      ACA10

                      IR

                      Nc ACA16

                      189

                      II

                      C, N

                      Orphan

                       

                      -

                      -

                      -

                      -

                      Exon+3'UTR

                      Nc ACA17

                      189

                      II

                      C, N

                      18S-Ψ1733

                      H

                      -

                      -

                      snoR88

                      -

                      IR

                      Nc ACA18

                      186

                      V

                      C, N

                      26S-Ψ2309

                      H

                      -

                      -

                      -

                      E2

                      IR

                      Nc ACA19

                      178

                      V

                      C, N

                      26S-Ψ1666

                      ACA

                      -

                      -

                      -

                      -

                      IR

                      Nc ACA20

                      160

                      V

                      N, R

                      26S-Ψ2228

                      ACA

                      -

                      snR84

                      -

                      Undet

                      IR

                      a All the box H/ACA snoRNAs were numbered according to the order of identification. b Len, cDNA length of the snoRNA. c Chr, chromosomal location of snoRNA gene. d Exp, expression situation. C, N, R, snoRNA was identified by cDNA library, northern blotting analysis, and reverse transcription analysis, respectively. e Gp, guide position. g IR, Intergenic region. The data for S. p snoRNAs were cited from Luo (2004) [38]. "Undet" indicates that the snoRNA has not been identified in the human genome although the corresponding modification site was detected. Abbreviation: S. p, S. pombe; S. c, S. cerevisia; A. t, A. thaliana; H. s, H. sapiens.

                      The snoRNA candidates identified by cDNA cloning or the snoscan program were subsequently confirmed by northern blot and/or reverse transcription analyses. The expression of 27 box C/D and all 20 box H/ACA snoRNAs were positively detected as expected (Figure 1 and 2). Among these snoRNAs, the sequence of CD31 snoRNA obtained from the cDNA cloning appears uncompleted; it corresponds to the second half of CD31 full-length which is further validated by the northern blotting.
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-515/MediaObjects/12864_2009_Article_2399_Fig1_HTML.jpg
                      Figure 1

                      Northern blot and RT analyses of box C/D snoRNAs from N. crassa. A. Northern blot analyses of box C/D snoRNAs. B. Reverse transcription analyses of box C/D snoRNAs generated from the computational screen. Lane M, molecular weight marker (pBR322 digested with Hae III and 5'-end -labeled with [γ-32P]ATP).

                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-515/MediaObjects/12864_2009_Article_2399_Fig2_HTML.jpg
                      Figure 2

                      Northern blot and RT analyses of box H/ACA snoRNAs from N. crassa. A. Northern blot analyses of box H/ACA snoRNAs. B. Reverse transcription analyses of the three box H/ACA snoRNAs overlaps with the computational screen. Lane M, molecular weight marker (pBR322 digested with Hae III and 5'-end -labeled with [γ-32P]ATP.

                      Together, through the combination of computational analysis and construction of the specialized cDNA libraries, 55 box C/D and 20 box H/ACA snoRNAs were identified and all the snoRNAs are denominated according to the order of identification (Table 1 and 2).

                      In most cases (86%) the C and D boxes in snoRNAs are highly conserved when compared to the consensus sequence (UGAUGA and CUGA, see Additional file 1). However, the C' and D' box are nonconserved and exhibit much more sequence-degeneracy than their C and D box counterparts. In some instances, the C' and D' box cannot be unambiguously inferred as a result of the absence of the functional region. Generally, the box C/D snoRNAs from N. crassa are similar to their metazoan and yeast counterparts in size. However, the sizes of box H/ACA snoRNAs from N. crassa are peculiar. Almost all of them are larger than 160 nt (Figure 2), reminiscent of some irregular box H/ACA snoRNAs in S. cerevisiae. These observations show that the N. crassa snoRNAs have their own unique sequence and structural features (see Additional file 2 and 3).

                      Functional properties of the N. crassa box C/D and box H/ACA snoRNAs

                      Based on the modification rules of snoRNAs [2], 55 box C/D snoRNAs from N. crassa were predicted to direct 71 methylations. These include 64 methylations on rRNAs which comprise 43 methyls on 26S rRNA, 20 methyls on 18S rRNA and one methyl on 5.8S rRNA (see Additional file 4A). The remnant seven methylations consist of four methyls on snRNAs and three methyls on tRNAs (see Additional file 4B and 4C). Furthermore, the structure and function elements of U14 which participate in the processing of pre-rRNA were unambiguously identified. Interestingly, two different methylated sites were predicted to be guided by the same functional element of a single snoRNA CD27. Two box C/D snoRNAs (CD53 and CD55) lack the sequences complementary to either rRNAs or snRNAs and therefore belong to orphan snoRNAs. Fourteen box H/ACA snoRNAs were predicted to guide 17 pseudouridine sites of rRNAs (see Additional file 5), and no pseudouridine sites on snRNAs were predicted. The remaining six box H/ACA snoRNAs were also classified into an orphan snoRNA family lacking functional region complementary to rRNA, tRNA or snRNA. A different modification pattern appears in N. crassa as compared to the two yeasts S. cerevisiae, and S. pombe (see discussion)[38, 39].

                      Interestingly, a novel snoRNA, CD29, possesses two guide elements that can form duplexes with U2 snRNA and 5.8S rRNA for 2'-O-methylation. Primer extension mapping of 2'-O-methylated nucleotides of the U2 snRNA and 5.8S rRNA in the presence of low concentration of dNTPs resulted in stop signals at the G32 and A43 residues, indicating that U2-A31 and 5.8S-A42 are methylated (Figure 3). We have identified cognates of CD29 in other filamentous fungi, however, these cognates only possess the guide sequence for the methylation of U2 snRNA. This suggests that CD29 evolves from the snoRNA with a single guide function. This is reminiscent of the human small Cajal body-specific RNAs (scaRNAs) that can guide modification of the RNA polymerase II-transcribed snRNAs such as U2 snRNA. The comparative analyses revealed that CD29 and its homologs in fungi have one functional element similar to that of human SCARNA9 which was first known as Z32 (GeneBank accession no. AJ009638), and therefore was homologous to this human scaRNA. In addition, we characterized a multi-function box C/D snoRNA, CD11, in N. crassa . CD11 has the potential to direct a methylation in U6 snRNA, and two methylations in 18S and 26S rRNAs, respectively (Figure 3). Interestingly, the CD11 is also partially similar to mgU6-47 in mammals [40], but possesses a novel function that can guide a N. crassa-specific methylation on 26S rRNA at A356.
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-515/MediaObjects/12864_2009_Article_2399_Fig3_HTML.jpg
                      Figure 3

                      Base-pairing model and verification of modification guided by CD11 (A) and CD29 (B). Black dots indicate nucleotides predicted to be methylated. Lane H, control reaction at 1.0 mM dNTP; Lane L, primer extension at 0.004 mM dNTP, and A, C, G and T lanes, the rDNA sequence ladder. Black triangles indicate potential methylation sites.

                      Genomic organization and expression of the snoRNAs in N. crassa

                      The genomic organization of the snoRNA genes in N. crassa exhibits great diversity. Among the 55 box C/D snoRNAs, forty five snoRNA genes are intron-encoded in protein-coding or non-coding host genes. The remaining nine were found in the intergenic regions with a putative polymerase II promoter upstream and appeared independently transcribed. Meanwhile, six gene clusters that only encode box C/D snoRNAs were identified from N. crassa . Interestingly, an exon-encoded snoRNA (CD6) was identified in the snoRNA gene cluster III in contrast to another two intron-encoded snoRNAs (CD9 and CD17) in the same cluster (Figure 5). Of 20 box H/ACA snoRNA genes, 16 are located in intergenic regions and two are intron-encoded. In particular, two snoRNA genes (ACA10 and ACA16) are located in the 3' UTR of two hypothetical protein genes, one of which is similar to phosphoglycerate mutase. Obviously, different strategies dominate in the expression of the two families of snoRNA genes in N. crassa .
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-515/MediaObjects/12864_2009_Article_2399_Fig5_HTML.jpg
                      Figure 5

                      Schematic representation of snoRNA gene clusters in N. crassa. The open and gray boxes represent snoRNAs and exons, respectively. The number below indicates the length (in nucleotides) of introns. Thinner lines indicate introns. Note: figure not drawn to scale.

                      In accordance with the mode of one snoRNA per intron in vertebrates [4], a large proportion of the box C/D snoRNA genes (45 of 55) are located within introns of the host genes. The distances from the intronic snoRNA genes to the 3' splice sites of introns, which has been proven to be important for the effective processing of intronic snoRNAs from their host mRNA precursors [41, 42], resemble those in D. melanogaster [32, 41, 42]. The distances from the snoRNA genes to the 5' splice sites appear to mainly be between 41 to 60 nt, similar to those in human[41] (Figure 4).
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-515/MediaObjects/12864_2009_Article_2399_Fig4_HTML.jpg
                      Figure 4

                      The distance distributions from the intronic snoRNA genes to the 3' and 5' splicing sites of host gene introns. The gray and black bars represent space lengths from the 3' and 5' splicing sites (SS), respectively, to the snoRNA genes.

                      Remarkably, five (cluster I to V) of the six box C/D snoRNA gene clusters arehighly conserved between yeast and N. crassa (Figure 5). Although these host genes were not well annotated for their introns and exons in the N. crassa genome, canonical intron splicing sequences were observed flanking every cluster of snoRNA genes. To further confirm this observation, the mature RNA transcripts were identified with the expected sizes by cloning and sequencing of RT-PCR products. It is worth noting that two snoRNA genes, CD16 and CD37, in the cluster V are validated to be co-transcribed by RT-PCR and sequencing, though each of the snoRNA genes in the cluster has a putative promoter upstream. Intriguingly, the putative promoter upstream of CD37, a homologue of U14, would play a role in guaranteeing and promoting the function of U14 that has been demonstrated vital in diverse eukaryotes. Our results further revealed that the genomic organization of the host genes for these five clusters is most like the UHG gene in animals. The host genes of Cluster I to V only contain short open reading frames with length ranging from 159 bp to 267 bp, suggesting the little potential for protein coding just like the gas 5 [43].

                      Unexpectedly, various alternative splicing events were found in the processing of polycistronic transcripts from the snoRNA gene clusters I and II by analyzing cDNA sequences from RT-PCR of the transcripts (Figure 6). In cluster I, two alternatively spliced transcripts, differing by the absence of exon 2 or exon 2 plus exon 3 were detected. The pattern of alternative splicing in the expression of cluster II was contingent on an alternative 3' splice site that allows the lengthening or shortening of exon 3.
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-515/MediaObjects/12864_2009_Article_2399_Fig6_HTML.jpg
                      Figure 6

                      Alternative splicing in the expression of snoRNA gene cluster I and II in N. crassa. The open and black boxes represent snoRNAs and exons, respectively. The number below indicates the length (in nucleotides) of exons and introns. Thinner lines indicate introns and dashed lines indicate splicing activities. Arrows indicate the primers used in RT-PCR analysis.

                      Discussion

                      High diversity of post-transcriptional modification predicted by snoRNAs in fungi

                      Identification of guide snoRNAs in diverse organisms can provide valuable information towards understanding RNA modification patterns and their function [18]. It is interesting to compare the pattern of modifications on target RNAs of N. crassa to those described in the two yeasts, S. cerevisiae and S. pombe. Among 71 methylations predicted by the guide snoRNAs in N. crassa, 32 represent the most highly conserved modifications shared by the multicellular fungi and the yeasts, and 31 (43.7%) are modifications that have not yet been reported in other fungi when compared with the two unicellular yeasts(Figure 7). In the yeasts, only ten and eight methylations are S. cerevisiae-specific and S. pombe-specific, respectively. Our results imply a more complex modification pattern in multicellular fungi than in unicellular yeasts. They also reveal the high diversity of post-transcriptional modification of RNAs in the fungus kingdom as it has been shown that about 40% of methylations are species-specific in a protozoan Trypanosoma [17]. The species-specific modifications highlight the different modification patterns and their peculiar importance. Although eliminating a single modification does not have a dramatic effect on the ribosome [44], loss of three to five modifications in an intersubunit bridge of the ribosome (helix 69) impairs growth and causes broad defects in ribosome biogenesis and activity [45]. On the other hand, early studies have demonstrated that ribosome modifications play roles in determining antibiotic resistance or sensitivity [15, 46]. Thus the species-specific modifications have potential use in finding therapeutic targets for prevention and treatment of diseases caused by some eukaryotic pathogens.
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-515/MediaObjects/12864_2009_Article_2399_Fig7_HTML.jpg
                      Figure 7

                      Venn diagram of the relationship of methylations in three fungi. The number of each part of the methylations is shown. Abbreviation: N.c, N. crassa; S. c, S. cerevisiae; S. p, S. pombe.

                      Another interesting observation in this study was the presence of duplexes between box C/D snoRNAs and tRNAs (tRNATrp and tRNALeu from N. crassa Database). Duplexes between tRNA and snoRNAs have been also found in C. elegans [24] and recently in Plasmodium falciparum [47]. tRNA modification guided by snoRNAs has been also reported in Archaea [23]. This study provides for the first time a prediction of fungal snoRNAs and their potential target sites in tRNAs, although these remain to be confirmed by further experiments.

                      Structural and functional evolution of snoRNAs in fungi

                      Our study demonstrates the extensive separation and recombination of functional regions occurring during the evolution of snoRNA genes in fungi. For instance, the CD5 snoRNA in N. crassa possesses two conserved guiding elements. In S. cerevisiae, however, the conserved function of CD5 is executed by two independent snoRNAs, snR72 and snR78, with a single functional element [48] (Figure 8). This suggests that CD5 may have evolved as a double-guide snoRNA through recombination of two different halves of two ancestral single-guide snoRNAs. The other possibility is that a gene duplication of a double-guide snoRNA gene in S. cerevisiae led to specialization of each paralog to only target one modification site followed by loss of the other guide element for both paralogs. Another example is CD50 and CD51 that carry a conserved guiding function for U24 and U24b in S. pombe, respectively. In contrast, the U24 in S. cerevisiae has two guiding functions. Comparative analyses revealed that the structure and function of U24 are well conserved among the budding yeast and the flowering plants A. thaliana and rice, but the homologues of the S. cerevisiae U24 exist as two independent snoRNAs in other distant eukaryotes, such as human and mouse [49]. This suggests that U24 snoRNA gene has evolved in two pathways, with one leading to a dual functional snoRNA gene and the other separating the guiding functions and giving rise to two independent snoRNA genes.
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-515/MediaObjects/12864_2009_Article_2399_Fig8_HTML.jpg
                      Figure 8

                      Alignment of homologous snoRNAs from three multicellular fungi and two yeasts. Conserved box elements are bold and boxed regions denote antisense elements. Stars indicate conserved nucleotides. Sp, S. pombe; Sc, S. cerevisiae; An, Aspergillus niger; Mg, Magnaporthe grisea; Nc, N. crassa .

                      It has been demonstrated the reciprocal evolutionary change between snoRNA complementary region and their rRNA target sequence in plants and nematodes[9, 24]. Our analyses indicate that co-evolution between snoRNAs and rRNAs exists widely in N. crassa (Figure 9) and plays an important role in preservation of phylogenetic conserved methylated sites of rRNAs which is essential for protein synthesis.
                      http://static-content.springer.com/image/art%3A10.1186%2F1471-2164-10-515/MediaObjects/12864_2009_Article_2399_Fig9_HTML.jpg
                      Figure 9

                      Coevolution between snoRNAs and their targets. A. Nucleotides of box C/D snoRNAs in the complementary region were changed in coordination with its target rRNA maintaining phylogenetical conservation of rRNA methylated sites. B. Nucleotides changed in box H/ACA snoRNAs respond to specific changes in the 18S rRNA of N. crassa . The nucleotides marked by black dot represent the 2'-O-methylation. Basepairs changed are indicated by arrows.

                      RIP may impact on the generation of snoRNA isoforms by gene duplication and transposition

                      SnoRNA gene isoforms or variants exist widely in diverse organisms, particularly in plants. For example, 97 box C/D snoRNAs with a total of 175 different gene variants were identified in the A. thaliana genome [50], and 346 gene variants encoding 120 box C/D snoRNAs were found in Oryza Sativa [9]. Compared with the plant snoRNAs, only a paucity of yeast snoRNA paralogs was detected because of a relatively small compact genome (~12 Mb for S. cerevisiae). The N. crassa genome (~ 40 Mb) is three-fold larger than that of the yeast; however, most snoRNA genes in this species are singleton. Why are the snoRNA genes devoid of isoforms in the N. crassa genome? It is known that a mutagenic process termed repeat-induced point mutation (RIP) has a profound impact on N. crassa genome evolution, which has greatly slowed the creation of new genes through genomic duplication and resulted in a genome with an unusually low proportion of closely related genes [51]. Of the predicted 10082 protein-coding genes, only six pairs (12 genes) share >80% nucleotide or amino-acid identities in their coding sequences [36]. RIP identifies duplications that are greater than ~400 bp (~1 kb in the case of unlinked duplications) and induces C:G to T:A during the sexual cycle [52, 53]. Early studies have provided clear evidence of retrotransposons inactivated by RIP [54, 55]. The analysis of the N. crassa genome sequence also revealed a complete absence of intact mobile elements [36]. Therefore the creation of new genes including snoRNA genes or their host genes through gene duplication and transposition seems to be impeded. It has been proposed that most, if not all paralogs in N. crassa duplicated and diverged before the emergence of RIP [51]. We have identified three U3 snoRNA gene variants, NcU3A, NcU3A-2 and NcU3A-3 in N. crassa (37). The sequence analysis revealed that these molecules have undergone nucleotide substitutions rather than RIP according to the calculation method previously reported [36]. In the case of CD46A and CD46B, we speculate that the two snoRNA gene isoforms may have duplicated and diverged before the emergence of RIP.

                      Alternative splicing in the expression of non-coding RNA genes with introns

                      It is well known that alternative splicing is an important and widespread process where one gene produces more than one type of mRNA which is then translated into different proteins in multicellular organisms [56]. Bioinformatic analysis indicates that 35-65% of human genes are involved in alternative splicing, which contributes significantly to human proteome complexity [57, 58]. However, alternative splicing is rarely reported for non-coding RNA genes which encode multiple introns. In this study, we identified several alternative splicing events that occurred in the processing of RNA precursors transcribed from the snoRNA gene cluster I and II of N. crassa. It has been reported that the mouse gas5 gene, a non-coding RNA and snoRNA host gene, had four alternative splicing transcripts [43]. Although different in snoRNA composition, the snoRNA gene clusters in N. crassa are most like UHG genes resembling gas5. Our results show that alternative splicing occurs frequently in the expression of snoRNA host genes in lower eukaryotes. This lends support to the concept that alternative splicing may be an ancient mechanism in regulating the expression of both protein-coding and non-coding RNA genes with introns. More work is necessary to elucidate the biological significance of the alternative splicing in the expression of non-coding RNA genes.

                      Conclusion

                      In this study, we report the first extensive identification of box C/D and box H/ACA snoRNAs from the filamentous fungus N. crassa using a combination of computational and experimental method. The repertoire characteristics, targets, genomic organization and the unique function of the N. crassa snoRNA genes were extensively compared with those of potential orthologues in close and distant organisms such as S. pombe, S. cerevisiae, A. n iger, M. grisea, A. thaliana and H. sapiens . Our results improve annotation of snoRNA genes in the N. crassa genome, an important model filamentous fungus, and provide insights into the characteristics and evolutionary significance of the snoRNA genes in the fungus kingdom.

                      Methods

                      Strains and Media

                      The N. crassa wild-type strain (As 3.1604, purchased from the China General Microbiological Culture Collection Center) was used for the construction of the cDNA library and all RNA analyses. The strain was grown in PSA medium (2% sucrose, 20% extract of potato) at 30°C. The Escherichia coli strain TG1 grown in 2YT (1.6% Bacto tryptone, 1% yeast extract, 0.5% NaCl) liquid or solid medium was used for cloning procedures.

                      Construction and screening of cDNA library

                      We prepared total RNA from N. crassa culture according to the guanidine thiocyanate-phenol-chloroform procedure described by Chomoczynski et al [59]. Small RNA (~20 μg) was fractionated by 50% PEG-8000 and 0.5 M NaCl. The construction of cDNA library were performed as described previously with minor modifications (see Additional file 6) [60]. After randomly sequencing clones, we employed dot hybridization to screen the colony PCR products with P47 and P48 as described by Liu et al. [37] We sequenced clones exhibiting the lowest hybridization signal.

                      Computational identification of box C/D snoRNA genes

                      Genomic sequences of N. crassa [36] available at http://​www.​broad.​mit.​edu/​annotation/​genome/​neurospora/​Home.​html (N. crassa assembly 7) were downloaded and searched for potential box C/D snoRNAs target rRNA/snRNA using snoscan [12] with default parameters. Methylated sites prepared for the snoscan included the conserved methylated nucleotides of S. cerevisiae (yeast snoRNA database), H. sapiens (snoRNA-LBME-db), and D. melanogaster [32]. The snoscan results were processed by an in-house developed perl program for candidate selection. A sequence with the following characteristics was considered as candidate: ① box C motif bit score ≥ 7.48, box D motif bit score ≥ 8.05, ② the guide bit score ≥ 18.65, the guide sequence and the target sequence can form a concatenated 10 bp duplex with at most 1 GU pair allowed, or can form a concatenated 9 bp duplex with high GC content. ③ if the guide region is adjacent to the D' box, the length of spaces between box C and guide sequence must be ≤ 20 bp. If the guide region is adjacent to the D box, the length of spaces between box C and guide sequence must be between 40 and 85 bp. ④ total sequence length between 75 bp and 130 bp, total overall bit score ≥ 20. The candidates within CDS region predicted by Broad/Whitehead Institute automatic gene calling software (a combination of manual annotation, FGENESH, GENEID, and GENEWISE) [36] were removed. The BLAST program [61] was used to search gene variants of all novel snoRNA genes to establish the snoRNA gene isoforms. About 1 kb of flanking sequences of the snoRNA gene candidates was searched further for possible box C/D snoRNA genes and additional non-canonical C/D gene candidates.

                      Northern blot analysis

                      An aliquot of 30 μg total RNA was separated by electrophoresis on an 8% polyacrylamide gel containing 8 M urea and electrotransferred onto nylon membrane (Hybond-N+; Amersham) using semi-dry blotting apparatus (BioRad). After immobilizing RNA using a UV cross-linker, northern blot hybridization was performed as previously described [49].

                      Reverse transcription and mapping of ribose methylation

                      Reverse transcription was carried out in a 20 μl reaction mixture containing 15 μg of total RNA and a corresponding 5'-end-labeled primer. After denaturation at 65°C for 5 min and then cooling to 42°C, 200 units of M-MLV reverse transcriptase (Promega) were added and extension carried out at 42°C for 1 hour. The cDNA was separated on an 8% polyacrylamide gel (8 M urea) and then analyzed with an imager.

                      The mapping of rRNA methylated sites was determined by primer extension at low dNTP concentrations as described previously [40, 62]. Briefly, the N. crassa 18S and 26S rDNA were amplified by PCR with the primer pair Nc18SF/Nc18SR and Nc26SF/Nc26SR, respectively, and then cloned into the pMD-18T vector (Takara). The plasmid DNA insert was directly sequenced with the same primer used for reverse transcription and run in parallel with the reverse transcription reaction on an 8% polyacrylamide gel (8 M urea).

                      RT-PCR analysis

                      15 μg of total RNA was reverse transcribed with 200 U of M-MLV reverse transcriptase (Promega) using the box C/D snoRNA gene cluster specific reverse primers (see Additional file 7) in a 20 μl reaction mixture as described above for reverse transcription and mapping of ribose methylations. The negative RT control was carried out without M-MLV reverse transcriptase. We designed two specific antisense oligonucleotides: the first reverse primer used in the reverse transcription reaction overlaps the last several nucleotides of the second reverse primer used in the PCR reaction to help avoid non-specific PCR products. After 1 h at 42°C, 2 μl of RT reaction was used for PCR amplification with the second reverse primer and the corresponding forward primer (see Additional file 7) in a final volume of 20 μl. The positive PCR control was performed on N. crassa genomic DNA with the same pair of primers. Negative PCR control was performed on 2 μl of the negative control RT reaction with the same pair of primers. The PCR program: 30 cycles of denaturation (30 s, 94°C), annealing (30 s, 50-55°C), and extension (1-2 min, 72°C), following by a final extension (10 min, 72°C). The PCR product was purified from a 1.5% agarose gel with the QIAquick Gel extraction Kit (QIAGEN) and cloned into pMD-18T vector (Takara) and transformed into the strain TG1 of E. coli . Positive clones were subsequently chosen for sequencing.

                      Oligonucleotides

                      Oligonucleotides used for construction of the cDNA library, northern blot analyses of novel snoRNAs and the primers for reverse transcription and RT-PCR experiments are not shown (see Additional file 7).

                      Database accession numbers

                      The sequences of all snoRNAs determined in this work have been deposited in the GenBank Nucleotide Sequence Databases under accession numbers EU780925 - EU780999 and EU526091-EU526095.

                      Abbreviations

                      snoRNA: 

                      small nucleolar RNAs

                      rRNA: 

                      ribosomal RNA

                      snRNA: 

                      spliceosomal nuclear RNA

                      tRNA: 

                      transfer RNA

                      UHG: 

                      U snoRNA host gene

                      SL RNA: 

                      spliced-leader RNA

                      pre-rRNA: 

                      precursor ribosomal RNA

                      bp: 

                      basepairs

                      dNTP: 

                      deoxyribonucleoside triphosphate

                      scaRNAs: 

                      small Cajal body-specific RNA

                      RIP: 

                      Repeat-Induced Point Mutation

                      gas5: 

                      growth arrest-specific 5

                      UTR: 

                      untranslated region

                      RT-PCR: 

                      reversed transcript PCR

                      cDNA: 

                      complementary DNA

                      CDS: 

                      coding sequence.

                      Declarations

                      Acknowledgements

                      We would like to thank Xiao-Hong Chen, Qiao-Juan Huang and Yi-Ling Chen for technical assistances. This research is supported by the National Natural Science Foundation of China (30570398, 30830066), the funds from the Ministry of Education of China and Guangdong Province (No. IRT0447, NSF05200303) and the National Basic Research Program (No. 2005CB724600).

                      Authors’ Affiliations

                      (1)
                      Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory for Biocontrol, Sun Yat-sen University
                      (2)
                      Centre National de la Recherche Scientifique (CNRS), UPR 2167, CGM

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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.

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